Genetic Analysis of Mental Retardation in...
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Genetic Analysis of Mental Retardation in Pakistan By Zehra Agha CIIT/FA08-PBS-005/ISB PhD Thesis in Biosciences COMSATS Institute of Information Technology Islamabad, Pakistan Spring, 2013
Transcript of Genetic Analysis of Mental Retardation in...
i
Genetic Analysis of Mental Retardation in
Pakistan
By
Zehra Agha
CIIT/FA08-PBS-005/ISB
PhD Thesis
in
Biosciences
COMSATS Institute of Information Technology
Islamabad, Pakistan Spring, 2013
ii
COMSATS Institute of Information Technology
Genetic Analysis of Mental Retardation in
Pakistan
A Thesis Presented to
COMSATS Institute of Information Technology, Islamabad
In partial fulfillment
Of the requirement for the degree of
PhD
(Biosciences)
By
Zehra Agha
CIIT/ FA08- PBS-005/ISB
Spring, 2013
iii
Genetic Analysis of Mental Retardation in Pakistan
A Doctoral Thesis submitted to the Department of Biosciences as partial
fulfillment of the requirement for the award of Degree of PhD (Biosciences).
Name Registration Number
Zehra Agha CIIT/FA08-PBS-005/ISB
Supervisor
Prof. Dr. Raheel Qamar (T.I.)
Tenured Professor of Biochemistry and Molecular Biology
Dean of Research, Innovation and Commercialization
Islamabad, Pakistan
Co-Supervisor
Prof. Dr. Hans van Bokhoven
Department of Human Genetics,
Radboud University Medical Centre,
Nijmegen, The Netherlands
Co-Supervisor
Dr. Maleeha Azam
Department of Biosciences, CIIT, Islamabad
COMSATS Institute of Information Technology (CIIT),
Islamabad, Campus. June, 2013
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Final Approval
This PhD thesis titled
Genetic Analysis of Mental Retardation in
Pakistan
By
Zehra Agha
CIIT/FA08-PBS-005/ISB
Has been approved
For COMSATS Institute of Information Technology, Islamabad
External Examiner: __________________________________________
Dr. Atika Mansoor
Chief Scientific Officer
Institute of Biomedical and Genetic Engineering, Islamabad
External Examiner: __________________________________________
Dr. Faheem Tahir
National Institute of Health, Islamabad
Supervisor: ________________________________________________
Prof. Dr. Raheel Qamar (T.I.)
Tenured Professor of Biochemistry and Molecular Biology
Dean of Research, Innovation and Commercialization, Islamabad
HoD: __________________________________________________
Prof. Dr. Raheel Qamar (T.I.)
Tenured Professor of Biochemistry and Molecular Biology
Department of Biosciences, CIIT, Islamabad
Chairman:__________________________________________________
Prof. Dr. Syed Habib Bokhari
Department of Biosciences, CIIT, Islamabad
Dean, Faculty of Science: __________________________________________
Prof. Dr. Arshad Saleem Bhatti (T.I.)
CIIT, Islamabad
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Declaration
I, Zehra Agha, CIIT/FA08-PBS-005/ISB hereby declare that I have produced the
work presented in this thesis, during the scheduled period of study. I also declare that
I have not taken any material from any source except referred to wherever due, that
amount of plagiarism is within the acceptable range. If a violation of Higher
Education Commission of Pakistan (HEC) rules of research has occurred in this
thesis, I shall be liable to punishable action under the plagiarism rules of the HEC.
______________________
(Zehra Agha)
(CIIT/FA08-PBS-005/ISB)
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Certificate
It is certified that Mr. Zehra Agha, CIIT/FA08-PBS-005 has carried out all the work
related to this thesis under my supervision at the Department of Biosciences, CIIT,
Islamabad and the work fulfills the requirement for the award of PhD Degree.
Date:_______________
Supervisor:
Prof. Dr. Raheel Qamar, T.I.
Tenured Professor of Biochemistry and Molecular Biology
Dean of Research, Innovation and Commercialization, Islamabad
Head of Department:
Prof. Dr. Raheel Qamar (T.I.)
Head
Department of Biosciences
CIIT, Islamabad.
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DEDICATION
My Affectionate Parents, My Dear Husband
&
My Wonderful Children
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ACKNOWLEDGEMENTS
All praises to Almighty ALLAH, the Omnipotent, the Most Compassionate and His
Prophet Muhammad (P.B.U.H) the most perfect ever born on the surface of the
earth, who is forever a beacon of guidance and knowledge for humanity as a whole.
I wish to express my sincere gratitude to everyone who has contributed to this work
and encouraged me along the way, especially my family who always support me in
every aspect of my career.
First and the foremost, thank to my worthy supervisor Prof. Dr. Raheel Qamar (T.I.)
Dean, Research, Innovation & Commercialization, COMSATS Institute of
Information Technology, Islamabad, who remained a source of inspiration for me
and for giving me the opportunity to work under his dynamic supervision.
My sincere thanks to Prof. Dr. Hans van Boekhoven, NCMLS, Radboud University,
The Netherland, who gave me a chance to work in his group. I am also grateful to Dr.
Zafar Iqbal who made my learning experience wonderful. I am also grateful to the
Higher Education Commission (HEC) Pakistan for their contribution in the
completion of this work by funding through their IRSIP and indigenous fellowship for
PhD programme.
I am indeed grateful to Dr. Maleeha Azam and Dr. Muhammad Ajmal for sharing
their expertise in human genetics and for their advice and guidance throughout this
work. I wish to register my deep sense of gratitude and sincere thanks to my senior
lab fellow Dr. Muhammad Imran for his guidance and help in my research work. I
appreciate the encouragements of all my colleagues and other friends especially
Humaira Ayub and Sobia Shafique. I feel at a loss of words and limitedness of
space to write their names but all those who have been in COMSATS I owe my
gratitude to them. I offer my humble gratitude to my dear Husband, affectionate
father, and my dear sisters and brothers. Further I cannot express my feelings in words
to thank my dearest Mother (late), without her prayers and love I would not have been
able to achieve this task. I thank to all the participants who donated blood for the
completion of this study.
Zehra Agha
CIIT/FA08-PBS-005/ISB
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ABSTRACT
Genetic Analysis of Mental Retardation in Pakistan
Genetic defects are estimated to account for more than 50% of Intellectual disability
(ID) cases, which is a highly heterogeneous genetic disorder with a prevalence of 1 to
2.5% in the World. A number of mutations in more than 450 different genes have
been found to be involved in the pathogenesis of Intellectual disability (ID) including
X linked ID as well as Autosomal dominant ID but very few data are available for
autosomal recessive syndromic as well as nonsyndromic ID.
In the current study sixteen different families (namely MRQ1, MRQ2, MRQ5,
MRQ8, MRQ11, MRQ12, MRQ14, MRQ15, MRQ16, MRQ17, MRQ18, MRQ19,
MRQ20, D1, PKMR71 and PKMR176) were selected for molecular analysis, which
also included the detailed clinical investigation of affected members. Diverse
methodologies were employed to find the genetic cause among the families such as
candidate gene analysis, microarray analysis and exome sequencing. Homozygosity
mapping of eleven families (MRQ1, MRQ2, MRQ5, MRQ11, MRQ12, MRQ14,
MRQ15, MRQ17, MRQ19, MRQ20 and D1) was performed using the Affymetrix
2.5K SNP microarray. Candidate gene analysis among the obtained homozygous
regions lead to the identification of three novel mutations in three known ID genes
RBBP8, BBS10 and TPO in families MRQ12, MRQ19 and MRQ18 respectively.
RBBP8 sequencing identified the mutation c.919A>G, p.Arg307Gly in family
MRQ12, while there was a 10bp deletion in exon 2 of BBS10, c.1958_1967del
(p.Ser653Ilefsx4) in family MRQ19 and a missense substitution c.14C>G, p.Ala5Gly
was found in the TPO gene. The latter was probably not the cause of ID in the family
MRQ18 due to low pathogenicity score. The data of Affymetrix 2.5K SNP microarray
was also analyzed for copy number variations in which three microdeletions of 607kb,
455kb and 444.26kb were found in three families i.e. MRQ12, D1 and MRQ5,
respectively. The heterozygous microdeletion of 607kb in family MRQ12
encompasses the exon 13-19 of gene NRXN1 including part of α- promoter as well as
the β- promoter of NRXN1, while the heterozygous microdeletion of 455kb in patient
x
of family D1encompasses first seven exons of the gene NRXN1 including the α-
promoter. Another heterozygous microdeletion of 444.26 kb was found in four
affected members from two branches of MRQ5 in the region of chromosome 15q11.1.
The region was highly polymorphic as reported in many previous studies hence the
microdeletion found in the current study was probably not involved in pathogenesis of
ID in family MRQ5.
In three recessive families MRQ11, MRQ14 and MR15 exome sequencing revealed
multiple homozygous and compound heterozygous variants, however, segregation
analysis by Sanger sequencing identified three novel variants in three novel genes
ZNF589, MLL4 and HHAT to be causative genes in these families. The variant in gene
ZNF589 in family MRQ11 was c.1604C>A, p. L319H, while the variant c.2456C>T,
p.P819H found in family MRQ14 was identified to be causing Kleefstra syndrome in
this family in a unique autosomal recessive mode of inheritance. A de novo
heterozygous variant c.1158G>C, p.W386C was found in family MRQ15 in HHAT.
The involvement of HHAT in nonsyndromic ID has not been reported previously,
however, it has been shown to cause holoprosencephaly in a mouse model. The
current study reveals that ID is a highly heterogeneous disorder and there probably are
many more genes which are involved in pathogenicity of this disorder and that the
advance techniques such as microarray analysis and exome sequencing are powerful
techniques to find the causative mutations in such genetic disorders.
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TABLE OF CONTENTS
1 Introduction…………………………………………………………………… 1
1.1 Intellectual Disability…………………………………………………... 1
1.1.1 Role of Nervous System in ID………………………………... 1
1.1.2 Classification of ID…………………………………………… 4
1.1.3 Prevalence…………………………………………………….. 4
1.1.4 Etiology of ID………………………………………………… 5
1.2 Diagnosis of ID………………………………………………………… 7
1.2.1 Clinical Evaluation ……………………………………………... 7
1.2.2 Molecular Diagnosis……………………………………………. 7
1.3 Role of Chromosomal Aberration in Pathogenesis of ID……………… 8
1.3.1 Numerical Chromosomal Abnormalities………………………... 8
1.3.2 Common Autosomal Trisomies …………………………............ 9
1.3.3 Structural Chromosomal Abnormalities…………………............ 9
1.3.4 Unbalanced Rearrangements……………………………………. 10
1.3.5 Cytogenetically Visible Deletions………………………………. 10
1.3.6 Cytogenetically Invisible Microdeletions………………………. 10
1.4 Trinucleotide Repeat Expansion……………………………………….. 11
1.5 Fragile X Syndrome……………………………………………………. 12
1.6 Role of Mitrochondrial Genome in Intellectual Disability…………….. 13
1.7 Metabolic Disorders…………………………………………………..... 13
1.8 Endocrine Hormonal Disorders………………………………………… 14
1.9 Epigenetic Modifications causing ID…………………………………... 14
1.10 Monogenic Causes of ID……………………………………………….. 14
1.11 Copy Number Changes in Single Gene………………………………… 15
1.12 X-linked ID…………………………………………………………….. 15
1.12.1 Molecular Pathways involved in XLID Genes……………….. 19
1.13 Autosomal Dominant Intellectual Disability …………………………... 20
1.14 Autosomal Recessive Intellectual Disability………………………….... 23
1.14.1 Autosomal Recessive Nonsyndromic ID………………........... 23
1.14.2 Autosomal Recessive Syndromic ID…………………………. 23
1.14.2.1 Bardet Biedle Syndrome…………………………. 23
1.14.2.2 Microcephaly Associated………………………… 26
1.15 Molecular Pathways Involved in ID…………………………………… 27
1.15.1 Genes of Glutamate Receptors and Excitatory Synapse …... 27
1.15.2 Genes of Synaptic Plasticity Pathway……………………... 28
1.15.3 Cell Adhesion Pathway…………………………………...... 28
1.15.4 Genes of MAGUK Family…………………………………. 28
1.15.5 Rho GTPase Pathway……………………………………… 30
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1.15.6 Genes of Enzymatic Pathway……………………………… 30
1.15.7 Housekeeping Genes……………………………………...... 31
1.15.8 Genes of Epigenetic Pathway……………………………… 31
1.15.9 Miscellaneous Pathways…………………………………… 32
1.16 Objectives………………………………………………………………. 32
2 Materials and & Methods……………………………………………………..
2.1 Clinical Diagnosis………………………………………………………. 35
2.2 Collection of Families and Blood Sampling……………………………. 36
2.3 Genomic DNA Extraction from Blood…………………………………. 36
2.4 FMR1 Screening………………………………………………................ 36
2.4.1 CGG RP PCR…………………………………………………….. 37
2.4.2 Capillary Electrophoresis (CE) Genotyping……………………... 37
2.4.3 Data Analysis…………………………………………………...... 37
2.4.4 Southern Blot Analysis…………………………………………… 37
2.5 Microarray Homozygosity Mapping……………………………………. 38
2.5.1 Steps for SNP array………………………………………………... 38 2.5.2 Sample Preparation………………………………………………... 38 2.5.3 Digestion of sample by Restriction enzyme………………………. 39
2.5.4 Ligation……………………………………………………………. 39 2.5.5 Polymerase Chain reaction……………………………………….. 39 2.5.6 Fragmentation……………………………………………………... 39 2.5.7 Labelling………………………………………………………….. 40 2.5.8 Targeted hybridization……………………………………………. 40 2.5.9 Data Analysis…………………………………………………….. 40 2.6 Candidate Gene Analysis and Mutation Screening……………............... 41
2.6.1 Sanger Sequencing……………………………………………….
2.6.2 In silico analysis of variants………………………………………
41
41 2.7 CNV Analysis…………………………………………………………… 42
2.8 Next Generation Sequencing…………………………………………… 43
2.8.1 Libaray preparation………………………………………………. 43
2.8.2 NGS Data Filtration……………………………………………… 44 2.8.2 Variant Screening………………………………………………… 45
2.9 X-Exome Sequencing…………………………………………………... 46
3 Results……………………………………………………………………..........
3.1 Results…………………………………………………………………... 54
3.2 Family MRQ1…………………………………………………………... 54
3.3 Family MRQ2…………………………………………………………... 57
3.4 Family MRQ5…………………………………………………………... 57
3.5 Family MRQ8…………………………………………………………... 64
3.6 Family MRQ11…………………………………………………………. 67
3.7 Family MRQ12…………………………………………………………. 71
3.8 Family D1………………………………………………………………. 78
3.9 Family MRQ14…………………………………………………………. 80
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3.1 Family MRQ15…………………………………………………………. 84
3.11 X-linked Families………………………………………………………. 87
3.11.1 Families…………………………………………………………. 87
3.12 Family MRQ17………………………………………….......................... 93
3.13 Family MRQ18………………………………………….......................... 93
3.14 Family MRQ19………………………………………….......................... 101
3.15 FamilyMRQ20…………………………………………........................... 105
4. Discussion………………………………………………………………............ 111
5. References……………………………………………………………………… 130
xiv
LIST OF FIGURES
Fig. 1.1 Different structures of brain with their functions……………….............. 3
Fig. 1.2 Diagrammatic representation and etiological classification of different
types of ID ……………………………………………………………… 6
Fig. 1.3 Some of the known genes for ID and their mechanisms……………….. 29
Fig.3.1 Pedigree of family MRQ1. …………………………………………….. 54
Fig.3.2 Homozygosity Mapper plot showing multiple the homozygous regions in
red color.…........................................................................................... 56
Fig.3.3 Pedigree of family MRQ2……………………………………………….. 58
Fig.3.4 Plot of Homozygosity Mapper showing weak homozygous regions in red
color………………………………………………………………………. 59
Fig.3.5 CNAG picture of Affymetrix 250k SNP array showing addition of one
extra copy of chromosome in chromosome 21… ………………………. 60
Fig. 3.6 Pedigree of family MRQ5. …………………………………………….. 61
Fig. 3.7 Homozygosity Mapper plot of MRQ5………………………………….... 63
Fig. 3.8 CNAG data of chromosome 15 of the four affected members of MRQ5
carrying the microdeletion……………………………………………… 64
Fig. 3.9 Pedigree of family MRQ8………………………………………………... 66
Fig. 3.10 Pedigree of family MRQ11……………………………………………... 67
Fig. 3.11 Homozygosity Mapper plot showing the multiple homozygous regions in
affected members of family MRQ11 in red color…………………….. 68
Fig. 3.12 Sequencing chromatogram of ZNF589 variant…………………………... 70
Fig. 3.13 Pedigree of family MRQ12…………………………………………….. 71
Fig. 3.14 Frontal picture of Proband of MRQ12………………………………….. 72
Fig. 3.15 Sequencing chromatogram of RBBP8 mutation among the family
members of MRQ12…………………………………………………….. 74
Fig. 3.16 Original data of CNV analysis derived from 250k Affymetrix SNP array
of proband from family MRQ12………………………………………… 75
Fig.3.17(A) Pedigree of family D1…………………………………………………... 75
Fig.3.17(B) Original data of Proband CNV analysis derived from Affymetrix 2.7
microarray analysis of family D1 showing deletion in the encircled
region………………………………………………….............................. 77
xv
Fig. 3.18 Pedigree of family MRQ14………………………………………………. 77
Fig.3.19(A) Photograph of MRQ14 proband indicating the facial features of Kleefstra
syndrome……………………………………………………… 79
Fig.3.19(B) The homozygosity mapper plot showing the multiple homozygous
regions in red color in family MRQ14…………………………………... 80
Fig. 3.20 The sequencing chromatogram of mutation in MLL4…………………….. 80
Fig. 3.21 Pedigree of family MRQ15……………………………………………... 81
Fig. 3.22 The sequencing chromatogram of variant in HHAT………………………… 83
Fig. 3.23 Pedigree of family
MRQ16………………………………………............................................ 84
Fig.3.24 Pedigree of family PKMR71…………………………………………… 86
Fig.3.25 Pedigree of family PKMR176………………………………………….. 89
Fig.3.26 Conservation analysis of CXORF58 variant p.R256Q from the software
Alamut (Bio interactive software)……………………………………….. 90
Fig.3.27 Conservation analysis of TCEANC variant p.Y312N from the software
Alamut (Bio interactive software)………………………………………. 94
Fig. 3.28 Pedigree of family MRQ17……………………………………….. 95
Fig. 3.29 The homozygosity Mapper plot of Affymetrix 250K SNP array showing
common homozygous regions in red color in affected members of family
MRQ17………………………………………………………………….. 96
Fig. 3.30 A Pedigree of family MRQ18……………………………………………. 97
Fig. 3.31 Sequencing chromatogram of variation found in TPO gene for all
members of family MRQ18……………………………………………... 98
Fig. 3.32 Pedigree of family MRQ19………………………………………………. 100
Fig.3.33 CT images of Proband …………………………………………………... 101
Fig.3.34 Sequencing chromatogram of 10bp deletion in exon 2 of
BBS10……………………………………………………………………… 104
Fig.3.35 Pedigree of family 20……………………………………………………...
105
Fig.3.36 Homozygosity Mapper plot of Affymetrix 250k array data showing weak
homozygous regions among affected members (in red
colour)………………………………………………………… ………….
106
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LIST OF TABLES
Table 1.1 Parts of the limbic system involved in different important
functions.…………………………………………………………. 2
Table 1.2 List of some known XLID genes (Adapted from Chiurrazi, et al.
(2008) and Gecz, (2009)…………………………………………… 16
Table 1.3 List of some known AD-ID genes and loci involved in syndromic
and nonsyndromic ID…………………………………………….... 21
Table 1.4 List of known loci and genes identified for nonsyndromic AR-ID 24
Table 2.1 Family MRQ14 homozygous and compound heterozygous
variants validation using Sanger sequencing and in silico
prediction…………………………………………………………..
46
Table 2.2 Family MRQ11 homozygous and compound heterozygous variant
validation using Sanger sequencing and in silico, prediction……... 48
Table 2.3 Family MRQ15 homozygous and compound heterozygous variant
validation using Sanger sequencing and in silico pathogenecity
predictions…………………………………………………………. 50
Table 3.1 Summary of families studied……………………………………… 53
Table 3.2 List of homozygous regions represented by starting and end
positions and rs numbers of SNPs…………………………………. 57
Table 3.3 List of variants, c DNA and amino acid changes in MRQ16,
PKMR71 and PKMR176………………………………………….. 87
Table 3.4 List of variants, segregation among all family members in
MRQ16…………………………………………………………….. 88
Table 3.5 List of variants segregation among all family members of
PKMR71…………………………………………………………... 92
Table 3.6 List of variants segregation among all family members of
PKMR176………………………………………………………….
93
Appendix
Appendix 1 List of primers for all coding exons of TPO………………………
Appendix 2
List of primers for exons of
NTF3………………………………………………………………
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Appendix 3 List of primers for all coding exon of NRXN1…………………………...
Appendix 4 List of primers for all coding exon of RBBP8……………………………
Appendix 5 List of Primers for all coding exons of HPRT……………………………
Appendix 6
List of primers for all coding exons of BBS10, which was sequenced in
7 fragments (1 for Exon 1, and 6 to cover the large Exon 2).
Appendix 7 List of primers for all coding exons of …………………………………..
Appendix 8 List of primers for all coding exons of RELN………………………...
Appendix 9 List of primers of NRXN1 copy number detection by qPCR…………
Appendix 10
Selected homozygous and compound heterozygous variants for the
family MRQ14, primer sequences, product sizes and annealing
temperatures…………………………………………………………..
Appendix 11
Family MRQ11 selected homozygous and compound heterozygous
variants, primer sequences, product sizes and annealing
temperatures…………………………………………………………...
Appendix 12
Selected homozygous and compound heterozygous variants of family
MRQ15, primer sequences, product sizes and annealing
temperatures………………………………………………………
Appendix 13
Selected homozygous and compound heterozygous variants of family
MRQ20, primer sequences, product sizes and annealingtemperatures.
Appendix 14 List of primers for variants of the family PKMR71………………….
Appendix 15 List of primers for the variants of the family PKMR176……………..
Appendix 16
Homozygous regions obtained among the affected members of family
MRQ14 and MRQ11……..........................................................
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LIST OF ABBREVIATIONS
AR-ID Autosomal recessive Intellectual Disability
AD-ID Autosomal Dominant Intellectual Disability
aa Amino Acid
AIDS Acquired Immuno Defieciency Syndrome
AD Alzheimer‟s Disease
ALS Amyotrophic Lateral Sclerosis
APS Ammonium Persulphate
Arg Arginine
ARNS-ID
Autosomal Recessive Non-Syndromic Intellectual
Disability
ARX Aristaless-Related Homeobox, X-linked
AS Angelman Syndrome
ATP Adenosine Tri Phosphate
BBS Bardet-Biedl Syndrome
BLAT Blast like Alignment Tool
bp Base Pair
CC2D1A Coiled-Coil and C2 Domains-Containing Protein 1A
CC2D2A Coiled-Coil and C2 Domains-Containing Protein 2A
CDG Congenital Disorder of Glycosylation
CDK5RAP2 Cdk5 Regulatory Subunit-Associated Protein 2
cDNA Complementary Deoxyribonucleic Acid
CENPJ Centromeric Protein J
CEP290 Centrosomal Protein, 290-Kd
CGH Comparative Genomic Hybridization
CK2 Casein Kinase II
CLDN14 Claudin 14
cM Centi Morgan
CMA Chromosomal Microarray Analysis
CNS Central Nervous System
Kb Kilobases
KCl Potassium Chloride
LB Lauria Broth
LDH Lactate Dehydrogenase
Leu Leucine
LIS1 Lissencephaly Sequence, Isolated
LOH Loss Of Heterozygosity
M Molarity
Mb Megabases
MCPH1 Microcephalin 1
mg Milligram
MgCl2 Magnesium Chloride
MKKS McKusick-Kaufman Syndrome
MKS1 Meckel Syndrome, Type 1
xix
ml Millilitre
mm Millimetre
mM Millimolar
MR Mental Retardation
MRI Magnetic resonance imaging
MRT Mental Retardation Autosomal
mRNA Messenger RNA
mtDNA Mitochondrial DNA
NaCl Sodium Chloride
NF1 Neurofibromatosis, Type I
ng Nanogram
NIBP NIK- And IKBKB-Binding Protein
NSID Non-syndromic Intellectual Disability
OPHN1 Oligophrenin 1
PAGE Polyacrylamide Gel Electrophoresis
PAH Phenylalanine Hydroxylase
PBS Phosphate Buffer Saline
PCR Polymerase Chain Reaction
PD Parkinson‟s Disease
pH Log of Hydrogen Ion Concentrations
PKU Phenylketonuria
Pol Polymerase
PNS Peripheral Nervous System
PRSS12 Protease Serine 12
PWS Prader–Willi Syndrome
RELN Reelin
qPCR Quantitative polymerase chain reaction
RNA Ribonucleic Acid
Rpm Revolutions Per Minute
rs Reference SNP
RT Reverse Transcription
SBMA Spinal and Bulbar Muscular Atrophy
SDS Sodium Dodecyl Sulphate
SNP Single Nucleotide Polymorphism
STS Sequence Tagged Site
STR Satellite Tandem repeat
T Thymine
T3 Tri-Iodothyronine
T4 Tetra-Iodothyronine
Taq Thermus Aquaticus
TBE Tris borate EDTA
TE Tris- EDTA
TEMED Tetra Methylethylenediamine
TM Melting Temperature
TMEM216 Transmembrane Protein 216
TMEM67 Transmembrane Protein 67
xx
TNF α Tumor Necrosis Factor Alpha
TRAPPC9 Trafficking Protein Particle Complex, Subunit 9
TUSC3 Tumor Suppressor Candidate 3
U International Unit for Enzyme Activity
UBE Ubiquitin-Conjugating Enzyme
UPD Uniparental Disomy
UTR Untranslated Region
UV Ultravoilet
VLDLR Very Low Density Lipoprotein Receptor
WHO World Health Organization
XLMR X-Linked Mental Retardation
α Alpha
β Beta
γ Gamma
μg Micro gram
μl Micro litre
XL-ID X linked Intellectual disability
0
Chapter 1
Introduction
1
1 Introduction
1.1 Intellectual Disability
Intellectual disability (ID) or Mental retardation (MR) is defined as a “condition of
arrested or incomplete development of the mind, which is specially characterized by
impairment of skills manifested during the developmental period, skills which
contribute to the overall level of intelligence, i.e. cognition, language, motor and
social abilities”(http://www.who.int/classifications/icd/en/). ID is a common disorder
of neural development that is defined on the basis of intelligence quotient (IQ) of 70
or below and developmental delay by 18 years of age in at least two behavior related
traits included in adaptive functioning such as self-defense and grooming including
dressing etc. (American Psychiatric Association. Task Force on DSM-IV. 2000).
1.1.1 Role of Nervous System in ID
The central nervous system consists of the brain and spinal cord, while the peripheral
nervous system contains the nerve cells (neurons). The control center of the body is
the CNS, which regulates the different functions of the organs and is responsible for
the thought process and control of emotions and behavior. The brain itself is located
in the cranial cavity and is made up of the forebrain and brainstem. Each part of the
human brain has its own special role in cognitive functions, where the forebrain is
involved in coordination of the body through various sensory and motor systems. The
brain stem is composed of the midbrain, pons, and medulla, the cerebrum is located at
the top of the brain and is divided in two hemispheres (right and left hemispheres).
The cerebellar cortex, basal ganglia and limbic system are located in the right
hemisphere which is divided in two separate lobes of which the frontal is involved in
the control of specialized motor learning, planning and speech. While functions such
as memory, emotions and learning are controlled by the limbic system, which is found
in the right hemispheres (Table 1.1). Figure 1.1 shows some important structures of
the brain, and their functions. Functions such as memory and learning whose defects
can be the cause of intellectual disability (ID) are not only restricted to the brain but
are also a result of long term potentiation (LTP) and long term depression (LTD), the
two processes are opposite to each other. LTP is the
2
Table 1.1. Parts of the limbic system involved in different important functions of the
body.
Parts of limbic
system Location Functions
Hippocampus Located in deep temporal lobe Use of memory to control
behavior
Hypothalamus Located below the thalamus
Involved in production of
emotional expressions such as
hunger, laughter, sexuality,
thirst, displeasure and offence.
Fornix and Para
hippocampus Small structures
Important connecting pathway
for limbic system
Thalamus
Large dual lobe composed of
grey matter located in deep
cerebral cortex
Controls sleep and awake
states and receives auditory,
somatosensory and visual
sensory signals
Cingulate Gyrus Medial side of brain
Involved in aggression,
response to pain and it
coordinated smell and sight
with pleasant memories of
previous emotions
Amygdala Anterior inferior region of the
temporal lobe
Generation of feelings of love,
friendship, affection, and
expression of mood
3
Figure 1.1. Different structures of brain with their functions.
(http://uwf.edu/jgould/resources.htm)
4
coincident activity of pre and post synaptic element, that results in long lasting
enhancement of chemical transmission (Abraham et al., 2002). This phenomena was
initially observed at the glutamatergic synapses between medial perforant path fibres,
originating from the entorhinal cortex, and granule cells in the dentate gyrus of the
hippocampus of the anaesthetized rabbit (Bliss et al., 1973), subsequently it has also
been studied widely in many species from mice (Bertrand et al., 1996) to monkey
(Urban et al., 1996). Different cellular, molecular and biological processes at the
neuronal level result in cognition and improper functioning of these, which leads to
intellectual dysfunctioning or mental retardation (Mandel et al., 2001). Furthermore, it
has been suggested that cognitive impairment is a consequence of defect in synapse
formation and plasticity, which is grouped under neuro-developmental disorders
(Fedulov et al., 2007). The functioning of different parts of the brain can be
understood more readily when any part of it becomes defective.
1.1.2 Classification of ID
Levels of ID are usually classified by the measurement of IQ (Intelligence Quotient).
According to the American association of ID there are five levels of severity of ID
(American Psychiatric Association. American Psychiatric Association. Task Force on
DSM-IV. 2000).
Profound ID (IQ ≤ 20)
Severe ID (IQ: 20-35)
Moderate ID (IQ: 36-51)
Mild ID (IQ: 52-67)
Borderline ID (IQ: 68-84)
1.1.3 Prevalence
The prevalence of severe and moderate ID in the developed world is 0.3-0.5% and
1.5-2%, respectively (Leonard and Wen, 2002). Maulik et al. (2011), reported the
highest rate of ID in low and middle income countries, which is because of a lack of
resources leading to malnutrition, cultural deprivation and poor status of health.
Another important reason identified in a number of studies is consanguinity, which is
one of the prime causes of reduced cognitive performance (Bashi, 1977). In Pakistan,
Durkin et al. (1998), found the prevalence of severe ID to be 1.9% and mild ID to be
5
6.5% which is in agreement with the prevalence reported by Mirza et al. (2009).
Bashir et al. (2002) attributed this high prevalence to a lack of maternal education,
complicated labor, neonatal infections, postnatal brain infections, injuries and
malnutrition.
1.1.4 Etiology of ID
The etiology of ID is diverse as shown in Figure 1.2; mainly two types of factors are
the major cause of ID, environmental and genetic, each of which contribute equally to
the disease etiology. In addition to these, single-gene mutations, prenatal infections,
premature birth and chromosomal abnormalities are also critical factors that can lead
to ID (Menkes et al., 2006). The causative factors can be established in 60-75% of
severe ID cases and 38-55% cases of mild ID. In addition, social and behavioral
factors such as destitution, dietary deficiencies, childhood illness (e.g. measles,
meningitis), alcohol and drug use by the mother, as well as severe stimulus
deprivation can be causative factors of ID (McLaren and Bryson, 1987). Apart from
these, accidental injury or other forms of acquired injuries particularly at crucial
periods of life are also the leading factors in causing ID. In the developed world the
highest rate of ID is seen due to fetal alcohol syndrome with an incidence rate of 1 in
100 live births, while the other major cause is Trisomy 21 (MIM 190685) with an
incidence rate of 1 in 800-1,000 live births (Brown, 2004). Important genetic factors
that are involved in the pathogenicity of ID are multiple, such as chromosomal
aberration, single gene disorders, epigenetic modifications, various metabolic factors,
mitochondrial factors and repeat expansion. Genetic ID can be divided into two major
types, which include syndromic and non-syndromic forms of the disease.
In syndromic ID, cognitive impairment is present with other clinical manifestations
including ophthalmological, otorhinolaryngological, orthopedic, skeletal, or other
defects. While in non-syndromic ID there is no other impairment present in subjects
except speech impairment due to inability of the ID patients to learn to speak, thus
this type of ID is also known as cognitive or learning impairment (Uyguner et al.,
2007).
6
Figure 1.2. Diagrammatic representation and etiological classification of different
types of ID (Basel-Vanagaite L. 2008).
Intellectual Disability
ID
Genetic
50%
Environmental
50%
Chromosomal
Aberrations Single
Gene
Metabolic Epigenetic Repeat
Expansion
Numerical
Aberration
Partial
Aberration
X linked
recessive
Autosomal
recessive
Microdeletions and Microduplications
Autosomal
dominant
X linked
dominant
Mitochon
drial
Injuries Infections Hypoxia during birth
7
1.2 Diagnosis of ID
The diagnosis of ID can be conducted using two major steps, a detailed clinical
evaluation followed by molecular diagnosis.
1.2.1 Clinical Evaluation
Initial diagnosis of ID involves clinical evaluation of the proband including the
gathering of comprehensive patient and family history, the prenatal, perinatal and
neonatal medical history of the proband and his/her IQ level assessment. Physical
examination can include ophthalmologic, otorhinolaryngologic, skeletal, radiologic or
neuro-anatomic tests (MRI and CT scan). In addition the patient is assessed for inborn
errors of metabolism including the testing for phenylketonuria, serum lactate levels,
hyper or hypothyroidism, etc. For the structural malformation of the brain complete
CT (Computed tomography) or MRI (Magnetic resonance imaging) scans of the brain
are taken. Also the complete blood picture, levels of amino acids, electrolyte balance
and serum creatinine kinase levels for dystroglycanopathies are usually obtained.
1.2.2 Molecular Diagnosis
The first step in molecular diagnosis involves the detection of chromosomal
abnormalities that are responsible for various syndromic forms of neuro-
developmental disorders. Cytogenetic techniques used for screening of chromosomal
aberrations are karyotyping, interphase fluorescent in situ hybridization, comparative
genome hybridization, in situ labeling and multiplex amplifiable probe hybridization
(Xu and Chen, 2003). Twenty eight percent of all ID cases have been shown to be
caused by chromosomal defects, which include numerical or gross chromosomal
abnormalities (poly or monosomy) or partial chromosomal abnormalities
(microdeletions and duplications) (Curry et al., 1997), these are usually responsible
for syndromic conditions with varied phenotypes ranging from mild to profound ID
(van Karnebeek et al., 2005). Apart from chromosomal aberrations other genetic
defects due to monogenic causes and epigenetic modifications are also involved in
causing ID which can be diagnosed by various techniques such as microarray analysis
and whole exome sequencing. In recent years, microarray analysis has replaced many
previously used conventional techniques such as linkage analysis by use of
8
microsatellite markers, this technique can analyze the whole genome for copy number
changes to identify deletions/duplications of complete chromosomes e.g., Down
syndrome (MIM 190685), defects in a chromosomal segment, or disease-specific
sequences e.g., deletion of 22q11 in Di George syndrome (MIM 188400)
(Antonarakis, 2001). Additionally arrays can be used for whole genome genotyping of
large cohorts, which can result in the identification of multiple loci responsible for
autosomal recessive ID (Najmabadi et al., 2007). Next Generation Sequencing is the
newest technique, which is replacing all known conventional techniques including
microarray analysis. It is a strong and economical diagnostic tool, which can be used
for both familial ID as well as for isolated patients (Rauch et al., 2012).
1.3 Role of Chromosomal Aberration in the Pathogenesis of ID
Genetic abnormalities due to chromosomal aberrations are very common for which
conventional karyotyping fails to detect imbalances of less than 3-5Mb (Gijsbers et
al., 2009). Combined analysis of karyotyping, FISH and other techniques can result in
the identification of 5-10% cases of unexplained ID (Stankiewicz et al., 2007;
Gijsbers et al., 2009). Fifteen percent of chromosomal aberrations are visible under
light microscope, majority of which are trisomy 21 (Down syndrome), others are
deletion in chromosome 5 (cri du chat syndrome MIM 123450) and chromosome 4
(Wolf-Hirsch horn syndrome, MIM 304150) (Chiurazzi, Oostra, 2000; Roizen and
Patterson, 2003). About 28% of ID cases are due to chromosomal abnormalities,
which are sometimes gross or numerical leading to poly or monosomy cases or are
sometimes partial losses, which result in microdeletions or microduplications (Curry
et al., 1997), in addition chromosomal aberrations can be structural. Following are
some of the different types of chromosomal aberrations causative of ID.
1.3.1 Numerical Chromosomal Abnormalities
The human diploid chromosomal complement consists of 46 chromosomes, while the
euploid number is 23. Multiples of the euploid state exists in nature, which can be
either diploid or sometimes due to defects at different stages of meiosis, can lead to
polyploidy state (triploid or tetraploid). Aneuploidy is the term used to describe
defective chromosomal numbers, where a missing or an extra copy of a specific
chromosome is present. Both aneuploid and normal diploid cells can co-occur in
9
individuals at the same time. In addition, mosaicism in individual is defined as more
than two different cell types being derived from a single fertilized egg or zygote,
which mostly occur in sex chromosome but also rarely in autosomes. Chimaerism is
distinct from mosaicism as it can be defined as a condition in which two distinct cell
lines are obtained from more than one fertilized egg. Down Syndrome (DS) and
Turner Syndrome (TS) (MIM 1639500) (45X) are both different forms of aneuploidy.
Where, DS is an autosomal trisomy and Turner is a sex chromosome monosomy, the
latter is currently the only known human monosomy (Frederick and Luthardt, 2001).
1.3.2 Common Autosomal Trisomies
With reference to ID there are two important autosomal trisomies, these are DS and
Trisomy 13 (Patau syndrome, MIM 266400). DS is the most commonly found (1/900)
viable autosomal trisomy in live births. DS children have multiple anomalies such as
ID, hypotonia, cardiac defects, gastrointestinal defects, respiratory tract illness,
Alzehmier, leukemia, in addition to having bulging eyes and webbed neck. Females
suffering from DS are usually fertile and can produce a child with a 50% chance of
inheriting the disease. Turner syndrome (single X chromosome) and Klinefilter
syndrome MIM 147390 (extra chromosome) patients do not show intellectual
abnormality but the patients of these syndromes may suffer developmental delays
(Giltay and Maiburg, 2010).
1.3.3 Structural Chromosomal Abnormalities
Structural rearrangements can produce balanced or unbalanced rearrangements, such
abnormalities involve the breakage of the chromosome at one end and its reunion
either to the same chromosome or with another chromosome. The rearrangements
could be balanced or unbalanced depending upon the disturbance it creates in the
amount of the genetic material, if there is no net change in the genetic material of the
chromosome than it would be a balanced rearrangement, while a net change would be
an unbalanced rearrangement such as Partial trisomy resulting in gain of genetic
material and partial monosomy for net loss of genetic material (Frederick and
Luthardt, 2001).
10
1.3.4 Unbalanced Rearrangements
Duplications, deletions or sometimes both, which can result as a consequence of
unbalanced rearrangements can be pathogenic. Deletions, duplications,
isochromosomes and ring chromosomes are all different forms of unbalanced
rearrangements. Deletions can be terminal deletion, interstitial deletion and proximal
deletions. Ring chromosomes are formed at the distal end of a chromosome, which
results in the loss of some chromosomal material and then rejoining of these breaks.
1.3.5 Cytogenetically Visible Deletions
One of the most common syndromes that is a result of autosomal deletion is Wolf–
Hirschorn (MIM 194190), in this syndrome the deletion is at the chromosomal region
4p16.3. The patients of this syndrome have clinical characteristics such as
developmental delay, growth arrest, midline facial defects, ID, microcephaly,
glabellum, cleft lip/ palate, cardiac anomalies, widely set eyes, wide nasal bridge
(Sheth et al., 2012a). Another such syndrome is Cri du chat (MIM 123450) where the
deletion is at 5p15.2 and the clinical manifestations include shrilled cry, broad set
eyes, dysmorphic chin, microcephaly, psychiatric disturbances and ID (Sheth et al.,
2012b). While in Langer–Giedion syndrome (MIM 156190) the deletion occurs at
8q24.11-q24.13, while the clinical manifestations include microcephaly, ID,
hypotrichosis, bulbous nose, dwarfism and exostosis (Pereza et al., 2012).
1.3.6 Cytogenetically Invisible Microdeletions
This category includes interstitial microdeletion, subtelomeric deletion and
microduplication. In the case of interstitial or intercalary microdeletions the anterior
part of the chromosome is found to be deleted. An example of an autosomal
interstitial microdeletion syndrome, which includes ID as a major symptom, is Prader
willi syndrome (PWS) (MIM176270), various genetic and phenotype complexities are
found in patients of this syndrome. The genetic cause of PWS identified till now, is
the loss of imprinted genetic material at chromosome 15q11–q13; the patients of this
syndrome usually have mild to moderate ID, autistic features, self-torturing habits,
tantrums, hyperphagia and obesity (Dykens et al., 2011). The Angelman syndrome
(MIM 105830), was identified by Magenis et al. (1987), who were able to
11
demonstrate deletion at 15q11-13 in two patients. Subsequent work on this syndrome
has shown that Angelman syndrome (MIM 105830) can be caused by a variety of
genetic mechanisms, which involve the imprinted region of the genome. The PWS
and Angelman syndrome are related to each other, as in the former the imprinted
region is paternal while in Angelman syndrome the imprinted region is maternal. The
other cause of Angelman syndrome is monogenic, which causes the loss of function
mutation in UBE3A gene that lies at the 15q11-13 locus (Magenis et al., 1987).
Miller-Dieker Smith Magenis syndrome (MIM 182290) is caused by microdeletion in
the region 17p11.2-p13.3, the clinical manifestations of which are lissencephaly,
microcephaly, small chin, growth arrest, cardiac abnormalities, flat mid face, broad
nasal bridge, short fingers and toes, ID, hyperactivity, dwarfism and specific
behavioral problems (Kirchhoff et al., 2007). Another microdeletion that causes
Williams Syndrome (MIM 194050) is located at locus 7q11.23, the clinical
characterization of the syndrome include cardiac defects, ID, constipated stool,
developmental retardation, isolated behavior, collagen vascular diseases (Kirchhoff et
al., 2007). In Beckwith–Wiedemann syndrome (MIM130650) the deletion responsible
for the pathogenecity is at 11p15.5, its clinical features include large tongue, tissue
and organ overgrowth, mild ID (Kirchhoff et al., 2007). The cat-eye syndrome (MIM
115470) is due to microduplication at 22pter-q11.2, its clinical features include
ophthalmic defects, imperforate anus, skin tags in front of the ears, constipated hard
stool, ID and various anomalies of the urinary and reproductive system. The most
common form of this duplication is an additional bisatellied and bicentric
chromosome 22, which is referred to as inv dup (22) (q11.2) (Frederick and Luthardt,
2001; Kirchhoff et al., 2007). Chromosomal defects are not the sole cause of
developmental delays and cognitive impairment; a number of neurodegenerative
disorders are known, which are the result of mitochondrial disorders, metabolic errors,
hormonal imbalances and repeat expansion.
1.4 Trinucleotide Repeat Expansion
Till now 22 neurological disorders have been identified, which are the result of repeat
expansions (La Spada and Taylor, 2010). Trinucleotide repeats are polymorphic and
present in the normal population, where they are inherited in a stable manner from
one generation to the next. The disorders caused by expansion of these repeats are
12
divided into two categories, one that are tolerable CAG expansions, which includes
the polyglutamine string in gene product while the other usually has a very long
stretch of CAG and are usually pathogenic. In such cases the severity and onset of the
disease depends on the location and the number of these triplet repeats present within
the genome. The CAG repeats involved in disease pathogenicity tend to increase in
length as they are transmitted from one generation to the next, which does not follow
the Mendelian inheritance rules. Repeat expansions can be amplified and analysed by
sequencing or sometimes by STR analysis (Shetty and Christopher, 2000). The most
prevalent form of ID inherited in X linked mode of inheritance is Fragile X syndrome.
1.5 Fragile X Syndrome (MIM 300624)
Martin and Bell (1943), were the first ones to define Fragile X syndrome MIM
300624, they reported it to be the leading cause of inherited ID. Subsequently a
number of studies have reported the incidence rate to be approximately 1 in 2,500 to
5,000 in men and 1 in 4,000 to 6,000 in women (Hirst et al., 1993; Coffee et al.,
2009). FMR1 mutations are mainly responsible for fragile X syndrome, this gene is
located on the X chromosome at locus Xq27.3, which overlaps with folate-sensitive
fragile site (Fryns et al., 1984). The primary genetic defect in fragile X syndrome is
an expanded set of CGG repeat, which is located in the 5' untranslated region. In the
normal population the repeat exists between 6 to 52 copies while in the families
having the polymorphic repeat range between 60-200, permutation spectrum is
observed and such families are highly prone to expand to full mutation resulting in
full blown manifestation of the disease (Shetty and Christopher, 2000). The clinical
features include several delayed milestones such as late crawling, late speech, late
sitting, late walking and late toilet training while with progression in age other signs
and symptoms develop such as a chest with a “hollow” look, strabismus, swollen
eyelids, elongated face – long forehead and jaw, hyper-extensible joints, flat feet (pes
planus), high arched palate, swollen testicles, hypotonia, enlarged ears, single palmer
Crease, mild to moderate ID, autistic features and hyperactivity (Bagni et al., 2012).
13
1.6 Role of Mitochondrial Genome in Intellectual Disability
Mitochondrial DNA (mtDNA) was first identified in 1963, later it was shown that 2-
10 copies of this small circular, supercoiled and double stranded DNA is present
within the matrix of all mitochondrion, which results in a total of 1000-10,000 copies
in every cell. The rate of mutation in mtDNA is 10 times higher than the nuclear
genome and hence mitrochondrial genome is more prone to damage. Various studies
have shown that the human central nervous system is mostly affected by defects in the
mitochondrial genome (Turnbull et al., 2010). Mutations in mtDNA have been
reported to be associated with disorders such as Alzheimer (MIM 104300),
Parkinson„s disease (MIM 168600) (Finsterer, 2007), and Alpers syndrome (MIM
203700) (Naviaux and Nguyen, 2004).
1.7 Metabolic Disorders
A few mental disorders are known with identified biochemical basis, but it is not yet
known that how abnormalities in these biochemical factors lead to dysfunctioning of
cognition or the brain (Kahler and Fahay, 2003). Although there are a few metabolic
disorders in which ID is also a profound clinical manifestation such as
Phenylketonuria (PKU) (MIM 261600), which is a result of defects in the liver
enzyme phenylalanine hydroxylase (PAH) that converts PHE (Phenylalanine) to
tyrosine, defects in this enzyme results in PKU. The PAH (MIM 612349) has been
shown to be primarily mutated in PKU patients and till date more than 400 causative
mutations have been identified in this gene (Eng et al., 2001). There are a number of
other disorders, which are caused by defects in the mitrochondrial genome such as
Urea Cycle Disorders, Hyperammonemias, Homocysteinuria, Cholesterol
Biosynthesis Disorders, Galactosemia, Hypothyroidism and Lysosomal Storage
Diseases, which is highly heterogeneous (Rohrbach and Clarke, 2007). For Glycine
Encephalopathy three genes have been identified (GLDC MIM 238300, AMT MIM
238310, GCSH MIM 238330) (Cao et al., 1994). Some of the disorders identified
recently due to mutations in mtDNA are Serine Biosynthesis Defects, Inborn errors
of purine–pyrimidine metabolism, Creatine Deficiency, Glucose Transporter
Deficiency, carbohydrate-deficient glycoprotein syndromes and Succinic
14
Semialedehyde Dehydrogenase and GABA Transaminase Deficiencies, these all
include ID as a major symptom (Kahler and Fahay, 2003).
1.8 Endocrine Hormonal Disorders
Endocrine hormones also play a significant role in the nervous system functionality
and cognitive development. Different disorders in association with ID have been
reported that result from dysfunctioning of the endocrine hormonal system e.g.
association of hypothalamus with cerebral gigantism (Fryssira et al., 2010), cerebral
dwarfism, Laurence-Moon-Biedl syndrome (MIM 209900) and Hypogonadotropic
hypogonadism with or without anosmia, a condition characterized by hypogonadism
due to impaired secretion of gonadotropins (Reddy, 1980).
1.9 Epigenetic Modifications causing ID
The phenomena of genomic imprinting results in the selected expression of a gene
from only one of the two parental copies. Epigenetic modifications such as DNA-
cytosine methylation, histone acetylation and methylation lead to the allele specific
expression of these imprinted genes. After fertilization the newly induced epigenetic
modifications in the germ cells are established as permanent change. Defects in the
imprinted region of chromosome 7 cause reduced growth, in chromosome 11 causes
Familial nonchromaffin paraganglioma and Beckwith-Wiedemann syndrome, in
chromosome 14 it causes Maternal and paternal UPD14 (uniparental disomy 14)
syndromes MIM 608149, while defects in chromosome 15 causes Prader-Willi
syndrome and Angelman syndrome (Chelly et al., 2006). The identified number of
transcriptional units known to be imprinted in human and mouse genome are 80
(http://www.mgu.har). Many syndromes, defective brain functions and ID are the
result of imprinted gene alterations or deregulation (Nicholls, 2000).
1.10 Monogenic Causes of ID
Studies of families with ID have contributed towards the understanding that single
gene defects can result in Mendelian inheritance of ID. In some of these families, the
trait was shown to be inherited in a X-linked mode where it was shown to be most
frequent in males. Mutations in a single gene may disrupt its function and may cause
15
many defects such as ID or a variety of phenotypes associated with ID, which are
dependent on the function of the mutated gene and the impact of the mutation on its
function. These groups of disorders are further categorized into different classes on
the basis of the mode of inheritance and phenotype. These classes include, X-linked
ID, Autosomal Dominant ID and Autosomal Recessive ID.
1.11 Copy Number Changes in Single Gene
It has been established that the human genome is highly variable from one person to
the other, where large number of duplications and deletions within the genome have
been found to be polymorphic (Sebat et al., 2004). Many such microdeletions and
microduplications are known to be responsible for ID and also syndromic ID. The
identification of regions containing copy number variations (CNV) that are large,
rare, encompasses the gene and are de novo in any patient would be of strong
pathogenic interest because such type of a combination is extremely rare in the
normal population (Conrad et al., 2006). Pathological duplications of LICAM and
MECP2 (Methyl CpG Binding Protein 2), have been reported in males with severe ID
and progressive syndromic neurological disorders (van Esch et al., 2005). Recently
pathogenic microdeletions in NRXN1 MIM 600565 and CNTNAP2 have been reported
to be causative of a wide spectrum of ID disorders such as epilepsy, schizophrenia,
specific ID and autism (Ching et al., 2010; Gregor et al., 2011).
1.12 X-linked ID (XL-ID)
It has been shown that ID ratio is particularly higher in males as compared to females
with a sex ratio of 1.4:1 for severe ID and 1.9:1 for mild ID. X chromosomal genetic
defect mostly account for this higher ratio of males with ID, according to one estimate
90% of ID patients are males (Ropers, 2006). Lisik and Sieron (2008), has reported
that 25% to 30% of ID in the population is due to X chromosome mutation.
Particularly because the human X chromosome contains excessively large number of
genes for learning and memory (Ross et al., 2005), which explain the male biasness
(Chelly and Mandal, 2001), however, carrier female may show milder symptoms,
possibly because of X-inactivation (Amos-Landgraf et al., 2006). Table 1.2 shows list
of some known genes involved in nonsyndromic XLID.
16
Table 1.2. List of some known genes involved in nonsyndromic XLID (Adapted from
Chiurrazi, et al. (2008) and Gecz, (2009).
NsXLID genes OMIM numbers Protein name
NLGN4 300427 Neuroligin 4
MID1 300552 Midin (midline 1 protein)
HCCS 300056 Homocytochrome 3 synthase
APIS2 602416 Oral facial digital syndrome 1 protein
NHS 300457
Fanconi anemia complementation group b
protein
CDKLS 300203
Adaptor related protein complex1, sigma 2
subunit
STK9 300672 Nance hooran syndrome protein
PDHSA1 300502 Pyruvate dehydrogenase (lipoamide) a 1
RPAS6KA3 300075
Ribosomal protein s6 kinase,90Kda
polypepetide 3
RSK2 303660
Ribosomal protein s6 kinase,90Kda
polypepetide 3
SMS 182290 Spermine synthase
ARX 300382 Aristaless- related homeobox protein
ILTRAPL1 300206 Interleukin receptor accessory protein like 1
CK 300831 Glycerol 3 phosphotransferase
DMD 300377 Dystrophin
OTC 300461 Omithine carbomolytransferase
TM4SF2 300096 Tetraspanin 7
BCOR 300485 BCL6 corepressor
ATP6AP2 300556
ATPase, H+transporting lysosomal accessory
protein 2
MAOA 309850 Monoamine oxidase A
NDP 300658 Nomin
ZNF674 300573 Zinc finger protein 674
ZNF41 314995 Zinc finger protein41
SYN1 313440 Synapsin
ZNF81 314998 Zinc finger protein 81
FTSJI 300494 Ftsj homolog 1
PORCN 300651 Drosophila porcupine homolog
PQBP1 300463 Polyglutamine-binding protein 1
KIAA1202/
SHR00M4 300579 KIAA1202 protein
JARIDTC/SMCX 314690 Junomji /ARID domain containing protein 1C
SMC1A/SMCIL1 300040
SMC1 structural maintainance of
chromosome 1-like 1-(yeast)
HADH2 300256
Hydrooxyacyl-coenzyme A dehydrogenase
type 2
17
Table 1.2. cont.
Gene OMIM number Protein name
HUWE1 300697
E3 ubiquitin-protein ligase HUWE1
(UREB1, ARFBP1)
PHF8 300560 PHD finger protein 8
FGD1 300546
FYVE, RhoEF and PH domain containing
protein 1
ARHGEF9 300429
Cdc42 guanine nucleotide exchange factor
(GEF) 9
OPH1N1 300127 Oliophrenin 1
DLG3 300189
Discs, large homolog 3 (neuroendocrine dig 3,
Drosophila)
MED12/HOPA 300188
Mediator of RNA polymerase II transcription ,
subunit 12 homolog
NLGN3 300336 Neuroligin3
SLC16A2/MCT8 300095
Solute carrier family 16, member 2
(monocarboxylic acid transporter 8)
KIAA2022 300524 KIAA2022 protein
ATRX 300032
Alpha thalasemia /mental retardation
syndrome X linked /X –linked helicase 2
ATP7A 300011
ATPase , CU++ transporting, Alpha
polypeptide
PGK1 311800 Phosphoglycerate kinase 1
BRWD3 300553
Bromodomain and WD repeat domain
containing protein 3
SRPX2 300642 Sushi-repeat containing protein X linked 2
TIMM8A 300356
Translocase of inner mitrochondrial
membrane 8 homolog A
NXFS 300016 Nuclear RNA export factor 5
PLP 300401 Protolipid protein 1
PRPSI 300661 Phosphoribosyl pyrophosphate synthase 1
ACSL4 300157
Acyl-CoA synthase long chain Family
member 4
PAK3 300142 P21(CDKNA1A)activated kinase 3
DCX 300121
Double cortin (double cortin )lissencephaly ,
X linked )
AGTR2 300034 Angiotensin II receptor , type 2
UBE2A 312180
Ubiquitin –conjugating enzyme E2A (RAD 6
homolog)
UPF3B 300298
UPF3 regulator of nonsense transcript
homolog B
NDUFA1 300078
NADH dehydrogenase (ubiquinone)1 α
subcomplex
LAMP2 309060 Lysosomal-associated membrane protein 2
CUL4B 300304 Cullin 4B
18
Table 1.2. cont.
Gene OMIM number Protein name
GRIA3 305915 Glutamate receptor ionotropic AMPA 3
OCRL1 300535
Phosphatidylinositol polyphosphate S
phosphatase
ZDHHC9 300646
Zinc finger , DHHC domain containing
protein 9
GPC3 300037 Glypican 3
PHF6 300414 PHD finger protein 6
HPRT 308000 Hypoxanithine phosphoribosyl transferase 1
SLC9A6 300231
Solute carrier family 9 member 6 sodium
hydrogen exchange 6 (NHE6)
ARHGEF6 300267
Rac/ cdc42 guanine nucleotide exchange
factor (GEF) 6
SOX3 313430
SRY (sex determining region Y) box 3
protein
FMR1 309550
Fragile X mental retardation protein 1
(FMRP)
FMR2 300823 AF4/FMR2 family , member 2 protein
IDS 300415 Iduronate 2-sulfatase
MTM1 300415 Myotubularin 1
SLC6A8 300036
Solute carrier family 6 (neurotransmitter
transporter, creatine), member 8
ABCD1 300371
ATP-binding cassette,subfamily D
(ALD),member 1
LICAM 308840 L1 cell adhesion molecule
MECP2 300005 Methyl CpG-binding protein 2
FLNA 300017 Filamin A, alpha (actin-binding protein 280)
RPL10 312173 Ribosomal protein L10
GD11 300104 GDP dissociation inhibitor 1
IKBKG 300248
Inhibitor of k light polypeptide gene enhancer
in B cells, kinase gamma
DKC1 300126 Dyskerin
MAGT1 300715 Magnesium transporter 1
ZNF711 314990 Zinc finger protein711
CASK 300172
Calcium calmodulin dependent serine protein
kinase
SYP 313475 Synaptophysin
19
XLID is a highly heterogeneous group of disorders, which is further subdivided into
syndromic (S-XLID) and nonsyndromic forms (NS-XLID). In S-XLID, ID is often
associated with other clinical symptoms, which are helpful in the identification of the
underlying molecular defects (Passos-Bueno et al., 1993). The fragile X syndrome
gene FMR1 is the most commonly mutated gene for XLID, the prevalence rate of this
syndrome is 1/4000 to 1/8000 (Hagerman, 2008). 215 monogenic XLID disorders
have been identified to date out of which 149 are S-XLID (51 out of the 149 are
neuromuscular syndromes) and 66 are NS-XLID. Moreover, 93 genes have been
identified for XLID out of which 53 are of S-XLID, 27 are NS-XLID and 13 are for
both S-XLID and NS-XLID (Chiurazzi et al., 2004). Many genes on the X
chromosome, which are involved in syndromes are also liable to cause NS-XLID such
as MECP2, mutations of which are causative for the pathology of Rett syndrome, and
are also known to be pathogenic in a number of NS-XLID. MECP2 once considered
to be lethal if mutated in males has now been shown to be the cause of mild and
profound ID and other associated symptoms in heterozygous conditions. This
suggests that mutations in heterozygous state in this gene have tolerable phenotype.
ARX (Aristaless Related Homeobox) is one of the most frequently mutated gene in
XLID. Mutations in ARX have been shown to be causative for seven distinct but
overlapping NS-ID and S-ID phenotypes (Kaufman et al., 2010). ARX encodes the
transcription factor for both activation and repression of genes, which are essential for
CNS development (Kaufman et al., 2010). The varying number of genes for XLID
disorders has pointed to the genetic heterogeneity of this disorder. X-linked disorders
are easier to be mapped due to the hemizygous state of males, there is only one X-
linked condition that differs from this mode of inheritance, this is EFID (epileptic fits
intellectual disability) or ID associated with epileptic fits, which is solely restricted to
females. In this disease, heterozygous females are affected while the hemizygous
males are found to be unaffected (Coppede et al., 2006).
1.12.1 Molecular Pathways Involved in XLID genes
Out of the 93 known genes of XLID, the protein encoded by these gene are present in
almost all cell compartments, where 30% of them are present in the nucleus, 28% in
the cytoplasm and 42% are present in other organelles. They are mostly involved in
various functions e.g. 19% of these proteins are involved in signal transduction and
20
22% work in the regulation of transcription. Other XLID genes encode proteins,
which work in different biological pathways such as protein synthesis (3%),
regulation of cell cycle, core metabolism (15%), DNA and RNA processing (6%) and
ubiquitin pathway (7%). All these pathways are involved directly or indirectly in
brain specific expression hence their involvement in the pathogenesis of ID
(Chiurazzi et al., 2008).
1.13 Autosomal Dominant Intellectual Disability (AD-ID)
Autsomal dominant intellectual disability (AD-ID) is a very rare condition because in
the cases of syndromic AD-ID subjects rarely give birth thus the disease is not passed
on to the next generation in the families. The genes which are thought to be involved
in AD-ID have largely been identified by the mapping of chromosomal breakpoints
among different types of inversions, deletions and duplications (Basel-Vanagaite,
2007). Recent observations suggest that in outbred Caucasian population, significant
numbers, of isolated cases of ID are found to occur due to de novo mutations
(Hamdan et al., 2011). In addition till date only 19 loci and genes, have been
identified in causing AD-ID. The MBD5 (Methyl-CpG Binding Domain Protein) on
2q23.1 lies in the locus MRD1 (MIM 156200), which has been previously identified
for NS-AD-ID but later MBD5 was also found to be mutated in four S-ID probands,
which were all missense mutations in S-ID patients (Wagenstaller et al., 2007).
Another locus for AD-ID is MRD2 on 9p24, which harbours the gene DOCK8
(Dedicator of cytokinesis 8); it was reported in two individuals by breakpoint
mapping of deletion and translocation, respectively. Two other loci MRD3 and
MRD4 also harbour two genes for AD-ID, CDH15 (Cadherin-15) and KIRREL3 (Kin
of IRRE-like protein 3). Bhalla et al. (2008) have reported 4 novel mutations in
CDH15 and 3 in KIRREL3 by sequencing these two genes in 600 individuals with
syndromic dominant ID. Table 1.3 shows the list of some known AD-ID genes
involved in syndromic as well as nonsyndromic ID.
Another important candidate for AD-ID is SYNGAP1 (Synaptic Ras GTPase-
activating protein 1), which is located at the locus MRD 5 and encodes SynGAP.
Initially this gene was found to be involved in learning and cognition, while studying
21
Table 1.3. List of some known AD-ID genes and loci involved in syndromic and
nonsyndromic ID.
Locus Name
OMIM entry
Number Genes Identified S/NS
Additional
features
MRD1 156200 MBD5 NS
MRD2 614113 DOCK8
S/NS Hyper-IgE
Recurrent
Infection
Syndrome
MRD3 612580 CDH15 NS
MRD4 612581 KIRREL3 NS
MRD5 612621 SYNGAP1 NS
MRD6 613970 GRIN2B NS
MRD7 614104 DYRK1A NS
MRD8 614254 GRIN1 NS
MRD9 614255 KIF1A S/NS spastic paraplegi
MRD10 614256 CANCNG2 NS
MRD11 614257 EPB41L1 NS
MRD12 614562 ARID1B NS
MRD13 614563 DYN1CH1
S/NS charcot-marie-
tooth disease,
axonal, type 2o
MRD14 614607 ARID1A NS
MRD15 614608 SMARCB1
S/NS malignant rhabdoid
tumor, somatic
MRD16 614609 SMARC4A S/NS malignant rhabdoid
tumor, somatic
MRD17 615009 PACS1 NS
MRD18 615074 GATAD2B NS
MRD19 615075 CTNNB1 S/NS
colorectal cancer
NO 602700 CREPPB
S/NS Rubenstien Taybi
Syndrome
NO 605894 EP300
S/NS Rubenstien Taybi
Syndrome
22
the effects of heterozygous mutation in mouse model. Sequencing of this gene in a
cohort of individuals led to the identification of heterozygous mutation in 3 unrelated
individuals with NS-ID (Hamdan et al., 2009a). GRIN2B is another probable
candidate for AD-ID located at the locus MRD6, which encodes for the NMDA
receptor, in this gene, four mutations have been identified in four unrelated
individuals, these mutations have confirmed that normal functioning of NMDAR
complex is important for the development of cognition. Of these four patients, two
individuals were found to carry chromosomal translocations overlapping GRIN2B
(Glutamate [NMDA] receptor subunit epsilon-2), which was identified by Endele et
al. (2010). Autosomal dominant mutations in SHANK2 (SH3 and multiple ankyrin
repeat domains protein 2) have also been reported in nonsyndromic AD-ID (Berkel et
al., 2010). Hayashi et al. (2009), have reported SHANK and HOMER (Homer protein
homolog 1) to be involvedin synaptic plasticity by forming a mesh like network
which produces a framework for the assembly of proteins at postsynaptic density. Van
Bon et al. (2011), identified a novel 52 kb deletion in DYRKIA (Dual specificity
tyrosine-phosphorylation-regulated kinase 1A) gene in a woman suffering from AD-
ID. Later Hamdan et al. (2011), identified two mutations in GRIN1 (Glutamate
Receptor, Ionotropic, N-Methyl D-Aspartate 1) resulting in NS-ID in locus MRD8.
These authors also identified a heterozygous c.296C>T, p.T99M substitution in
KIF1A (Kinesin-like protein) gene in locus MRD9. Blair et al. (2011), have reported a
novel locus for syndromic AD-ID on chromosome 3p, where the affected individuals
suffered from epilepsy with generalized seizures variably associated with ID. De novo
mutations in STXBP1 (nonsense, p.R388X; splicing, c.169+1G>A) in two patients
with severe mental retardation and nonsyndromic epilepsy have been reported by Fadi
et al. (2009). Rubinstein Taybi syndrome another known syndrome with autosomal
dominant mode of inheritance of ID has been shown to be associated with mutations
in two genes CREPPB (CREB Binding Protein) and EP300 (E1A binding protein
p300). Cathy et al. (2002), have reported 10% of the individuals to have CREPPB
microdeletions while 30% to 50 % individuals were found to carry other heterozygous
mutations in CREPPB, while only 3% individual in their study had mutations in
EP300. Rubinstein Taybi syndrome was found to mostly occur as de novo in families
(Stevens, 1993). Tsurusaki et al. (2012), identified 2 mutations in the SMARCB1 gene
23
in the locus MRD15 in 4 patients with S-ID and mutation 1636-1638 del AAG in
SMARC4 at the locus MRD16 in a patient suffering from nonsydromic AD-ID.
1.14 Autosomal Recessive Intellectual Disability (AR-ID)
Autososmal recessive inheritance is a plausible mode of inheritance for genetic
defects resulting in ID. So far most of the mutations identified in XLID are missense
as well as nonsense but autosomal recessive as well as dominant ID have been found
to have truncating mutations. Autosomal recessive ID is further divided into
autosomal recessive nonsyndromic ID (NS-ARID) and syndromic ARID (S-ARID).
1.14.1 Autosomal Recessive Nonsyndromic ID
Autosomal recessive nonsyndromic ID (NS-ARID) is a highly heterogeneous
disorder, to date only 11 genes have been shown to be involved in the pathogenecity
of NS-ARID (Table 1.4). Thus the deep interests of geneticist, as more than 450 genes
in the human genome are thought to play a role in cognitive development (van
Boekhoven, 2011). Some of the other candidate genes which were identified by
Najamabadi et al. (2011), for NS-ARID families out of 272 families of different
phenotypes were MED13L, PRKRA, SLAC2A1, ABCD6, ADRA2B, ASCC3, ASCL1,
C11ORF46, C12ORF57, C8ORF41, C8ORF86, CASP2, CCNA2, COQ5, EEF1B2,
ELP2, ENTPD1, FASN, HIST3H3, INPP4A, NDST1, PECR, PRMT10, PRRT2,
RALGDS, RGS7, SCAPER, TRIMT1, UBR7 and ZNF526. Recently ANK3, which was
previously known to be associated with bipolar disorder, schizophrenia and
psychiatric disorders has been found to be involved in the pathogenecity of ID with
autism and sleeping disorder (Iqbal et al., 2013).
1.14.2 Autosomal Recessive Syndromic ID
There are multiple syndromes which manifest ID as a major clinical symptom. Some
of these, which were studied in the current work are described below.
1.14.2.1 Bardet Beidl Syndrome
Bardet–Biedl Syndrome BBS [OMIM 209900] is a rare genetically heterogeneous
autosomal recessive ciliopathy characterized by primary features such as retinitis
pigmentosa, postaxial polydactyly, obesity, ID, and hormonal & renal dysfunctions.
24
Table 1.4. List of known loci and genes identified for nonsyndromic AR-ID
Locus
Cytogenetic
band
OMIM
entry
Gene
identified Type of mutation
MRT1 4q26 249500 PRSS12
4BPDEL,1350ACGT(Molinari et
al., 2002)
MRT2 3p26.2 607417 CRBN
p.R419X(Higgins et al., 2000)
dbSNP: 121918363
MRT3 19p13.12 608443 CC2D1A
IVS13-16DEL(Basel-Vanagaite et
al., 2006)
MRT4 1p21.1-q13.3 611107
MRT5 5p15.31 611091 NSUN2
p.Q227Y(Abbasi-Moheb et al.,
2012), Q372Y(Khan et al., 2012),
c.IVS5A>C,GLN679
c.IVS1 G>C(Martinez et al., 2012)
MRT6 6q16.3 611092 GRIK2
DEL/INV EX7-11(Motazacker et
al., 2007)
MRT7 8p22 611093 TUSC3
P.Q55Y(Garshasbi et al., 2011)
Ibp INS:787C(Molinari et al., 2008)
MRT8 10q22 611094
MRT9 14q11.2-q12 611095
MRT10 16p12.2-q12.1 611096
MRT11 19q13.2-q13.3 611097
MRT12 1p34.1 611090 ST3GAL3 p.D370Y(Hu et al., 2011)
MRT13 8q24.2 613192 TRAPPC9
p.R475Y(Mochida et al., 2009),
c.1422C>T, p.R475X(Mir et al.,
2009)
c.1708C>T, p.R570Y(Philippe et al.,
2009)
MRT14 19q13.12 614020 TECR p.P182L(Caliskan et al., 2011)
MRT15 9q34.3 614202 MAN1B1 p.R334C(Rafiq et al., 2011)
MRT16 9p23-p13.3 614208
MRT17 11p15 614207
MRT18 6q23.2 614249 MED23 p.R617Q(Hashimoto et al., 2011)
MRT19 18p11.3
614343(Ab
ou Jamra et
al., 2011)
No gene has been identified yet
MRT20 16p12.2-q12.1
611096(Ab
ou Jamra et
al., 2011)
MRT21 11p15
MRT22
25
Table 1.4 cont.
Locus Cytogenetic band OMIM entry
Gene
identified Type of mutation
MRT23 11p13-q14
614344(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
MRT24 6p12.2-q12
614345(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
MRT25 12q13.11-q15
614346(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
MRT26 14q11-q12
614346(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
MRT27 15q24.1-q26.1
614340(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
MRT28 6p26-q27
614347(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
MRT29 4q27-q28.2
614333(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
MRT30 6q12-q15
614342(Abou
Jamra et al.,
2011)
No gene
identified
No mutation
identified
26
The secondary features may include hepatic fibrosis, language deficits, behavioral
traits, facial dysmorphism, dental anomalies, developmental delay and cardiac
abnormalities. To date 17 genes have been identified to cause BBS including BBS1
(11q13) (Beales et al., 1999), BBS2 (16q21) (Kwitek-Black et al., 1993), BBS3 (3p12-
q13) (Chiang et al., 2004), BBS4 (15q22.3) (Chiang et al., 2004), BBS5 (2q31)
(Mykytyn et al., 2001), BBS6 (20p12), BBS7 (4q27), BBS8 (14q32.11), BBS9 (7p14),
BBS10 (12q21.2), BBS11 (9q33.1), BBS12 (4q27) (Hjortshoj et al., 2008), BBS13
Table 1.4. List of known loci and genes identified for nonsyndromic AR-ID. (17q23) ,
BBS14 (12q21.3), BBS15 (2p15), BBS16 (1q43) (Chen et al., 2011; Schaefer et al.,
2011) and BBS17 (LZTFL1) (3p21.31) (Marion et al., 2012).
1.14.2.2 Microcephaly Associated Syndromes
In Microcephaly syndromes, the clinical features other than microcephaly
differentiate them from one another. Often a characteristic feature other than
microcephaly marks a specific syndrome associated with ID (Woods et al., 1992).
Seckel syndrome (MIM-210600), Filippi syndrome (MIM-272440) and Jawad
syndrome (MIM-251255) are three different syndromes in which microcephaly, ID,
dwarfness and digital anomalies are common. A „bird-headed‟ peculiar facial feature
is the hallmark of Seckel syndrome while digital anomalies and severe ID are
distinctive marks of Filippi syndrome (Filippi 1985; Goodship et al., 2000; Borglum
et al., 2001; Kilinc et al., 2003; Sharif and Donnai 2004), while polydactyly,
synpolydactyly, café de laute spots on the skin are distinguishing features of Jawad
syndrome (Hassan et al., 2008). For Seckle syndrome five loci have been identified
i.e. SCKL1, where the causative mutation is in ATR (ataxia telangiectasia and Rad3
related) which has been shown to produce alternative splicing site (Borglum et al.,
2001), SCKL2 has been linked to the chromosome region 18p11.31-q11.2 (O'Driscoll
et al., 2003), SCKL3, to the chromosomal region 14q23-q24 (Goodship et al., 2000)
and SCKL4 which has been linked to the locus 13q12.2 where a mutation in the
CENPJ (Centromere protein J) gene has been reported (Griffith et al., 2008). In
addition the SCKL5 locus, which is linked to 15q21 has been identified to contain
mutations in CEP152 (Centrosomal protein of 152 kDa) (Kalay et al., 2011). For
Filippi syndrome no gene has yet been identified (Filippi, 1985). Qvist et al. (2011),
have reported that one of the causative genes for both Jawad syndrome and Seckle
27
syndrome is RBBP8 besides the other causative genes for Seckle syndrome (ATR,
CENPJ, and CEP152)
1.15 Molecular Pathways Involved in ID
ID is a highly diverse heterogeneous group of cognitive disorders, till date causative
mutations in more than 450 genes have been identified, such genetic defects account
for more than 50% cases of syndromic and nonsyndromic ID (van Bokhoven, 2011).
As the number of causative genes increase, the number of biological pathways
involved in cognitive development also increase. It has been shown by different
investigators that the ID causative genes actually share common molecular pathways.
In the section below brief description of some of the important genes and their
biological pathways are summarized.
1.15.1 Genes of Glutamate Receptors and Excitatory Synapses Pathway
In the human brain, the glutamate receptor ion channels mediate fast
neurotransmission at the excitatory synapses. These are in distinct subfamilies such as
AMPA, Kainate and NMDA, differing in their functional properties (Gielen, 2010).
Many of the genes identified in ID are linked to ionotropic glutamate receptor and
excitatory synapses such as GRIK2 (Glutamate receptor, ionotropic kainate 2), which
encodes GLuR6 a subunit of a Kainate receptor (KAR).
Similarly mutations in GRIN2B (Glutamate [NMDA] receptor subunit epsilon-2) that
encodes the protein NR2B, which is the subunit of the NMDA receptor (NMDAR),
has been shown to be the causative factor of ID in 6 NS-ID patients.
SYNGAP encodes SynGAP, a GTPase activating protein that is part of the NMDAR
complex, which is involved in binding to the NR2B subunit, mutations in this gene
have also been shown to cause ID (Kim et al., 2005). While another gene of this
pathway i.e. DLG3 (Discs, Large Homolog 3), which is also the part of NMDAR
complex has been found to be mutated in multiple families with NS-ID (Tarpey et al.,
2004; Pavlowsky et al., 2010).
28
1.15.2 Genes of Synaptic Plasticity Pathway
Synaptic plasticity is a very important process involved in cognition, learning and
memory. NLGN4 (Neuroligins 4) and Neurexins are the synaptic genes, among them
NLGN4 has been shown to be involved in neuronal cell adhesion and synaptogenesis
by interacting with glutamatergic postsynaptic proteins and NRXN/NLGN complexes.
While a number of mutations in OPHN1 (Oligophrenin-1) have been reported in
probands of S-ARID (Nadif et al., 2009), this protein also plays an important role in
synaptic plasticity and maturation by encoding an activity-dependant protein, which is
important for stabilizing the AMPARs (α-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid receptor).
Two other genes i.e. PRSS12 (Protease, Serine, 12) and FMR1 (Fragile X Mental
Retardation 1) are both also involved in neuronal plasticity. PRSS12 encodes
neurotrypsin, which is a synaptic protease; it is activated by NMDAR and works to
adhere to agrin at the neuromuscular junction synapse and in CNS. A number of
mutations in this gene have been shown to cause NS-ID (Matsumoto-Miyai et al.,
2009). While FMRP (Fragile X Mental Retardation 1 Protein) is encoded by FMR1,
which is an established gene for fragile X syndrome, it functions in neuronal plasticity
by working as a repressor of local protein translation (Bagni and Greenough, 2005).
Figure 1.3 shows the network of genes, identified in regulation of synaptic plasticity.
1.15.3 Cell Adhesion Pathway
CDH15 (Cadherin 15) is another candidate gene of ID, which is localized in the brain
and skeletal muscles, defects in this gene reduces the adhesion of cells by more than
80% (Bhalla et al., 2008).
1.15.4 Genes of MAGUK family
Genes of MAGUK family (Membrane-Associated Guanylate Kinases) such as CASK
(Calcium/calmodulin-dependent serine protein kinase) and IL1RAPL1 (Interleukin 1
receptor accessory protein-like 1) encode synaptic proteins. CASK acts as a Mg2+
independent neurexin kinase, and forms networking of many proteins from different
29
Figure 1.3. Schematic representation of known ID genes and their involvement in
synaptic plasticity and transmission. The figure elaborates the networking of different
molecular signaling pathways such as organization of postsynaptic-membrane protein
complexes, controlling presynaptic vesicle cycling, and organization of postsynaptic-
membrane protein, control of chromatin structure and gene expression and regulation
of cytoskeleton dynamics. The figure has been adapted from Vaillend et al. (2008).
30
groups at the cellular junctions, many ID patients have been reported with mutations
in this gene (Mukherjee et al., 2008), while defects in IL1RAPL1 are known to be the
causative factor in a number of autism and NS-ID probands. Mutations in this gene
results in defective localization of the MAGUK family protein i.e. PSD-95(DLG4)
(Connector enhancer of Kinase suppressor of Ras 1) which is essential for regulation
of functioning and structuring of NMDA receptors, signaling proteins and ion channel
regulation.
1.15.5 Rho GTPase Pathway
Rho GTPases are another very important pathway in NS-ID as well as S-ID. In this
pathway RALGDS (Ral Guanine Nucleotide Dissociation Stimulator) and CNKSR1
homozygous missense and frameshift mutations, respectively have been identified by
Najamabadi et al., (2011). Protein encoded by RALGDS stimulates the disassociation
of GDP from the Ras-related RalA and RalB GTPases, thus allowing GTP binding
and activation of the GTPases. The GTPases regulator belongs to a distinct pathway
of genes, which have been reported to be involved in NS-ID. CNKSR1 is another
candidate gene for NS-ID, it is a Rho effector, which acts as a scaffold protein and
mediates networking between the Ras and Rho GTPase signaling pathways.
Mutations in FGD1 (FYVE, RhoGEF and PH domain containing 1), OPHN1, PAK3
(P21 protein Cdc42/Rac-activated kinase 3) and ARHGEF6 (Rac/Cdc42 guanine
nucleotide exchange factor (GEF) 6) all have been shown to be involved in causing
ID (Ramakers, 2002), as these regulate the cascade of events through the Rho
signaling pathways such as cytoskeleton assembly, vesicular trafficking in dendritic
spines etc. These are all crucial steps, defects in which can result in impairing
different processes, which are essential for cognitive functioning.
1.15.6 Genes of Enzymatic Pathway
Mutations in some genes have been identified in causing ID, which belong to different
enzymatic pathways such as MAN1B1 (Mannosidase, alpha, class 1B, member 1),
NSUN2 (NOP2/Sun RNA methyltransferase family, member 2), ST3GAL3 (ST3 beta-
galactoside alpha-2, 3-sialyltransferase 3) and TECR (Trans-2,3-enoyl-CoA
reductase). MAN1B1 has been identified to be mutated in NS-ID cases, it encodes for
endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase. This
31
enzyme belongs to a group of enzymes, which functions to mature N-linked glycans
in the secretory pathway (Rafiq et al., 2011).
Khan et al. (2012), identified mutation in NSUN2 in a Pakistani inbred family; these
investigators reported the expression of NSUN2 protein in the cortical and brainstem
neurons of 3-day-old and 3-month-old mice. The most striking localization of this
protein was in the nucleoli of Purkinje cells of the cerebellum, specifically adjacent to
the dense heterochromatic regions.
ST3GAL3 was identified by Hu et al. (2011), to cause NS-ID, this gene encodes beta-
galactoside-alpha-2, 3-sialyltransferase-III, a Golgi resident membrane protein that
forms the sialyl Lewisa
(Carbohydrarte antigen), epitope on glyocoproteins. These
glycoproteins form the glycocalyx, which is composed of sialic acid that act as key
determinants of a variety of cellular recognition and communication processes. Moon
and Horton. (2003), reported that TECR was a trans-2, 3-enoyl-CoA reductase and
they showed it to be implicated in NS-ID.
1.15.7 Housekeeping genes
Several mutations in housekeeping genes such as LARP1 (La ribonucleoprotein
domain family, member 1), LDMA, KDM6 (Lysine (K)-Specific Demethylase 6A),
HISTIH4B, HIST3H3, TRIMT1, EEF1B2, ADRA2B, POLR3B, C11ORF46, UBR7,
ZCCHC8 and ABCD6 have been reported by Najamabadi et al. (2011). All these
genes have been shown to be involved directly or via other pathways in the regulation
of DNA replication, transcription, translation and post-translational events. One of the
house keeping gene such as RBBP8 (Retinoblastoma protein 8) has been shown to be
involved in DNA repair mechanism and it has been reported by Qvist et al. (2011) to
be involved in causing S-ID.
1.15.8 Genes of Epigenetic Pathway
One of the important pathways implicated to be involved in cognition is based on
genes that encode regulators of chromatin structure and of chromatin mediated
transcription regulation. To date more than 20 “epigenetic ID genes” have been
identified and this number is increasing with time as the availability of modern
techniques for diagnosis increases. A prominent example is the EHMT1, encoding
euchromatin histone methyltransferase 1, which carries heterozygous mutations in
32
25% of patients with a recognizable ID disorder, classified as Kleefstra syndrome. It
has been reported that four genes MBD5, MLL3, SMARCB1 (SWI/SNF related,
matrix associated, actin dependent regulator of chromatin, subfamily b, member 1),
and NR1I3 (Nuclear receptor subfamily 1, group I member 3) encode epigenetic
regulators. All of them have been shown to be involved in chromatin modification,
which is ultimately involved in cognition and other brain development processes (van
Bokhoven, 2011).
1.15.9 Miscellaneous Pathways
Some of the genes involved in ID belong to different pathways such as CACNA1G (Calcium channel, voltage-dependent, T type, alpha 1G subunit) and TRAPPC9
(Trafficking protein particle complex 9). CACNA1G encodes the protein, which is a
calcium ion channel and has a critical role in GABA (gamma amino butyric acid)
receptor-mediated spike and wave discharges in thalamocortical pathways, many
mutations have been identified in NS-ID patients in this gene (Hu et al., 2005;
Najmabadi et al., 2011). While TRAPPC9 binds NIK and IKK-β and plays a central
role in the neuronal NF-kappa-β signaling pathway and mutations found in this gene
have been reported to be pathogenic and resulted in causing NS-ID as well as
postnatal microcephaly and syndromic ID (Hu et al., 2005).
Most of the genes identified till date have been located using conventional linkage
analysis, homozygosity mapping, CNV analysis, array technology and next generation
sequencing in addition to CGH hybridization, FISH and other valuable traditional
techniques. The genes are from various pathways that are involved in different
functions, which indicate that not only the genes expressed in the brain are involved
in cognition but genes from multiple pathways including housekeeping as well as
enzymatic and structural pathways can also be implicated in cognition in addition to
their other vital functions.
1.16 Objectives
The current study was conducted in order to identify the genetic basis of Mental
Retardation/Intellectual Disability in the Pakistani population. In this regard various
genes involved in cognition were identified using techniques such as microarray
33
analysis, Next Generation Sequencing (Exome sequencing). It is hoped that these
findings will be helpful in designing different strategies of gene therapy, for the
patients suffering from ID and will be helpful in decreasing, the disease burden in the
society.
Chapter 2
Material and Methods
35
2 Material and Methods
In the present study sixteen consanguineous families presenting with ID were
identified from different areas of Pakistan. Initially, autosomal as well as X-linked
families with atleast one affected individual were selected. The affected members of
the different families were suffering from both syndromic and non-syndromic ID.
2.1 Collection of families
The families were identified by visiting different special schools and disability
centers. Each family was visited at their residence to evaluate the affected as well as
unaffected members. The parents were interviewed in detail to determine the
complete prenatal, perinatal, postnatal, and other developmental history of the
affected individual in order to exclude environmental and accidental causes of ID.
The assessment of ID was done by evaluating the learning disabilities, cognitive
impairment and I.Q assessment by using a special questionnaire (amended and
translated version of Wechsler scale). The study was duly approved by the Ethics
Review Committee of COMSATS Institute of Information Technology, Islamabad,
and Radboud University Medical Centre Nijmegen, The Netherlands.
2.1.1 Clinical Diagnosis
The clinical diagnosis was done in two steps, initially a detailed profile of the affected
member was obtained including weight and height at birth. In addition the detailed
clinical features were noted including the head circumference, involvement of any
structural or facial dysmorphism, digital anomalies, ectodermal and epidermal
dysmorphism. Complete family structure of affected members was also noted to
determine the mode of inheritance of the disease. Children with complicated labor and
hypoxia at birth were excluded from the study.
The initial screening was followed by a detailed evaluation of the proband by a
clinician who tested the IQ as well as other developmentally delayed milestones of the
proband. In addition other clinical data were obtained including serum lactate, serum
electrolyte, complete blood picture, thyroid testing by hormonal profiling of T3/T4
and CT (computed tomography) scan or MRI (magnetic resonance imaging). Some
biochemical tests for the measurement of amino acid, creatine and creatinine level;
36
and urine profile were also obtained, where required to exclude metabolic disorders
associated with ID. Eye examination was performed in the case of ciliary disorders
like BBS.
2.2 Collection of Families and Blood Sampling
The proband were identified from different areas of Pakistan and from National
Institute of Handicap, Islamabad and National Institute of Training for Disabled,
Islamabad. After obtaining informed written consent of the guardian of the proband,
blood was drawn through venipuncture from all available family members and kept in
acid citrate dextrose (ACD) vacutainers at 40C till further use.
2.3 Genomic DNA Extraction from Blood
The gDNA was isolated from whole blood using a standard phenol chloroform
method as described by Sambrook and Russell (2006). Briefly the method consisted
of lysis of red blood cells (RBC) followed by removal of cellular debris and protein
digestion using Proteinase K. The digested proteins were removed by extraction with
phenol/chloroform and isoamyl alcohol. Precipitation of gDNA was done by using
isopropanol and sodium acetate followed by washing with 70% ethanol. Finally the
gDNA was resuspended in 1X TE buffer (pH 8.0) and stored at -20˚C till further use.
The gDNA was quantified by separation on 1% agarose gel in comparison with a
known concentration of λ-HinD-III DNA ladder. The gel picture was obtained using a
Gel documentation system and the software Bio Cap MW software V11.01 (Vilber
Lourmat, Cedex, France).
2.4 FMR1 Screening
The families with only males affected with ID were further screened for the
determination of the repeat length by using a three-primer CGG repeat
primed FMR1 PCR method. A total of six families were screened for FMR1. The
protocol consisted of the following steps:
37
2.4.1 CGG RP PCR
CGG RP PCR was performed using the kit Amplide XTM
FMR1 PCR kit (Asuragen,
Inc. Austin, TX) to calculate the number of CGG repeats in FMR1 gene. PCR
mastermix was prepared by using 11.45 µL GC-Rich AMP Buffer, 1.5µL of 6-
carboxyfluorescein (FAM)-labeled FMR1 reverse primer (FAM-
AAGCGCCATTGGAGCCCCGCACTT) and unlabelled forward primer
(CAGCTCCGTTTCGGTTTCA) and 0.05 µL GC-Rich Polymerase Mix. 40ng of
gDNA probands was mixed with 13 µL of mastermix and amplified using ABI 9700
PCR (Applied Biosystems, Foster City, CA). Amplification cycles consisted of initial
denaturation at 98 °C for 5min followed by 25 cycles of 97 °C for 35 sec, 62 °C for
35 sec, and 72 °C for 4 min; and a final extension at 72 °C for 10 min. After
amplification the samples were stored at 4 °C to 30 °C till further analysis.
2.4.2 Capillary Electrophoresis (CE) Genotyping
The repeat length was determined by capillary electrophoresis (CE) using a 3130XL
Genetic Analyzer (Applied Biosystems) as per the manufacturer‟s recommendations
using a ROX 1000 size ladder (Asuragen, Inc).
2.4.3 Data Analysis
Gene Mapper software (version 4.0; Applied Biosystems) was used to analyze the
data obtained from the CE. The FMR1 alleles were categorized according to the
American College of Medical Genetics guidelines for normal (<45 CGG repeats),
intermediate (45-54 CGG repeats), pre mutation (55-200 CGG repeats), and full
mutation (>200 CGG repeats).
2.4.4 Southern Blot Analysis
Southern Blot analysis was performed for the proband of family MRQ 8, who had the
full mutation allele, to confirm the fragment size. The proband‟s DNA was digested
with EcoRI and NruI followed by separation on a 0.8% agarose/Tris acetate EDTA
(TAE) gel, after which partial depurination was done, followed by denaturation of
samples. DNA sample was then transferred in 10X standard saline citrate (SSC) to a
charged nylon membrane (Roche Diagnostics, Basel, Switzerland) using a vacuum
38
transfer apparatus (Vacuum Blotter 785; Bio-Rad, Hercules, CA). A 1-kb DNA ladder
(Invitrogen, Carlsbad, CA) was used as a size standard. The cross-linking of
membranes was done with a UV Cross linker (Fisher Scientific, Pittsburgh, PA) and
the membrane was hybridized overnight at 42°C in roller bottles (Isotemp, Fisher
Scientific) in Dig Easy Hybridization Buffer (Roche Diagnostics) with the FMR1
genomic probe StB12.3 (McConkie et al., 1993), labeled with Dig-11-dUTP (PCR
Dig Synthesis Kit; Roche Diagnostics). After denaturation, the membrane was
blocked with Cot1 DNA (Invitrogen) and the filters were washed in 2X SSC/1% SDS
followed by 1× SSC/0.1% SDS at 65°C. The membrane was then processed using the
detection buffer according to the manufacturer (Roche Diagnostics) and X-ray film
(Super RX; Fuji Medical X-Ray Film, Bedfordshire, UK) was exposed to the
membrane. Analysis of the CGG repeat number on the Southern blot was done using
the Tech FluorChem 8800 Image Detection System (Alpha InfoTech, San Leandro,
CA).
2.5 Microarray Homozygosity Mapping
All families were genotyped on high resolution platform GeneChip 250K Nsp array
(Affymetrix, Santa Clara, CA), while family D1 was analyzed using 270K cytogenetic
array to identify the homozygous regions among them.
2.5.1 Steps for SNP array
The 250K Nsp array is highly sensitive and is capable of genotyping more than
250,000 SNPs, in addition it also contains 474,642 SNP probes for CNV analysis. The
microarray experiment was performed in 96-well plate and consisted of the following
steps.
2.5.2 Sample preparation
First the concentration of pre extracted gDNA was analyzed by measuring the
concentration with a nanodrop spectrophotometer (Isogen, Life Science, De Meern,
and The Netherlands). Purified DNA with concentration of 50 ng/μL was subjected to
subsequent analysis. 5 μL of each DNA sample was aliquoted in each well of 96 well
plate.
39
2.5.3 Digestion of samples by restriction enzymes
During this particular step the samples are subjected to digestion using the NspI
restriction enzyme. The digestion mastermix was prepared in 2.5mL tube, each
mixture contained AccuGENE® Water 11.5 μL, NE Buffer 2 (10X) 2 μl, BSA (100X;
10 mg/mL) 0.2 μL and NspI (10 U/μL) 1 μL. The mastermix was then aliqouted to the
sample of DNA contained in the 96 well plate. The plate was placed in a thermal
cycler and heated at 37ºC for 120 minutes followed by heating at 65 ºC for 20 minutes
and finally was held at 4 ºC.
2.5.4 Ligation
During this stage the Ligation mastermix was prepared by adding Adaptor NspI
(50μM) 0.75μL, T4 DNA ligase buffer (10X) 2.5μL and T4 DNA ligase (400U/μL)
2μL per reaction in 2.5mL tube. 5.25μL ligation master mix was aliquot to previously
digested DNA. The program for ligation step consisted of heating at 16ºC for 180
minutes followed by heating at 70ºC for 20 minutes and the held at 4ºC. After ligation
step the samples in the 96 well plate were diluted with 75 µl of ultrapure water.
2.5.5 Polymerase Chain Reaction (PCR)
During this step PCR was performed by using the PCR mastermix that contained
ultrapure water 39.5 μL, TITANIUM Taq PCR Buffer (10X) 10 μL, GC-Melt (5M)
20 μL, dNTP (2.5 mM each) 14 μL, PCR Primer 002 (100 μM) 4.5 μL, TITANIUM
Taq DNA Polymerase (50X) 2 μL and then 90μl master mix was added to the 96 well
plate which already contained the 10μl ligated DNA. The DNA was then amplified in
a thermal cycler and the following steps, preheating of chamber, stage1(denaturation):
94ºC for 3 minutes (1 cycle), (Annealing) stage 2: 94ºC for 30 sec, 60ºC for 45 sec
and 68ºC 15 sec, total number of cycles were 30. Final extension was done at 78 ºC
for 7 minutes.
2.5.6 Fragmentation
This step consisted of fragmenting of DNA by DNaseI. The reaction mixture for
fragmentation contained 5 μl of fragmentation buffer (DNaseI buffer) to 96 well plate
containing amplified purified PCR products. The total volume in each well was 50 μl
(45 μl PCR product: 5 μl fragmentation buffer). At this step the diluted fragmentation
40
reagent 5 μl was added to the mixture of fragmentation buffer and sample. The plate
was then loaded on a thermal cycler and heated at 37 ºC for 35 minutes and 95 ºC for
15 minutes.
2.5.7 Labeling
During this step the fragmented DNA was subjected to 3' end labeling by using
Terminal deoxynucleotidyl transferase (TdT). The labeling mastermix contained TdT
Buffer (5X) 14 μL Terminal deoxynucleotidyl transferase (TdT) (30 mM) 2 μL, and
TdT enzyme (30 U/μL) 3.5 μL. Total of 19.5 μL of reaction mixture was aliqouted to
each well of the 96 well plate. The plate was then heated in a thermal cycler at 37ºC
for 4 hours and 95ºC 15 minutes. This step was followed by targeted hybridization.
2.5.8 Targeted Hybridization
The step consisted of preparing the hybridization mixture which contained MES
(12X; 1.25 M) 12 μl, Denhardt‟s Solution (50X) 13 μL, EDTA (0.5 M) 3 μL, HSDNA
(10 mg/ml) 3 μL, OCR, 0100 2 μL 220 μL, Human Cot-1 DNA (1 mg/ml) 3 μl,
Tween-20 (3%) 1 μL, DMSO (100%) 13 μl and TMACL (5 M) 140 μL. The
hybridization mixture was then heated at 95 ºC for 10 minutes ,followed by a holding
step at 49 ºC. The hybridized samples were loaded on the Affymetrix 250 K array
platform (Affymetrix, Santa Clara, CA) for processing. The chips were washed,
stained and finally scanned by using GeneChip scanner 3000 7G (Affymetrix, Santa
Clara, CA).
2.5.9 Data Analysis
For CNV determination the Affymetrix 250K SNP array data were analyzed using
Copy Number Analyzer for GeneChip (CNAG)
(http://www.genome.umin.jp/CNAG_DLpage/files/CNAG), while the Affymetrix
Genotyping Console (version 2.0) was used to obtain the genotype calls. The data
were analyzed using the online tool Homozygosity Mapper
(http://www.homozygositymapper.org/) to determine the common homozygous
intervals among affected family members. The regions of homozygosity of at least 1
Mb were further analyzed by visual inspection. All data were mapped using the
Human Genome Build hg19 release Feb. 2009.
41
2.6 Candidate Gene Analysis and Mutation Screening
The array analysis provided good homozygous intervals in some of the families,
where the genes which were already known for ID were sequenced in order to
determine the pathogenic mutations among them. List of families and their
homozygous regions obtained from Homozygosity mapper and candidate genes
screened among them are listed in Appendix 17. Besides array analysis exon and
exon-intron junctions of candidate genes such as HPRT1 and TPO were sequenced
based upon the phenotype match of the affected members with particular syndromes.
2.6.1 Sanger Sequencing
Sequencing primers were designed by Exon Primer tool of UCSC Genome Browser
(http://genome.ucsc.edu/) and by Primer3 version 0.4.0,
(http://frodo.wi.mit.edu/primer3/). The primer sequences and their product sizes are
given in Appendix 1-8. Briefly the sequencing of different genes consisted of the
following steps: the exons and exon intron boundaries were amplified using a thermal
cycler (Gene AMP, PCR System 9700, Applied Biosystems) using 0.25mM dNTPs,
1X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl), 2.5mM Mg+2
using MgCl2
0.5μM of each primer, 2.5U Taq polymerase (Fermentas Life Sciences, Ontario,
Canada) and 50ng gDNA. The thermal profile consisted of initial denaturation at 95˚C
for 5 min followed by 30 cycles of amplification at 95˚C for 1 min, 57˚C for 30 sec
and 72˚C for 45 sec, a final extension was also carried out at 72˚C for 5 min. After
amplification the samples were separated on 2% agarose gel, visualized under UV
transillumination and documented using the gel documentation system (Vilber
Lourmat).The PCR products obtained were then purified for sequencing, using the
GeneJET TM PCR purification kit (Fermentas, Life Sciences). The purified products
were then sequenced using the dye-termination chemistry (BigDye Terminator,
version 3 on a 3730 or 2100 DNA analyzer; Applied Biosystems) and the same
primers as were used in the PCR amplification. The sequencing data was analyzed
manually by using the tool Finch TV (http://www.geospiza.com/
Products/finchtv.shtml) and VectorNTI Advance 11 software (Invitrogen Life
Technologies).
2.6.2 In-Silico Analysis of Sequence Variants
In case a mutation was found in a particular gene it was further analysed for its
pathogenic impact by in silico analysis using the online tools Alamut Biointeractive
42
http://www.interactive-biosoftware.com, which gave the conservation of the amino
acid change in different species. The tools used were phyloP (Phylogenetic profiling
conservation score analysis, PhastCons scores, allele frequency in public databases
(dbSNP and Exome Sequencing project)) as well as the Grantham distance. The
Polyphen (Polymorphism Phenotyping v2) (http://genetics.bwh.harvard.edu/pph2/)
and Sift (Sorting intolerance from tolerance) score (http://sift.jcvi.org) as well as
Align GVGD, and Mutation Transfer scores were also calculated from Alamut
Biointeractive software to detect the damaging effect of the mutation. Various online
tools such as HSF (Human Splicing Finder), MaxEntScan (Maximum Entropy scan),
NNSplice (Splice site prediction by neural networks) and GeneSplicer,
(cbcb.umd.edu/ software/ Gene Splicer/) were used to determine the impact of the
canonical splice site mutations.
2.7 CNV analysis
Copy number variations were analyzed manually by investigating the data obtained
from CNAG tool. The consistent decrease of less than -3 in the log ratio five times
consecutively in the data were considered standard for microdeletion in a particular
region of the chromosome, while consistent increase of more than 3 in the log ratio,
five times consecutively in the data were considered to be standard for the presence of
double copy number. Confirmation of exact copy number in any particular region was
done by Syber green based qPCR analysis. The primers for qPCR were designed
using the online software Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/
primer3_www. cgi). The reaction employed the reference gene CFTR and the
quantification of the candidate gene was done relative to it. All the amplification
reactions were performed using the ABI Prism 7500 detection system (Applied
Biosystems). The thermal profile consisted of initial denaturation at 95 °C for 10 min
which was followed by 35 cycles of amplification at 95 °C for 15 sec, 60 °C for 1
min. Subsequently, the PCR products were subjected to a linear temperature transition
from 60 °C to 95 °C and the melting curves of Syber Green fluorescence was obtained
using the ABI Prism 7500 software (Applied Biosystems). The DNA CNV
differences for gene were calculated using the protocol of Livak and Schemittgen.
(2001). In the current study, two pathogenic CNVs encompassing NRXN1 were
identified in family MRQ12 and D1 with the help of the Affymetrix 2.5K array data
43
and 2.7K Cytogenetic array data, respectively. The lists of primers for NRXN1 qPCR
are given in Appendix 9.
2.8 Next Generation Sequencing
Families in which no true homozygous regions were identified by array analysis were
subjected to Exome Sequencing using the Next Generation sequencing (NGS)
platform. This technique can be highly advantageous in the identification of novel
genes, when used in combination with homozygosity mapping data, and conducting
targeted exome sequencing of the identified linkage regions. In addition, this
technique is also suited for the identification of compound heterozygous mutations. In
this technique exonic sequences of all genes are first enriched and then sequenced. p
robands of different families (MRQ11, MRQ14, MRQ15 and MRQ20) were selected
for exome sequencing, which was performed on a 5500XL sequencing platform from
Life Technologies (Carlsbad, CA). The protocol consisted of enrichment of the
exomes of probands of families MRQ11, MRQ14, MRQ15 according to the
manufacturer‟s protocol using Sure SelectXT Human All Exon v.2 Kit (50Mb),
containing the exonic sequences of approximately 21,000 genes from Agilent
Technologies, Inc. (Santa Clara, CA). Life Scope software v2.1 from Life
Technologies was used to map the reads along the hg19 reference genome assembly.
The DiBayes algorithm, with high-stringency calling, was used for single-nucleotide
variant calling, while the small Indel Tool was used to detect small insertions and
deletions.
2.8.1 Library Preparation
Briefly the enrichment was carried out by shearing the DNA sample to produce
fragments of 150-200 bps, ends of these fragments were repaired to produce blunt
ends, which were phosphorylated at the 5‟ end, adenine overhangs were then added to
the blunt-ended DNA fragments with the help of the Klenow fragment of DNA
polymerase. Indexing-specific adaptors were then ligated to these fragments, which
were then PCR amplified followed by hybridization to the enrichment probe library,
which contained biotinylated ribonucleic acid (RNA) probes. Hybridized fragments
were selected by streptavidin coated magnetic beads, and RNA probes were then
44
digested and the enriched DNA was eluted to be used in further downstream
processes. For DNA sequencing the SOLiD TM sequencer from Life Technologies, a
ligation mediated di-base detection system along with a set of five universal
sequencing primers were used. In the first step universal sequencing primers were
annealed, these bind to adapter sequences, this was followed by annealing and ligation
of the 8-mer probe, which contained two colour-coded bases and a fluorescent label at
the 3‟ end. Fluorescence detection followed cleavage and removal of three bases at
the 3‟ end of the 8-mer probe along with the fluorescent label. Each universal primer
was then offset by a base followed by probe annealing, ligation, fluorescence
detection and cleavage of fluorescent label to detect the incorporated bases. The DNA
template sequence was then finally retrieved by decoding the sequences of the
detected colours. Sequencing reads were then aligned against a reference assembly to
find the variants using LifeScope™ Genomic Analysis Software v2.5.1. The exome
sequencing data were filtered to narrow down the number of variants to determine the
identity of the causative mutation.
2.8.1 NGS Data Filtration
The data filtration process consisted of a number of steps; initially thousands of
variants were obtained of which quite a few were located in exonic sequences while
others were in canonical splice sites. The first filtering step was based on excluding
all variants, which were found in dbSNP (v132) and an inhouse database, which
consisted of 177 exomes based on healthy individuals and patients of extremely rare
disorders. Minimum cut off value for variant sequence reads was set upto five. This
step further reduced the number of variants to hundreds, most of which were non-
synonymous changes and altered transcripts. As there was a recessive pattern of
inheritance in the families under study, the data were filtered to retain only
homozygous changes (with 70% variant calls) and compound heterozygous (20%-
80% variant calls). The next step of filtering the data was Binary Sequence
Alignment/Map format (BAM file) investigation, which was conducted manually to
validate the presence and minimal percentage (20% for heterozygous and 70% for
homozygous variants) of all variant reads. Manual validation was conducted because
the number of reads in automatically generated output file often varies from the actual
number of reads in BAM file, this step further reduced the number of variants to
45
mostly homozygous changes in recessive diseases and some compound heterozygous
changes.
2.8.2 Variant Screening
The variants obtained for all families after the final filtration step were further
analyzed with Alamut Biointeractive software to obtain the phyloP , PhastCons
scores, Grantham distance , where a score of more than 2.0 for the former was
considered to be pathogenic, while for the latter a distance of more than 80 was taken
to be pathogenic, information on variant scoring alogrithams such as Align GVGD,
SIFT, Mutation Transfer, Polyphen2 and Splice site predictions. Thus the variants
with less than 2 Phylop score and less than 80 Grantham distance were excluded,
which resulted in 5 to 11 homozygous variants and 3 to 4 compound heterozygous
variants in different families. In addition the homozygous regions obtained after array
analysis of the respective families were also checked in the exome data for the
presence of any pathogenic variant in the respective proband. The validation and
segregation of Exome data were conducted by sequencing the particular variant of the
gene in the families. The primers were designed for different variants in the gene
using the online software Primer3 (http://frodo.wi.mit.edu/). The primer sequences
and their product sizes for the variants of all the four families are given in Appendix
10-13. The list of variants c. DNA as well as protein position and the scores of
different pathogenicity prediction software are listed in Table 2.2 to 2.4.The
amplification by PCR and purification of PCR products for sequencing was done as
described above. affected family members detail clinical data was not available but
generally their symptoms were same as that of proband. The family members were
genotyped using 250K array but no homozygous region were found (Fig 3.36). Hence
Exome sequencing was performed, which revealed 19 homozygous variants and 22
compound heterozygous variants. Filtration on the basis of phyloP score and
Grantham distance left only eight variants, six homozygous and two compound
heterozygous. Segregation of all the variants was tested by Sanger Sequencing among
all family members but none of them segregated with the disease in the family, no
further analysis was performed to investigate the cause of ID in the family MRQ20.
46
2.9 X-Exome Sequencing
In addition to autosome sequencing, X-exome analysis was also carried out for
identifying the pathogenic mutations in genes located on the X-chromosome, which
was performed same as that of Next generation Sequencing. Families with X-
chromosome mode of inheritance including MRQ16, PKMR176 and PKMR71 were
selected for X-exome sequencing. The X-exome data were also filtered as described
above for the autosome exome sequencing. For the variants identified in the X-exome
sequencing the primers were designed and segregation checked as described above.
The primers and their product sizes are given in Appendix 14 and 15.
The detailed raw data will be submitted to Higher Education Commission thesis
repository for public.
47
Table 2.1. Family MRQ11 homozygous and compound heterozygous variant validation using Sanger sequencing and in silico, prediction.
Gene
(RefSeq id) Protein function
cDNA
change
Amino acid
change PhyloP
Grantham
distance SIFT
Mutation
transfer Polyphen Zygosity
Segregation
in Family
SMOX
(NM_175839)
Oxidation
reduction 1604C>A Ser535Tyr 5.409 144 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
TAS1R2
(NM_152232)
Signal
transduction 971C>T Gly324Asp 5.23 94 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
ATP11A
(NM_015205)
ATP biosynthetic
process 64G>T Asp22Tyr 5.14 160 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
ADORA2B
(NM_000676)
Positive
regulation of
cAMP
biosynthetic
process
590T>C Ile197Thr 4.87 89 Deleterious Disease
causing
Probably
damaging Homozygous
Not
segregating
ZNF589
(NM_016089)
Regulation of
transcription,
DNA-dependent
956T>A Leu319His 2.965 100 Deleterious Disease
causing
Probably
damaging Homozygous Segregating
ZNF502
(NM_033210)) Unknown 746G>A Asp820AspFs 5.11 1000 Deleterious
Disease
causing Probably
damaging Homozygous
Not
segregating
ADHFE1
(NM_144650)
Oxidation
reduction 955A>G le319Val 3.51 29 Tolerated
Disease
causing Probably not
damaging Heterozygous
Not
segregating
ADHFE1
(NM_144650)
Oxidation
reduction 1172C>T Thr391Ile 1.26 89 Tolerated
Disease
causing Probably
damaging Heterozygous
Not
segregating
CMYA5
(NM_153610) Unknown 7472T>C Ile2491Thr 2 89 Deleterious
Disease
causing Probably
damaging Heterozygous
Not
segregating
CMYA5
(NM_153610) Unknown 8534G>A Arg2845Lys 0.51 26 Tolerated
Not
disease
causing
Probably not
damaging Heterozygous
Not
segregating
DCHS1 Calcium- 3017C>T Arg1006His 2.55 29 Deleterious Disease Probably Heterozygous Not
48
(NM_003737) dependent cell-
cell adhesion
causing damaging segregating
DCHS1
(NM_003737)
Calcium-
dependent cell-
cell adhesion
1265T>A Tyr422Phe 4.91 22 Deleterious
Disease
causing Probably
damaging Heterozygous
Not
segregating
DPAGT1
(NM_001382.3)
UDP-N-
acetylglucosamine
metabolic process
951G>C Ser317Arg 4.39 110 Deleterious
Disease
causing Probably
damaging Heterozygous
Not
segregating
DPAGT1
(NM_001382.3)
UDP-N-
acetylglucosamine
metabolic process
38A>T Ile13Asn 4.86 149 Deleterious Disease
causing
Probably
damaging Heterozygous
Not
segregating
DENND2C
(NM_001256404) Unknown 1129T>C Lys377Glu 3.608 56 Unknown Unknown Unknown Heterozygous
Not
segregating
NM, mRNA accession number; PhyloP, Phylogenetic P-values; Polyphen, Polymorphism phenotyping; SIFT, Sorting intolerance from tolerance
49
Table 2.2. Family MRQ14 homozygous and compound heterozygous variant validation using Sanger sequencing and in silico pathogenecity
predictions
Gene
(NM id) Protein function cDNA change
Amino acid
change
PhyloP
score
Grantham
distance SIFT
Mutation
transfer Polyphen Zygosity
Segregation
in Family
NME7
(NM_013330) Unknown 38C>T Arg13Gln 5.53 43 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
PPP1R9A
(NM_01166160)
Nervous system
development 1387C>T Pro463Ser 5.29 74 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
DYM
(NM_017653)
Dyggve-melchior-
clausen disease,
223800 (3); smith-
mccort dysplasia,
1205A>T p.Leu402* 4.571 1000 Unknown Unknown Unknown Homozygous Not
segregating
DLG1
(NM_004087)
Cell to cell
adhesion, nervous
system
involvement
574T>C Ile192Val 4.518 29 Deleterious Disease
causing
Probably
damaging Homozygous
Not
segregating
KMT2B (MLL4)
(NM_014727) Unknown 2456C>T Pro819Leu 4.429 98 Deleterious
Disease
causing
Probably
damaging Homozygous Segregating
EHMT2
(NM_006709)
Chromatin
modification,
biological process
and nervous system
involvement
1151C>T Arg384Gln 3.503 43 Deleterious Disease
causing
Probably
damaging Homozygous
Not
segregating
VAV2
(NM_001134398)
Nervous system
phenotype 2495A>G Met832Thr 3.138 81 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
PLCD4
NM_032726)
Intercellular
signaling cascade 1885C>T Leu629Phe 2.9 22 Deleterious
Disease
causing
May be
damaging Homozygous
Not
segregating
ZNF227
NM_182490)
Regulation of
transcription,
DNA-dependent
956C>T Thr319Ile 2.715 89 Deleterious Polymorphism May be
damaging Homozygous
Not
segregating
50
SMEK1
(NM_032560) Unknown 2239A>C Ser747Ala 3.03 98 Tolerated
Disease
causing
Not
damaging Homozygous
Not
segregating
UGT8
(NM_001128174) CNS development 359A>G Asn120Ser 2.47 46 Tolerated
Disease
causing
Not
damaging Homozygous
Not
segregating
DNAH17
(NM_173628)
Microtubule-based
movement, ciliary
or flagellar motility
12599A>C Val4200Gly 4.821 109 Deleterious Unknown Probably
damaging Heterozygous
Not
segregating
DNAH17
(NM_173628
Microtubule-based
movement, ciliary
or flagellar motility
12267C>T Met4089Ile 5.974 10 Deleterious Unknown Probably
damaging Heterozygous
Not
segregating
SACS
(NM_014363) Protein folding 10291C>G Val3431Leu 4.266 32 Deleterious
Disease
causing
Probably
damaging Heterozygous
Not
segregating
SACS
(NM_014636) Protein folding 5461A>G Cys1821Arg 2.631 180 Deleterious
Disease
causing
Probably
damaging Heterozygous
Not
segregating
TEP1
(NM_007110)
Telomere
maintenance via
recombination
Lys1174Glnfs
*16 3519>C 2.109 1000 Unknown Unknown Unknown Heterozygous
Not
segregating
TEP1
(NM_007110)
Telomere
maintenance via
recombination
Pro606Leu 1817G>A 3.26 98 Tolerated Polymorphism Not
damaging Heterozygous
Not
segregating
NM, mRNA accession number; PhyloP, Phylogenetic P-values; Polyphen, Polymorphism phenotyping; SIFT, Sorting intolerance from tolerance
51
Table 2.3. Family MRQ15 homozygous and compound heterozygous variants validation using Sanger sequencing and in silico prediction.
Gene
(NM id) Protein function
cDNA
change
Amino acid
change PhyloP
Grantham
distance SIFT
Mutation
transfer Polyphen Zygosity
Segregation
in Family
SF3B3
(NM_012426) RNA splicing 82C>A Gln28Lys 6.119 53 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
LARGE
(NM_004737)
N-acetylglucosamine
metabolic process] 251C>G Ser84Thr 5.72 58 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
HHAT
(NM_001122834)
Multicellular
organism
development
1158G>C Trp386Cys 5.433 215 Deleterious Disease
causing
Probably
damaging Heterozygous
Present in
affected only
NEDD4
(NM_198400)
Protein
ubiquitination during
ubiquitin-dependent
protein catabolic
process
872C>T Gly291Glu 2.915 98 Deleterious Polymorphism Probably
damaging Homozygous
Not
segregating
PLCH1
(NM_001130960) Cell division IVS13+1C>T no 4.407 0 Unknown Unknown Unknown Homozygous
Not
segregating
PDS5B
(NM_015032)
Intercellular
signalling cascade IVS25-1G>A no 6.172 0 Unknown Unknown Unknown Homozygous
Not
segregating
WASF1
(NM_003931)
Protein complex
assembly IVS4+1C>T no 5.63 0 Unknown Unknown Unknown Homozygous
Not
segregating
DENND2A
(NM_015689) Unknown 54G>C Pro297Arg 2.342 110 Deleterious
Disease
causing
Probably
damaging Homozygous
Not
segregating
HMCN1
(NM_031935) Response to stimulus 7163G>A Gly2388Glu 4.013 98 Deleterious
Disease
causing
Probably
damaging Heterozygous
Not
segregating
HMCN1
(NM_031935) Response to stimulus 13190G>A Arg4397Gln 0.754 43 Tolerated
Disease
causing
Probably
not
damaging
Heterozygous Not
segregating
MED13L
(NM_015335)
Regulation of
transcription from 1447G>T Pro483Thr 3.577 38 Tolerated
Disease
causing
Probably
not Heterozygous
Not
segregating
52
RNA polymerase II
promoter
damaging
MED13L
(NM_015335)
Regulation of
transcription from
RNA polymerase II
promoter
740A>G Leu247Pro 2.644 98 Tolerated Disease
causing
Probably
not
damaging
Heterozygous Not
segregating
ZNF772
(NM_001024596)
Regulation of
transcription, DNA-
dependent
1145T>G Glu382Ala 2.468 107 Tolerated Disease
causing
Probably
not
damaging
Heterozygous Not
segregating
ZNF772
(NM_015335)
Regulation of
transcription, DNA-
dependent
878G>T Pro293His 1.828 77 Deleterious Polymorphism Probably
damaging Heterozygous
Not
segregating
NM, mRNA accession number; PhyloP, Phylogenetic P-values; Polyphen, Polymorphism phenotyping; SIFT, Sorting intolerance from tolerance
Chapter 3
Results
54
3.1 Results
Sixteen recessive families with individuals suffering from syndromic ID, non-
syndromic ID and X-linked ID were analyzed in the current study. Different
techniques were used to identify the genetic cause in these families (Table 3.1).
Out of the 16 families, mutations in five families (MRQ8, MRQ12, D1, MRQ18 and
MRQ19) in five different genes (RBBP8, NRXN1, TPO, BBS10 and FMR1) for ID
were identified by Microarray and candidate gene analysis. Further in three families
(MRQ11, MRQ14 and MRQ15) pathogenic mutations were identified by Exome
sequencing in three novel genes. For the remaining eight families (50%) no
pathogenic mutation was identified in any of the candidate genes. Results of the
solved and unsolved families are given below.
3.2 Family MRQ1
Five members of family MRQ1 (Fig. 3.1) were sampled (III:1: Father, III:2: Mother,
IV:1: proband affected son, IV:2 and IV:3: unaffected daughters) from Rawalpindi,
Pakistan. The family was highly consanguineous in which, the two female daughters
were unaffected with normal milestones while the affected proband (IV:1) was an 8
year old boy. The boy was born with uncomplicated labor after an uneventful 38
weeks pregnancy, his birth weight, height and head circumference were not available
but overall he seemed normal at birth. No hypoxia was noticed after birth, and he did
not suffer any postnatal infection. He had delayed milestones and only started walking
and speaking by the age of 4 years. At the time of examination at the age of 8 years
the boy was weak with a height of 38 inches and weight of 45lbs. In addition he could
only speak a few meaningful words and had some dental anomalies. Neurological
examination showed that his IQ was in the range of moderate ID (IQ: 36-51). His CT
scan did not show any structural abnormality in the brain, neither were any of his
biochemical levels elevated or below the standard levels. He had a shy demeanor but
no cardiac, respiratory, genital and other structural anomaly; thus the family was
classified as non-syndromic ID.
55
Table 3.1. List of techniques used for screening of the families.
Family ID Techniques
MRQ1 250k Affymetrix Microarray and Fragile X screening
MRQ2 250k Affymetrix Microarray
MRQ5 250k Affymetrix Microarray and Sanger sequencing for candidate genes
MRQ8 Fragile X screening
MRQ11 250k Affymetrix Microarray, Exome and Sanger sequencing
MRQ12
Fragile X screening, 250k Affymetrix Microarray and Sanger
sequencing for candidate genes
D1 2.7 Cytogenetic Microarray, Sanger sequencing for candidate genes
MRQ14
250k Affymetrix Microarray, Fragile X screening, Exome and Sanger
sequencing
MRQ15 250k Affymetrix Microarray, Exome and Sanger sequencing
MRQ16 Fragile X screening, X-Exome and Sanger sequencing
MRQ17 250k Affymetrix Microarray and Sanger sequencing for candidate gene
MRQ18 Sanger sequencing for candidate gene
MRQ19 250k Affymetrix Microarray and Sanger sequencing for candidate genes
MRQ20 250k Affymetrix Microarray, Exome and Sanger sequencing.
PKMR71 Fragile X screening, X-Exome and Sanger sequencing
PKMR176 Fragile X screening, X-Exome and Sanger sequencing
56
MRQ1
Figure 3.1. Pedigree of family MRQ1. The females are represented by circles, and
squares are for males while filled squares are for affected males. The diagonal lines
across the symbols represent the expired family members. Generations are
represented by Roman numerals while individuals within a generation are
symbolized by Arabic numerals. Consanguinity is represented by double line in the
pedigrees.
57
The FMR1 screening gave normal CGG repeats >45 in the proband. The family was
analyzed using the Affymetrix 250k SNP microarray, which revealed multiple
homozygous regions in the affected member (Fig 3.2), while those regions were
heterozygous in all the unaffected members. Table 3.2 shows the list of homozygous
regions obtained from the online tool Homozygosity Mapper, none of these regions
contained any known ID genes, and therefore further work on this family was not
conducted.
3.3 Family MRQ2
The family MRQ2 was consanguineous (Fig 3.3) and was collected from Khyber
pakhtunkhwa, Pakistan. Five members of the family were sampled (I:1: father, I:2:
mother, II:1: affected proband daughter, II:2 and II:3: unaffected daughters). The
proband‟s clinical data at birth were not available but her mother reported an
uneventful 39 week pregnancy ending in normal delivery. At the time of sampling at
8 years of age, the proband had a short height of 32 inches with weight of 50lbs,
where her physical examination revealed that she had visible proptosis and pterygium
colli, which are hallmarks of Down syndrome. Her milestones were delayed, as
reported by her parents, and her neurological examination did not reveal any other
abnormality except a low IQ, which was in the range of moderate ID (IQ: 36-51). The
family was analyzed using Affymetrix 250K SNP microarray platform. The data
obtained from genotyping using the Genotype console (version 2.0) were analyzed
using the Homozygosity mapper, which did not reveal any homozygous regions (Fig
3.4). The copy number variation data were obtained from the software Copy Number
Analyzer for GeneChip (CNAG). This revealed the presence of an extra copy of
chromosome 21, thus classifying the patient as trisomy 21, the variation in copy
number occurred de novo (Fig 3.5).
3.4 Family MRQ5
The highly consanguineous five generation family MRQ5 (Fig 3.6) was ascertained
from Rawalpindi, Pakistan. Two branches, one from the fourth generation and the
other from the fifth generation were available for sampling. Seven members were
sampled from one branch (IV:1: mother, IV:2: father, V:2 and V:4 unaffected
58
MRQ1
Figure 3.2. Homozygosity Mapper plot showing multiple the homozygous regions in
red color.
59
Table 3.2. List of homozygous regions represented by starting and end positions and
rs numbers of SNPs.
Chromosome
number Physical position of SNPs RS numbers of SNPs
Size of the
regions
(Mb)
2 81171004-87765002 rs7583676-rs41465348 6.5
2 88350889-102105846 rs4972168-rs974374 13.7
2 213033044-215084937 rs6736654-rs918352 2.0
2 211017099-213027539 rs1990513-rs16847808 2.0
5 20198524-26039266 rs6451727-rs11954990 5.5
5 30849338-36600633 rs9292407-rs7730144 3.6
6 75838897-81696551 rs9343272-rs9449206 5.8
6 81705012-86011423 rs13216306-s476613 5.0
7 139347318-143040227 rs9690319-rs2887121 3.6
7 143748364-157112363 rs4407791-rs6459750 13.3
10 19774177-22937071 rs11010136-rs1614387 3.1
10 30019507-43620551 rs11007719-rs2742236 13.6
10 43670805-50824619 rs7092548-rs3810950 7.1
11 125296401-131882807 rs10893378-rs1940033 6.5
12 96651259-98686502 rs2032774-rs1480077 2.3
12 98726255-109330196 rs972987-rs7980554 5.6
12 109546114-115176627 rs2515913-rs2062715 5.6
17 59545329-71940218 rs873363-rs8075246 12.3
17 72011807-79001300 rs9646391-rs8072124 6.9
18 10217527-14940733 rs12326687-rs786031 4.7
18 15069726-23411752 rs12605904-rs1612695 8.3
16 6587247-7535456 rs1476979-rs11077191 9.8
13 41543494-43748496 rs9532691-rs7984876 3.9
60
MRQ2
Figure 3.3. Pedigree of family MRQ2, in the pedigree females are represented by
circles, squares are for males. While filled circle is for affected female. Generations
are represented by Roman numerals while individuals within a generation are
symbolized by Arabic numerals. Consanguinity is represented by double line in the
pedigree.
61
MRQ2
Figure 3.4. Plot of Homozygosity Mapper showing weak homozygous regions in red
color.
62
Trisomie Chr 21 !
Figure 3.5. CNAG picture of Affymetrix 250k SNP array. In the upper and lower
panel the line deviating from „0‟ plane is showing addition of one extra copy of
chromosome 21.
63
MRQ5
Figure 3.6. Pedigree of family MRQ5. The circles represent females while squares are
for males. The filled circles indicate affected females and filled squares are represent
affected males. The diagonal lines across the symbols indicate the deceased
individuals. The double line in the pedigree shows the consanguineous marriage. The
proband is indicated by arrow.
64
daughters, V:3 and V:6 affected daughters, V:5:affected son) and six members were
sampled from the other branch (IV:1: mother, IV:2: father, V:2 and V:4 unaffected
and V:7 unaffected daughters). The affected member‟s at birth clinical data were not
available but they were all born after an uneventful 39 weeks pregnancy without any
complicated labor. None of them suffered from any infection or postnatal injury.
They all were aggressive and were unable to perform any essential life functions
independently. In addition they all had IQ in the range of profound ID (IQ<20). One
of the affected member from the second branch (V:8 ) was selected for further clinical
examination as all affected members had the same phenotype. The boy was eighteen
years old, he had spasticity, and ataxia and was also reported to suffer from epileptic
seizures. His behavior was self injurious and aggressive. His biochemical tests
including blood complete picture, serum lactate, serum electrolyte, and T3/T4
hormone were all in the normal range. His neurological exam by CT scan showed
mild prominent ventricular system, otherwise there was no other anomaly seen in the
brain.
Affymetrix 250K SNP array analysis was done for all the affected members, followed
by homozygosity mapper analysis, which did not reveal a common homozygous 12
(Fig 3.7). CNAG analysis revealed one of the affected member (V:9) to be
genetically a chimera, composed of two different types of cells. In addition CNAG
analysis revealed a heterozygous deletion of 444.26Kb in 15p11.1-q11.1 including the
centromeric region in four affected members (Fig 3.8). The deletion region was found
to be highly polymorphic and has been also reported previously in normal individuals;
hence the pathogencity of ID in this family cannot be linked to this region only. For
candidate gene analysis all the coding exons of gene NTF3 found in the homozygous
region of chromosome 12 were sequenced but no pathogenic mutation was identified
in any of the exon.
3.5 Family MRQ8
The family MRQ8 belonged to Baluchistan, Pakistan. The family had a number of
affected members but only one branch could be sampled (II:1: father, II:2: mother,
65
MRQ5
Figure 3.7. Homozygosity Mapper plot of MRQ5, showing weak homozygous region
on chromosome 12 of 0.2Mb in affected members in red color.
66
Figure 3.8. CNAG data of chromosome 15 of the four affected members of MRQ5
carrying the microdeletion. The CNV is represented by the circled area among all four
members.
67
III:2: affected daughter, III:1 and III:3: affected sons, III:4 and III:5: unaffected sons
and III:6: daughter in law) (Fig 3.9). The family had a high ratio of affected male
members, while only one of the female was affected, but her phenotype was less
severe. The sampled branch had three members affected with IQ range of mild ID
(IQ: 52-67). The three affected members had elongated face, strabismus, autistic
behavior and scoliosis. The FMR1gene was screened in the proband (III:3) by using
the three primer CGG repeat primed FMR1 PCR method. The CGG repeat expansion
was >200 in the proband, full mutation was thus detected, and the affected members
of family MRQ8 were confirmed to suffer from Fragile X mental retardation
syndrome.
3.6 Family MRQ11
The inbred family MRQ11 with two affected children (Fig 3.10) was sampled from
Attock, Pakistan. The sampled members were (III:2: mother, IV:1, IV:4 & IV:5
unaffected daughters, IV:2: affected son, IV:3: affected daughter). The two affected
members had similar phenotype and both were born after an uneventful 39 weeks
pregnancy. The labor was normal and they did not suffer from any postnatal hypoxia.
Both of them had delayed milestones including speech and motor functions. They
were weak with below average weight and height (23 kg and 114 cm for affected
son) and (22kg weight and 88cm height of the affected daughter). They had
strabismus, hypotonia, and had IQ in the range of moderate ID (IQ:36-51). The
neurological examination by CT scan did not show any anomaly of the brain, in
addition all their biochemical tests were also in the normal range. The family was
analyzed by Affymetrix 250K Microarray SNP analysis, which gave some true
homozygous regions in the affected members (Fig. 3.11), but no previously identified
gene for ID was found in those regions. CNAG analysis also did not reveal any
pathogenic CNVs. Therefore exome sequencing was performed to obtain the variants
in the proband (IV:2), which gave 38 homozygous and 30 compound heterozygous
68
MRQ8
I:1 I:2
II:1 II:2 II:3 II:4 II:5 II:6 II:7 II:8 II:9
III:1 III:2 III:3 III:4 III:5 III:6 III:7 III:8 III:9 III:10 III:11 III:12 III:13 III:14 III:15 III:16 III:17 III:18 III:19
V
Figure: 3.9. Pedigree of family MRQ8. In the pedigree females are represented by
circles, squares are for males. While filled squares represent affected males. The
diagonal lines across the symbols are for deceased individuals. Generations are
represented by Roman numerals while individuals within a generation are symbolized
by Arabic numerals. Consanguinity is represented by double line in the pedigree.
69
MRQ11
Figure 3.10. Pedigree of family MRQ11. In the pedigree females are represented by
circles, squares are for males. While filled circle is for affected female and filled
square represents affected male. The diagonal lines across the symbols are for
deceased individuals. Generations are represented by Roman numerals while
individuals within a generation are symbolized by Arabic numerals. The arrow
indicates the proband. Consanguinity is represented by double line in the pedigree. In
addition the segregation of ZNF589 variant is also shown in the pedigree. The +/+
represents homozygous wild type, M/M is for homozygous mutants and +/M is for
heterozygous carriers.
70
MRQ11
Figure 3.11. Homozygosity Mapper plot showing the multiple homozygous regions in
affected members of family MRQ11 in red color.
71
changes after filtration. The homozygous regions identified from SNP data were also
considered to filter the variants. The data were further filtered in order to get the most
relevant changes responsible for pathogenicity of ID. For this purpose variants with
phyloP > 2.0 and Grantham distance of > 80 were selected, which gave only seven
homozygous variants and four compound heterozygous variants. For the validation
Sanger sequencing was performed to determine the segregation of the particular
variants among all family members. Of all the variants the gene ZNF589 variant,
c.956T>A, at the position Chr3:4167990-4167990 (Hg19, built March, 2009) was
found to segregate with the disease in the family. The chromatograms of Sanger
sequencing for the affected and unaffected members are shown in Figure 3.12. This
ZNF589 variant had a phyloP score of 2.98 and Grantham distance of 99. The gene
has not been reported previously for ID and the GO (Gene ontology database)
www.geneontology.org/ describes the gene ZNF589 to be involved in DNA
dependent regulation of transcription.
3.7 Family MRQ12
The family MRQ12 is a consanguineous Pakistani family (Fig. 3.13) from Azad
Kashmir, Pakistan. The parents are first cousins, at the time of sampling the mother
was 72 years old with learning disabilities, epilepsy, and was also suffering from
insulin dependent diabetes mellitus since the age of 22 years. The father had normal
intellectual functioning, throughout his life, but at the age of 76 years he had
symptoms of Parkinson‟s disease but with no family history of the disease. The
mother gave birth to six children while one pregnancy was spontaneously terminated
at 4 months due to growth arrest of the male fetus. Four of the six children were
intellectually normal without any congenital abnormality. One of her sons (IV: 2) died
at the age of 18 years from leukemia but he did not have ID and/or any psychiatric
disorder. The two youngest sons (IV-5 and IV-6) were both affected.
The proband IV:5 was born after 40 weeks of an uneventful pregnancy.
Microcephaly was noted at birth but detailed clinical data of birth weight and height
of the proband and other affected brother were not available. The proband started
walking at the age of 11 months and speaking at the age of 4 years. At three months
of age his intellectual weakness was noted due to delayed milestones, at age 10 years
he had symptoms of progressive hyponychia (Fig. 3.14).
72
M: ZNF589,c.956T>A; p.Leu319His
Figure 3.12. Sequencing chromatogram of ZNF589 variant. The red arrow in the
upper figure is for heterozygous variation, the middle figure is for wild type and the
bottom one is indicating the homozygous mutant variant.
73
MRQ12
I:1 I:2 I:3 I:4
II:1 II:2 II:3 II:4 II:5 II:6
III:1 III:2
IV:1 IV:2 IV:3 IV:4 IV:5 IV:6 IV:7
R+/M
Ndel/+
R+/M
N+/+
RM/M
Ndel/+
RM/M
Ndel/+
R+/M
N+/+
Figure 3.13. Pedigree of family MRQ12. In the pedigree females are represented by
circles, squares are for males. Gray filled circle indicates the mildly affected female,
while filled squares represent affected males. The diagonal lines across the symbols
are for deceased individuals. The small square with a diagonal line indicates prenatal
death. Generations are represented by Roman numerals while individuals within a
generation are symbolized by Arabic numerals. The arrow indicates the proband.
Consanguinity is represented by double line in the pedigree. In addition the
segregation of mutation of RBBP8 and NRXN1 CNV is also shown in the pedigree.
The R is for RBBP8 while N is for NRXN1, while +/+ represents homozygous wild
type, M/M is for homozygous mutants and +/M is for heterozygous carriers. The del/+
is heterozygous carrier of CNV.
74
Figure 3.14. The top left panel is frontal picture of the proband of MRQ12 at the age
of 40 years, while the top right panel shows the picture of the hand with complete
hyponychia of the finger nails. The bottom figure is MRI scan of the different regions
of the brain (a) normal MRI image (b, c) MRI of tuboflair images of the brain of
MRQ12‟s proband showing diffuse white matter. The red arrows in the figures are
pointing towards the area of the brain where abnormal white matter deposition can be
seen.
75
Upon clinical evaluation at the age of 40 years the proband had a short stature and
obesity (height 162 cm and weight 76 kilograms). He had symmetrical microcephaly,
head circumference was 46cm, and he had dysmorphic facial features with synophrys,
a prominent irregular shaped long nose and a short philtrum. He also had deeply set
eyes and bilateral convergent strabismus. His ears were rotated posteriorly and he had
a highly pitched and shrill voice. On his fingers and toes complete hyponychia was
observed, he also suffered from hyperhidrosis. His gait was normal without ataxia and
he could visualize things normally, and speak normally but his comprehension was
poor, he couldn‟t count and write. Cardiological evaluation, metabolic screen,
complete blood count (CBC) and liver function tests were all normal, while his brain
MRI revealed accentuated white matter of the cerebrum and the cerebellum (Fig. 3.14
b,c), suggestive of an underlying white matter disease (WMD) of the brain. Otherwise
the ventricle and cortical sulci were normal with no focal mass lesion or midline shift
and no abnormal collection or hemorrhage was noticed.
Fragile X screening test was proved negative for the family MRQ12 proband.
Genotyping of the two affected sibs of MRQ12 by 250K array revealed the only true
homozygous region 18p21.2-q12.2 that overlaps the known locus SCKL2 for Seckle
and Jawad syndrome, which carries the RBBP8 gene previously known to be involved
in the pathology of both syndromes. In order to find the mutation all coding exons 2-
19 of the gene RBBP8 were sequenced, which revealed a homozygous change in
exon 11 c.919A>G, p.Arg307Gly. In silico analysis revealed that the change was at -2
to 5 donor splice site position within exon 11. The substitution co-segregated with the
disease in the family in a recessive manner (Fig. 3.15), the mother (III: 2), Father (III:
1) and daughter (IV:1) were heterozygous for the substitution while the two sons with
the severe phenotype were homozygous for the substitution (IV:5 and IV:6). The
250K array CNV data also revealed a 607 kb deletion in the region (chr2:502, 10-508,
17 Mb UCSC Human Genome Browser version 19) of chromosome 2p16.3 (Fig.
3.16), resulting in a heterozygous deletion of exon 13 to exon 19 as well as the β-
promoter of NRXN1, which was confirmed by qPCR in all patients of the family. The
deletion was inherited by the affected sons in a dominant manner from the mother
(III: 2) who suffered from mild ID and epilepsy, (IV: 5 and IV: 6), while both father
(III:1) and daughter (IV:1) had normal copy number of NRXN1. No other mutation on
76
Figure 3.15. Sequencing chromatogram of RBBP8 mutation among the family
members of MRQ12.
77
Figure 3.16. Original data of CNV analysis derived from 250k Affymetrix SNP array
of proband from family MRQ12 showing heterozygous deletion in the encircled
region (chr2:502,10-508,17 Mb UCSC Human Genome Browser version 19).
78
the other allele of NRXN1 was detected to be transmitted from the parental
chromosome.
3.8 Family D1
Patient II:4 in the family D1 (Fig 3.17A) is a female born to non-consanguineous
Dutch parents. When referred for clinical genetic evaluation at the age of 28-years,
mild ID was noted, while her elder sister had a normal phenotype. The family had two
abortions (both females) during early weeks of pregnancy, while the proband was
born after a pregnancy of 42 weeks. The pregnancy was complicated by maternal
hypertension, birth weight was low-normal (2.89kg, 2.3-5th
percentile). Gross motor
development was normal, but speech development was delayed, with the first words
being spoken after the age of 2 years. She went to a special education school for
children with ID. Her behaviour was shy and introvert and she was an anxious child.
Medical problems included a primary amenorrhea due to gonadal dysgenesis,
diagnosed at the age of 16 years, which was treated with hormonal replacement
therapy. Since the age of 16 years she had recurrent episodes of loss of consciousness.
Computer tomography of the brain and cardiac evaluation did not reveal any
abnormalities. Based on the clinical presentation, a probable psychogenic cause was
considered.
Upon physical examination she had a height of 173.5 cm, weight of 70 kg and a head
circumference of 53.3 cm. She had minimal facial dysmorphic features including
deeply set eyes and synophrys. Her feet were flat, long and narrow with long toes and
she had long fingers. A metabolic screen could not explain her ID.
SNP array analysis of the proband of D1 with the Affymetrix 2.7K cytogenetic array
platform revealed a ~455 kb loss in the chromosomal region 2p16.3 (50,87-51,39 Mb,
Hg19, built March 2006), encompassing the α-promoter of NRXN1 including the first
exon (Fig 3.17 B). Array analysis in both parents showed no deletion, indicating that
the deletion in this patient had occurred de novo. Sequencing of the other allele of the
NRXN1 gene of the patient did not reveal any other pathogenic mutation in the
patient. Analysis of the FSHR (Follicle stimulating hormone receptor) MIM 136435,
in the proband revealed known polymorphisms but no mutation that could explain the
gonadal dysgenesis, this gene was sequenced because it is located near the distal
breakpoint of the deletion so it was hypothesized that a position effect of the deletion
79
Family D1
(A)
(B)
Figure 3.17. (A) Pedigree of family D1, The circles represent female while the square
represents the male. The filled circle indicates the affected female. The small circles
with diagonal lines indicate the spontaneous abortions. (B) Original data of proband
CNV analysis derived from Affymetrix 2.7 microarray analysis of family D1 showing
deletion in the encircled region 2p16.3 (50,87-51,39 Mb, Hg 19, built March 2006).
80
of FSHR in combination with a mutation on the other allele could have led to the
gonadal dysgenesis.
3.9 Family MRQ14
The highly inbred family MRQ14 with three affected children with ID was sampled
from Multan, Pakistan. Four different branches were sampled from this family
because the different branches were found to suffer from different genetic disorders,
including ID (V:8, V:9,V:10 affected sons, V:11 unaffected daughter, V:12
unaffected son, IV:4 unaffected mother and IV:5 unaffected father). The three
affected members had similar phenotype and were born after an uneventful 39 weeks
pregnancy with a normal labor and no postnatal hypoxia. The proband (V:8) had
dysmorphic facial features such as flattened nose bridge, with widely spread eyes; the
teeth were also widely spaced and he suffered from hypotonia (3.19A). The Proband
suffered from speech and motor delay, at the time of sampling his age was 16 years,
while his weight was 23 kg and he had a short height of 82 cm. He suffered from
hypertonia. The three affected boys had IQ in the range of profound ID (IQ<20). They
all were not able to perform any essential functions of life independently. The
neurological examination by CT scan did not reveal any anomaly of the brain. All the
biochemical tests were also in the normal range. FMR1 screening showed negative
results for the proband. Genotyping with the Affymetrix 250K Microarray SNP
analysis revealed some common homozygous regions among the affected members
(Fig 3.19B), but the CNAG analysis did not reveal any microdeletion or duplication;
the homozygous regions also did not harbor any known gene for ID. Therefore exome
sequencing was performed to obtain the variants in the proband. After filtration, 53
homozygous and 23 compound heterozygous changes among different genes were
obtained. The data were further filtered in order to get the most relevant changes
which could be the causative factor in the pathogenicity of ID in the family. For this
purpose the variants with phyloP > 2.0 and Grantham distance > 80 were selected,
which resulted in only twelve homozygous variants remaining and three compound
heterozygous variants. Sanger sequencing was performed for the validation and
determination of segregation of the particular variants in the family. Of all the variants
the gene MLL4 variant, c.2456C>T, p.P819L, lying at the position Chr19:36212705-
36212705 (Hg19, built March 2009) was found to completely segregate with the
disease in the family (Fig 3.20). MLL4 variant had a phyloP score of 4.429 and
81
MRQ14
Retinitis Pigmentosa Intellectual Disability Leber Congenital Amaurosis
I:1 I:2
II:1 II:2 II:3 II:4 II:5 II:6 II:7 II:8
III:1 III:2 III:3 III:4 III:5 III:6 III:7 III:8 III:9 III:10 III:11 III:12 III:13
IV:1 IV:2 IV:3 IV:4 IV:5 IV:6 III:14
V:1 V:2 V:3 V:4 V:5 V:6 V:7 V:8 V:9 V:10 V:11 V:12 V:13 V:14 V:15 V:16 V:17 V:18 V:19 V:20
M/M M/M M/M +/M +/M
+/M+/M
Figure 3.18. Pedigree of family MRQ14. In the pedigree the females are represented
by circles, squares are for males. While filled circle is for affected female and filled
squares represent affected males. The diagonal lines across the symbols are for
deceased individuals. Generations are represented by Roman numerals while
individuals within a generation are symbolized by Arabic numerals. The arrow
indicates the proband. Consanguinity is represented by double line in the pedigree. In
addition, the segregation of mutation of MLL4 is also shown in the pedigree. The
symbol M/M is for homozygous mutant and +/M is for heterozygous carriers. No
genetic analysis has been performed yet for RP and LCA phenotypes in respective
branches of the family.
+/M
82
MRQ14
Figure 3.19. (A). Photograph of MRQ14 proband indicating the facial features of
Kleefstra syndrome. Figure 3.19. (B). The Homozygosity Mapper plot showing the
multiple homozygous regions in red color in family MRQ14. For details of
homozygous regions, see appendix 16.
83
Figure 3.20. The sequencing chromatogram of mutation in MLL4. The arrows indicate
mutant (top), Homozygous wild type (middle) and the heterozygous carrier of variant
of MLL4.
84
Grantham distance of 98. The gene has not been reported previously for ID but the
GO (Gene ontology) database (www.geneontology.org/) describes the gene. MLL4
mouse phenotype with abnormally low levels, to be involved in aberrant somite
development, open neural tube defects, embryonic lethality during organogenesis and
increased apoptosis (http://www.geneontology.org/). 200 age matched randomly
selected, ethnicity matched control population were also sequenced to determine the
frequency in the general population but the variant was not found in any of the control
samples. Segregation of the variant was also determined among all family members
belonging to different branches to see whether the variant is involved in pathology of
ID only, or also the other genetic defects in the family, but the variant segregated only
in the ID branch, in addition the change was found to have been inherited from the
heterozygous paternal grandfather (III:6), the maternal grandparents were not
available for screening of the change but it is suspected that the other allele was
inherited from one of the maternal grandparents because the family is inbred.
3.10 Family MRQ15
MRQ15 was one of the out bred families in the panel, which was collected from
Punjab, Pakistan. Five members of the family were sampled (I:1: father, I:2: mother,
II:3: affected daughter, II:5: unaffected son and II:8:affected son) (Fig 3.21). The two
affected members were evaluated for their IQ, which was in the range of profound ID
IQ<20. They both suffered from motor and speech delay, and at the time of
examination at the age of 10 year (II:8) and 14 years (II:3) they could speak only
some meaningful words and could recognize only their parents and siblings. They
were both not able to perform any important functions of life independently; but no
structural anomaly was present and their CT scan also did not show any brain
malformation. Genotyping by 250K array did not reveal any homozygous region in
the affected members. Hence the NGS (Exome sequencing) was used to identify the
genetic cause of ID in this family. After final filtration using phyloP score and
Grantham distance, seven homozygous variants in different genes and four compound
heterozygous variants were identified, all were further tested by Sanger sequencing
for segregation in the family members but none of them segregated except the
heterozygous change in HHAT (Hedgehog acetyl transferase) MIM 605743 (Fig
3.22). The variant present in the two affected members as a heterozygous change
85
MRQ15
I:1 I:2
II:1 II:2 II:3 II:4 II:5 II:6 II:7 II:8
II:9
+/+ +/+
+/++/M +/M
Figure 3.21. Pedigree of family MRQ15. In the pedigree females are represented by
circles, squares are for males. While filled circle is for the affected female and filled
square represent the affected male. The diagonal lines across the symbols are for
deceased individuals. Generations are represented by Roman numerals while
individuals within a generation are symbolized by Arabic numerals. The arrow
indicates the proband. In addition the segregation of de novo mutation of HHAT is
also shown in the pedigree. The symbol +/+ represents homozygous wild type and
+/M is for heterozygous affected members.
86
Figure 3.22. The sequencing chromatogram of variant in HHAT. The arrow is
indicating heterozygous de novo change in two affected siblings and homozygous
wild type member of family MRQ15.
87
The identified HHAT variant (c.1158G>C and p.W386C) had a phyloP score of 5.43
and the Grantham distance was 215. This gene has not been reported previously for
ID but has been previously shown to be involved in the development of multicellular
although it was not present in any of the parents and the unaffected daughter. DNA of
the other members was not available for testing.organ, while a mouse phenotype with
low levels of gene expression was found to have decreased motor neuron number,
oligodactyly, abnormal spinal cord interneuron morphology and various other
abnormalities of the nervous system, hence this variant is suspected to be involved in
the pathology of ID in the family MRQ15.
3.11 X-linked families
Three families (MRQ16, PKMR71, PKMR176) were suspected to have X-linked
mode of inheritance because of the high number of affected males in the families,
hence the FMR1 screening was performed but in none of the families the repeat length
was found to exceed the normal range i.e. <45 CGG repeats.
3.11.1 Families MRQ16, PKMR71 and PKMR176
Families MRQ16, PKMR71 and PKMR176 were sampled from the rural areas of
Punjab, all three families had only male affected members. The affected members had
IQ range of <20 hence they fall in the category of profound ID.
The initial clinical examination of the family MRQ16 (Fig 3.23) by the neurologist
classified the family to suffer from Lesch Nyhan syndrome because three affected
boys (IV:12, IV:13, IV:14, IV:15) had neurological and behavioral disturbances
including self- mutilation, and hyperuricemia. So based on this assumption the
candidate gene HPRT1 was tested first, however, no pathogenic change in the coding
exons of the gene was found. The family was further screened by X-exome analysis to
determine if there was any pathogenic variant on the X chromosome, after filtration
some variants were obtained (Table 3.3) but none of these variants segregated with
the disease in the family (Table 3.4).
The affected members of PKMR71 (Fig. 3.24) and PKMR176 (Fig. 3.25) were
classified to suffer from non-syndromic ID because they did not suffer any other
dysmorphic feature except that they were unable to speak any meaningful words
88
MRQ16
Figure 3.23. Pedigree of family MRQ16. In the pedigree females are represented by
circles, squares are for males. The filled squares represent affected males. The
diagonal lines across the symbols are for deceased individuals. Generations are
represented by Roman numerals while individuals within a generation are symbolized
by Arabic numerals. The underlined branch is the sampled branch. while the arrow
indicates the proband.
89
Table 3.3. List of variant genes, cDNA and amino acid changes in MRQ16, PKMR71
and PKMR176 respectively.
Family name Variant genes cDNA change Amino acid change
MRQ16
FAM47A c.1130 G>A p.A377V
ITGB1BP2 c.143 T>G p.V48G
PIR c.730 C>T p.E244K
TEX11 c.943 C>T p.E315K
ZCCHC5 c.602 A>G p.L201P
PKMR71
Cxorf58 c.766 G>A p.R256Q
DGAT2L6 c.338 T>A p.F451
IGBP1 -224T>A acceptor
TCEANC c.382 T>A p.Y 312N
SLC10A3 c.478 C>T p.G22S
PKMR176
CCDC22 c.628 A>G p.E239G
MID2 c.716 A>G p.N323S
TMEM164 c.298 G>A p.V144I
90
Table 3.4. List of variant genes, segregation among all family members in MRQ16.
Individual
ID
FAM47A ITGB1BP
2
PIR TEX11 ZCC
HC5
Status of
Affection
p.A377V p.V48G p.E244K p.E315K p.L20
1P
c.1130
G>A
c.143 T>G c.730
C>T
c.943
C>T
c.602
A>G
IV:11 A/G G C/T C G Affected
IV:12 A/G G C C G Affected
IV:13 A/G G T C G Affected
IV:15 A/G G C C G Affected
IV:10 G G C C G Normal
III:3 A G T C G Normal
III:4 A G T C G Normal
91
PKMR71
I:1 I:2
II:1 II:2 II:3 II:4 II:5 II:6 II:7 II:8
Figure 3.24. Pedigree of family PKMR71. In the pedigree females are represented by
circles, squares are for males. While filled squares are for affected males. Generations
are represented by Roman numerals while individuals within a generation are
symbolized by Arabic numerals.
92
PKMR176
I:1 I:2
II:1 II:2 II:3 II:4 II:5 II:6 II:7 II:8
Figure 3.25. Pedigree of family PKMR176. In the pedigree females are represented by
circles, and squares are for males. While filled squares are for affected males.
Generations are represented by Roman numerals while individuals within a generation
are symbolized by Arabic numerals.
93
because of the profound ID. In the family PKMR71, five variants were obtained from
X exome analysis (Table 3.3), among all these variants the segregation analysis
(Table 3.5) revealed the two variants CXORF58 (c.333G>A, p. R256Q) and TCEANC
(c. 382T>A, p.Y312N) to segregate in a recessive manner in the family. But in silico
analysis revealed the CXORF58 variant to be not conserved in 11 species as the
change was found to be present in two mammals, the rat and dolphin (Fig. 3.26).
While the TCEANC variant was moderately conserved among 11 species (Fig. 3.27)
but had very low phyloP score (0.21), also the Grantham distance between the two
amino acids Asparagine and Tyrosine is very large i.e. 143 for the change p.Y312N.
Hence it was concluded that both the variants could not be causative of the strong
phenotype in the affected members of PKMR71. In the family PKMR176, three
variants were identified from X exome analysis (Table 3.3) but all these three variants
did not segregate in the family when tested by Sanger sequencing (Table 3.6).
3.12 Family MRQ17
The highly inbred family MRQ17 (Fig 3.28) was sampled from the area of North
West area of Pakistan. The affected members (IV:3, IV:12 and IV:13) were born after
an uneventful 39 week pregnancy, they all displayed microcephaly, short stature,
psychomotor retardation, profound ID and muscle spasticity. The brain CT scan could
not be performed because consent was not given by the parents. However, based upon
symptoms they were suspected to suffer from lissencephaly.
All the affected members were genotyped by 250K array, which revealed a
homozygous region of 8.44MB on Chr7: 95492611-103983576 (Fig 3.29) and no
chromosomal aberrations were detected from CNAG analysis. The gene RELN
(Reelin) MIM 600514, already known for lissencephaly resides in the region hence all
its coding exons were sequenced but no pathogenic mutation was found in any of the
65 exons. The family was not analyzed any further.
3.13 Family MRQ18
The inbred family MRQ18 (Fig 3.30) was sampled from the area of Rawalpindi,
Pakistan. Only the affected child (IV:4) was found to have mild ID (IQ< 65)
withcongenital hypothyroidism as well as some dental anomlies. He was 9 years age,
94
at Table 3.5. List of variant genes segregation among all family members of
PKMR71.
Individuals
ID
Cxorf58 DGAT2L6 IGBP1 TCEANC SLC10A3 Status of affection p.R256Q p.F451 acceptor p.Y 312N p.G22S
c.766
G>A
c.338
T>A
c.-224
T>A
c.382
T>A
c.478
C>T
II:1 G/A G/A T A/T C Affected
II:2 A A A A C/T Affected
II:3 A A T A C Affected
II:4 A A A A T Normal
II:5 G T T T C Normal
I:1 G A A T C Normal
I:2 G G/A T A/T C/T Normal
95
Table 3.6. List of variant genes segregation among all family members of PKMR176.
Individuals
ID
CCDC22 MID2 TMEM164 Status of
affection p.E239G p.N323S p.V144I
c.628 A>G c.716 A>G c.298 G>A
I:1 A A C Normal
I:2 A A/G C Normal
II:1 A/G A T Affected
II:2 A A C/T Affected
II:3 A A C Affected
II:4 A A C/T Normal
II:5 A G C Normal
II:7 A A T Normal
96
Figure 3.26. Conservation analysis of CXORF58 variant p.R256Q from the software
Alamut (Bio interactive software). The indicated region in the outline shows the
variation not to be conserved.
Human
Gorilla
Orangutan
Macaque
Marmoset
Tarsier
Rabbit
Dog
Dolphin
Wallaby
97
Figure 3.27. Conservation analysis of TCEANC variant p.Y312N from the software
Alamut (Bio interactive software). The indicated region in the outline shows the
variation to be moderately conserved.
Human
Gorilla
Orangutan
Macaque
Marmoset
Tarsier
Rabbit
Dog
Chicken
Frog
98
MRQ17
IV:1 IV:2 IV:3 IV:4 IV:5 IV:6 IV:7 IV:8 IV:9 IV:10 IV:11 IV:12 IV:13
III:1 III:2 III:3 III:4 III:5 III:6
II:1 II:2 II:3 II:4 II:5 II:6 II:7 II:8 II:9 II:10
I:1 I:2 I:3 I:4
Figure 3.28. Pedigree of family MRQ17. In the pedigree females are represented by
circles, and squares are for males. While filled circles are for affected females and
filled squares represent the affected males. The diagonal lines across the symbols are
for deceased individuals. Generations are represented by Roman numerals while the
individuals within generation are symbolized by Arabic numerals. The arrow
indicates the proband. Consanguinity is represented by double line in the pedigree.
99
MRQ17
Figure 3.29. The homozygosity Mapper plot of Affymetrix 250K SNP array showing
the only true homozygous region of 8.44Mb region (chr7: 95492611-103983576) in
red color in affected members of family MRQ17.
100
MRQ18
Figure 3.30. Pedigree of family MRQ18. In the pedigree females are represented by
circles, squares are for males. While filled circles are for affected female and filled
square represents the affected male. The diagonal lines across the symbols are for
deceased individuals. Generations are represented by Roman numerals while
individuals within a generation are symbolized by Arabic numerals. The arrow
indicates the proband. Consanguinity is represented by double line in the pedigree. In
addition the segregation of TPO mutation is also shown in the pedigree. The symbol
+/+ represents homozygous wild type, M/M is for homozygous mutant and +/M is for
heterozygous carriers.
101
At the time of evaluation, he had a short stature and his weight was also below the
normal range, detailed clinical data were not available.All the coding exons of the
candidate TPO were analyzed by Sanger sequencing in the affected member as well
as unaffected members of family, and a variant i.e. g.1418194G>C, p.Ala5Gly in
exon 2 of the gene was identified (Fig 3.31). The variant segregated in a recessive
manner among the family member as both the parents were heterozygous carriers and
the daughter was wild type homozygous while the only affected son was homozygous
carrier of the mutation. The in silico analysis revealed the change to be conserved in
14 species with a phyloP score of -1.17 and minor effect of the amino acid change
was noted with a Grantham distance of only 60, the SIFT analysis revealed the change
to be tolerated. No further analysis regarding detection of chromosomal aberration
was performed on the family MRQ18.
3.14 Family MRQ19
MRQ19 was a consanguineous Punjabi Pakistani family (Fig. 3.32), which presented
with characteristic features of BBS. The family had two affected male siblings and
one normal female child. The proband (IV:1) is the eldest male child of first cousin
parents, he was born at full term after an uneventful pregnancy. Obesity and
polydactyly in both hands were noted at birth. His milestones were delayed, he had
myopia, learning disabilities, he learned to walk at the age of 3 years and began to
speak at 4 ½ years. At the time of the clinical examination (age 15 years) his behavior
was shy, introvert and he was anxious, his face had a moon like appearance due to
obesity (height: 156cm, weight: 75kg). At the time of the eye exam he had complaints
of night blindness for several years, whereas his visual acuity was 6/60 and he had CF
< 1m (counting finger vision), in addition he had astigmatism and myopia.
Funduscopy revealed pigmentary degeneration, which confirmed retinitis pigmentosa.
His brain CT scan showed no intracranial hemorrhage, mass effect or midline shift.
However, benign vascular calcification was noted in the bilateral basal ganglia. Subtle
non-specific tiny parenchyma calcification near the vertex was seen (Fig 3.33), no
other abnormal density was present in the brain parenchyma. One supernumerary digit
was observed bilaterally having a small metacarpal, proximal and distal phalanx (Fig
3.33), while the soft tissues were unremarkable. The metabolic screen and renal
function tests showed normal range of different parameters. However, all the liver
102
TPO, M: c.14C>G, p.Ala5Gly
Figure 3.31. Sequencing chromatogram of variation found in TPO gene for all
members of family MRQ18.
103
MRQ19
I:1 I:2 I:3 I:4
II:1 II:2 II:3 II:4
III:1 III:2 III:3 III:4 III:5 III:6
IV:1 IV:2 IV:3
+/M +/+
+/M +/M
+/MM/M +MM
Figure 3.32. Pedigree of family MRQ19. The females are represented by circles,
while the squares represent males. The diagonal lines across the symbols indicate the
deceased individuals. The filled squares represent the affected males. The Generations
are represented by Roman numerals while individuals within a generation are
symbolized by Arabic numerals. The arrow indicates the proband. Consanguinity is
represented by double line in the pedigrees.
104
Figure 3.33. CT images of Proband. A) Non contrast CT image showing a diffuse
symmetrical parenchyma calcification involving dentate nuclei. B) Non contrast CT
image showing dense calcification involving basal ganglia and thalamus. C) Non
contrast CT image showing calcification in grey white matter interface of frontal lobe
on both sides while the other figure is the X-ray image of hands showing postaxial
polydactyl.
105
enzymes were raised, where the Serum Glutamate Pyruvate Transaminase (SGPT)
was 140 U/L (reference range 0-35 U/L), Serum Glutamic- Oxaloacetic Transaminase
(SGOT) was 66 U/L (reference range 5-43 U/L) and Alkaline phosphatase was 369
U/L (reference range 44-147 U/L). In addition, the boy had hypogonadism with a
micropenis and undescended testis. The other affected sibling of the proband was also
male; he was the third child of his parents. The other affected sibling of the proband
was also male; he was the third child of his parents, born after a normal pregnancy of
40 weeks. At the time of the clinical assessment he was 4 years old, he had night
blindness and his vision was deteriorating gradually. He was also obese and had a
small penis with undescended testis. He started speaking a few words and walking at
the age of 4 years. The patient could not be taken to the diagnostic center for further
clinical assesment. Genotyping of the affected members revealed two regions on
chromosome 12q21.31-12q21.2 and one on chromosome 16q21 containing known
genes BBS10, BBS14 (CEP290) and BBS2, respectively. Sequencing of these genes
revealed a homozygous deletion of 10 nucleotides in BBS10 c.1958_1967del
(p.Ser653Ilefsx4) exon 2 (Fig 3.34), in silico analysis revealed that the change caused
substitution of serine by isoleucine due to the frameshift that also causes the creation
of a premature termination codon after four amino acids. Segregation analysis in the
family showed that the identified mutation completely co-segregated with the
phenotype, where the father (III:4), mother (III:5), paternal grandfather (II:1), paternal
grandmother (II:2) and unaffected sister (IV:2) were all heterozygous for this
mutation. Both the affected brothers (IV:1, IV:3) had the carry homozygous mutation,
while the DNA of the other family members was not available for carrier testing.
Besides this no other mutation was found in BBS2 or BBS14 genes.
13.15 Family MRQ20
The inbred family MRQ20 (Fig 3.35) was identified from Sheikhupura, Pakistan. The
proband‟s (II:4) detailed clinical data at birth was not available but the postnatal
period was found to be normal as reported by the mother. At the time of the clinical
examination at the age of 22 years, she had IQ in the range of profound ID <20. She
could not speak any meaningful words and had motor delay; she also could not
perform any life functions independently. In addition she was hypertonic and ataxia as
106
Figure 3.34. Sequencing chromatogram of 10bp deletion in exon 2 of BBS10 in
proband and affected brother (IV:1 and IV:3), wild type homozygous paternal
grandmother and heterozygous unaffected paternal grandfather (II:2 and II:1).
Heterozygous unaffected mother and father (III:5 and III:4) and unaffected
heterozygous sister of proband (IV:2).
IV:1
IV:3
II:2
II:1
III:5
IV:2
III:4
107
MRQ20
Figure 3.35. Pedigree of family MRQ20, in the pedigree females are represented by
circles, squares are for males. While filled circles are for affected females and filled
squares represent the affected males. The diagonal lines across the symbols are for
deceased individuals. Generations are represented by Roman numerals while
individuals within a generation are symbolized by Arabic numerals. The arrow
indicates the proband. Consanguinity is represented by double line in the pedigree.
108
Figure 3.36. Homozygosity Mapper plot of Affymetrix 250k array data did not show
any true homozygous regions among affected members (only a few weak regions
shown in red were observed).
109
noted in her gait. Her neurological examination by CT scan reported cerebellar
atrophy because of prominent folia of cerebellum with widening of sulci. Other
affected family members detail clinical data was not available but generally their
symptoms were the same as that of the proband. The family members were genotyped
using 250K array but no homozygous region were found (Fig 3.36). Hence Exome
sequencing was performed, which revealed 19 homozygous variants and 22
compound heterozygous variants. Filtration on the basis of phyloP score and
Grantham distance left only eight variants, six homozygous and two compound
heterozygous. Segregation of all the variants was tested by Sanger Sequencing among
all family members but none of them segregated with the disease in the family, no
further analysis was performed to investigate the cause of ID in the family MRQ20.
Chapter 4
Discussion
111
4 Discussion
Intellectual disability is a major health problem, which in the Western World affects 1
in 75 child by 10 years of age, where the males are more likely to suffer than females
(1·6:1). The disease is usually characterized by functional impairment and lifelong
need for support and mediation. It has been shown that two third of the ID patients are
mildly affected with IQ in the range of 50-70 while a third are severely affected with
IQ less than 20 (Gecz and Haan, 2012). The disease consumes enormous amount of
public health resources, while the information about the disease burden is mostly
based on information that is gained from studies conducted in the developed countries
(Maulik et al., 2011). ID is a highly heterogeneous neurological disorder with extreme
but relatively unexplored genetic heterogeneity. Recently some biochemical markers
to detect the level of activity of certain pathway have been established to determine
the genetic basis of ID, which can be defined as the condition resulting due to
pathogenic genetic mutation (Raymond, 2006). Till date mutations in over 450 genes
have been implicated in ID, which only represents a minor part of all ID genes that
have been predicted to be causative of the disease (van Bokhoven 2011; Inlow and
Restifo, 2004; Schuurs-Hoeijmakers et al., 2011). The sheer number of ID genes
presents a complication for the identification of the genetic defect in individual
families and isolated cases. Also in their clinical presentation, ID disorders show a
large phenotype variability. In addition a plethora of associated features can be seen in
a range of ID syndromes. Other neurological features, such as autism, epilepsy,
ADHD (Attention deficit hyperactivity disorder) and behavioral anomalies are
particularly common (van Bokhoven, 2011), and for mutations in some genes, some
of these disorders can be seen in various combinations.
ID of genetic nature can also occur due to chromosomal defects, for example repeat
expansion in different parts of the gene including the promoter, UTR, coding region,
as well as due to numerical or partial aberrations, epigenetic changes such as
imprinting defect and single gene defect, which involves autosomal and X-linked
causes (Basel-Vanagaite et al., 2006). Diagnosis of the genetic cause can be highly
challenging as 30-50% of the cases remained unsolved before techniques became
112
available to detect small chromosomal abnormalities such as duplications,
submicroscopic deletions or copy number changes. Homozygosity mapping is a
highly effective approach used to identify pathogenic defects among the genes in ID
patients from inbred families. Till date homozygosity mapping has been employed
successfully for mapping more than 30 autosomal recessive ID loci among Pakistani
and Iranian families (Rafiq et al., 2010; Kuss et al., 2011). Diagnosis of small copy
number changes among sporadic cases has increased by 14 % due to the introduction
of genome wide profiling as well as the introduction of NGS technologies, which
have a high capacity to improve diagnosis because of their unbiasness, their
systemization and their ability to scan the whole genome at very high resolution and
at very low cost. So using the NGS platform in combination with array analysis can
be helpful in making the diagnosis more effective in benefiting the families with
affected members (Cooper et al., 2011).
In the current study, sixteen families segregating autosomal recessive and X-linked
neurodevelopmental and cognitive impairment, from various regions of Pakistan with
extensive inbreeding, were collected to determine the underlying genetic cause by
employing different techniques including homozygosity mapping and Exome
sequencing. Clinically the families were highly diverse with a range of different
phenotypes. The IQ of the patients among this panel of families was mostly less than
20. The probable genetic cause was identified in eight out of the sixteen families,
while eight families remained unsolved. Among the solved families, pathogenic
variants were identified in different genes in family MRQ2, MRQ8, MRQ11,
MRQ12, D1, MRQ14, MRQ15, MRQ18 and MRQ19.
Trisomy21
Genotyping of the family MRQ2 with 250K SNP array revealed no homozygous
regions but the CNAG analysis showed the proband to contain an extra chromosome
21 making it a case of trisomy 21 (MIM 190685), which is a highly prevalent
syndrome among different ID syndromes usually classified as Down syndrome. A
triplicate of any chromosome always contributes to serious genetic defects; trisomies
are the cause of one quarter of pregnancy losses in the form of spontaneous abortions
or miscarriages. DS is found in three out of 1000 live births, the basis of this form of
aneuploidy is always the error at the level of meiosis in either the maternal or paternal
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germ cells resulting in the production of an extra copy of a particular chromosome.
The 21q22-21 region, which has been established to elicit DS has been shown to be
due to the duplication in the proximal region of 21q22.3 (Rahmani et al., 1989),
which is responsible for the pathogencity of DS. The maximum size of this region
detected by linkage mapping was 3000kb and the minimum size detected by pulse
field gel electrophoresis analysis was 400kb (Rahmani et al., 1989). Chromosome 21
has been studied extensively and it is now known to harbor many genes, defects
among which have shown to be involved in causing several cognitive disorders such
as amyotrophic lateral sclerosis, familial Alzhemier disease, leukemia and above all
the most prevalent ID syndrome DS (Onodera and Patterson, 1997). Identification of
trisomy in the family MRQ2 by GeneChip 250k SNP array has further validated the
effectiveness of this genotyping strategy in the diagnosis of genetic cause of ID
disorder.
FMR1
Gene specific FMR1 PCR was performed on the probands of families suspected to
have X-linked mode of inheritance among which the family MRQ8 proband was
found to contain the CGG repeat length of > 200 copies, which is suggestive of full
mutation in the patient. The CGG repeats are highly polymorphic in nature, the
normal alleles contain 5-44 copies that are inherited stably from parents to the
offsprings, the grey zone alleles are the ones which have 45-54 copies and pre
mutation alleles carry 45-200 copies, which are always unstable and can evolve into
full mutation during maternal transmission, the length of the repeat is thus decisive of
risk of transmitting the mutation (De Rubeis and Bagni., 2011). According to Olga et
al. (2007), Fragile X Syndrome is one of the common forms of ID syndromes; it is
mainly caused by expansion in the triplet repeat sequence CGG, which is found at the
5‟ UTR of the FMR1 gene located on the X chromosome. The higher length of this
triplet sequence causes hypermethylation and subsequent silencing of the FMR1 gene.
The product of FMR1 gene is FMRP which is a selective RNA-binding protein, which
acts as a negative regulator of translation by associating with the ribosomes. The
FMRP is found to act in synaptic plasticity by controlling the synthesis of certain
proteins, which are encoded by mRNAs found in dendrites (Penagarikano et al.,
2007). In highly specialized cells such as neuron, protein syntheses occur in all parts
such as the soma, axon, dendrites and also at synapses where translation factors,
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polyribosomes, and specific mRNAs have been detected at the post synaptic sites. The
protein synthesis at synapses actually regulates the storage of long term memories
involved in cognitive functions. The functions of synaptic plasticity such as long term
potentiation induced by BDNF (Brain derived neurotrophic factor) in the
hippocampus and long term depression induced by mGlu-LTD ( metabotropic
glutamate receptor) also depend upon the local protein synthesis. In addition protein
FMRP has been implicated in both basal as well as local protein synthesis as depicted
by studies that FMRP represses translation both in vitro and in vivo (Hou et al., 2006;
Park et al., 2008). Hence the involvement of FMR1 defect has been established to
impair the function of synaptic plasticity, which is important for the mediation of
cognitive development.
BBS10
BBS is a rare heterogeneous developmental disorder including ID, for which to date
17 genes have been identified, in which many mutations have been reported in
different studies, which are the cause of BBS (Marion et al., 2009). The current study
defines a novel pathogenic deletion mutation in BBS10, which will be helpful in
further delineating the causative BBS mutations. The phenotype in the current family
is identical to that described previously (Green et al., 1989), except that in addition to
the classical features the proband had benign vascular calcification of the bilateral
basal ganglia, which is a characteristic feature of idiopathic basal ganglia calcification
(Fahr disease; [OMIM 213600]), a rare dominant neurological disorder with
characteristic deposits of calcium and resultant cellular apoptosis of the basal ganglia
and cerebral cortex, regions of the brain which control movement (Dai et al., 2010).
In addition to the neurological features, in the proband the liver function was also
aberrant with elevated levels of SGPT, SGOT and Alkaline phosphatase. To date 73
mutations (http://www.hgmd.cf.ac.uk) in the BBS10 (C12ORF58; [MIM 610148]
have been reported. The 10 nucleotide deletion in BBS10 in the current study in a
Pakistani family (MRQ19) is a novel mutation, which has not been reported
previously. BBS10 is a group II chaperonin protein belonging to the Homotrimeric
Cation Channel (TRIC) family located within the basal body of the primary cilium of
differentiating preadipocytes (Seo et al., 2010). It is the part of the BBS/CCT complex
composed of MKKS, BBS10, BBS12, TCP1, CCT2, CCT3, CCT4, CCT5 and CCT8.
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Lack of BBS6, BBS10 and BBS12 in cultured cells has been shown to be the cause of
failure of the assembly of a functional BBSome, which is the primary cause of BBS in
patients with mutations in these genes (Seo et al., 2010). In the current family
MRQ19 the 10 nucleotide deletion in exon 2 results in a frame shift, which causes a
premature termination at the downstream fourth amino acid, which could result in a
nonfunctional protein that would ultimately prevent the assembly of the proteins in
the CCT/BBSome complex. The inhibition of BBSome assembly can be the cause of
pathogenesis of BBS in the patients. Obesity is a cardinal feature of BBS, it has been
shown that BBS10 protein is located within the basal body of the primary cilium and
its inhibition of expression can impair ciliogenesis, which would ultimately lead
towards an increase in phosphorylation of glycogen synthase kinase 3 pathway
(GSK3) and induce the peroxisome proliferator-activated receptor nuclear
accumulation hence increasing adipogenesis, which would lead towards increased
accumulation of fats and ultimately towards obesity (Marion et al., 2009). As BBS10
protein dysfunction can impair a long cascade of reactions, it is thus postulated that
the mutation present in family MRQ19 produces a truncated protein due to frame shift
and absence of the BBS10 protein expression at the basal body of the primary cilium,
which would activate the pathway of impaired and pathogenic adipogenesis.
The mutation present in the affected members of MRQ19 is predicted to lead to loss
of function of the BBS10 protein, by producing a truncated protein and/or nonsense-
mediated RNA decay. A similar disruptive effect has been previously predicted for
the Cys91fsX95 mutation, which accounts for almost half of all BBS10 patients
(Stoetzel et al., 2006). In general, the phenotype in the MRQ19 family is similar to
that of the reported BBS10 cases, except for the bilateral calcification of the basal
ganglia. In addition in a Danish study, BBS10 truncating mutations were associated
with a severe phenotype, including polydactyly, hypogenitalism, retinitis pigmentosa
and renal aberrations, yet brain calcification was not reported (Hjortshoj et al., 2010).
Given the highly consanguineous structure of the MRQ19 family, it is possible that
the bilateral basal ganglia calcification is due to a homozygous mutation in another as
yet unknown gene. Alternatively, basal ganglia calcification could also be a feature
associated with reduced penetrance in BBS10. Pleiotropic effects and oligogenic
inheritance are well known in BBS (Badano et al., 2006). Interestingly, experiments
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with zebra fish show that the simultaneous suppression of BBS10 protein grossly
exaggerated the expression of the phenotypes (Stoetzel et al., 2006). Thus, there is a
possibility that the severe brain phenotype in patients from the MRQ19 family is
because of genetic interaction with DNA variants at another (BBS) locus. This
possibility is strengthened by the consanguineous structure of the family as well as the
observed SNP array homozygosity at the BBS2 and BBS14 loci. In conclusion, it is
suggested that the severe phenotype for BBS patients in family MRQ19 could be due
to the homozygous defect c.1958_1967del (p.Ser653Ilefsx4) in BBS10. The findings
may have implications for genetic counseling in this family, which might have an
earlier onset of retinal disease and calcification of the brain ganglia and lower risk of
renal disease, which may imply a more favorable prognosis.
RBBP8 and NRXN1
In the present investigation a consanguineous Pakistani family (MRQ12) was studied
with segregating congenital microcephaly syndrome whose clinical symptoms are
largely similar with Seckle and Jawad syndrome. The only true homozygous region
the affected sibs share overlapped with the SCKL2 locus of Seckle and Jawad
syndrome that harbours the RBBP8 recently identified in a study by Qvist et al.
(2011). In their study the authors had predicted that in both diseases (Seckle and
Jawad syndromes) the RBBP8 mutations lead to the production of shorter transcripts
which yields a C-terminally truncated form of RBBP8 that partially hampers DSB
(double strand breaks) resection and ATR (ataxia telangiectasia and Rad3-related
protein) activation (Qvist et al., 2011). Where ATR orchestrates cellular responses to
DNA damage and replication stress, thus the complete loss of ATR function can lead
to chromosomal instability and cell death (Fang et al., 2004).
In the family MRQ12 a novel substitution was identified in RBBP8 exon 11:
c.919A>G, p. Arg307Gly. In silico analysis using Alamut software (www.interactive-
biosoftware.com) shows that the amino acid at this position (Arg) is highly conserved.
The physiochemical difference between the two amino acids (Arg) and (Gly) is
moderate with a Grantham score of 125. Another fact that suggests the particular
change to be pathogenic is that the change actually lies close to the 5‟ donor splice
site i.e. at -2 position within exon 11. Krawczak et al. (2007), have reported that the
disease-associated mutations found in the donor splice-sites clustered more closely
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around the exon/intron junction, with 64% of the 5‟splice site mutations in the Human
Gene Mutation Database (HGMD) affecting the obligate GT dinucleotide. Mutations
outside the GT are particularly prevalent at positions -2, -1, and +3 to +6. Further they
add that predicting the effects of changes in the consensus 5‟ region near the
intron/exon boundaries is thought to be relatively straightforward. Although in the
current study functional analysis was not conducted to detect the impact of the change
at the 5‟donor splice site but from studies of Qvist et al. (2011), it is evident that the C
terminally truncated short of RBBP8 produces a shorter protein that cause the cells to
respond suboptimally to endogenously arising DNA damage, which lowers the
apoptotic threshold in the patients cells and causes reduction in their proliferative
potential resulting in retarded growth in such patients. Our results are in agreement
with these previously reported findings as the mutation in family MRQ12 is also
located in the C- terminal part of the RBBP8 protein and this change can result in the
production of an abnormal protein, which could led to the observed defects, as RBBP8
acts together with the MRN (MRE11-RAD50-NBS1) complex to promote DNA end
resection and the generation of single-stranded DNA, which is critically important for
homologous recombination repair (Yuan.and Chen, 2009). Based on these factors it is
purposed that the mutation identified in MRQ12 is responsible for the severe
phenotype of the two sibs as this region bears an MRN interaction domain (Sartori et
al., 2007) that is crucial for DNA-end resection. Although a second MRN interaction
point has been found in the N-terminal part of the protein (Yuan et al., 2009), the C-
terminal region is essential for RBBP8-mediated activation of MRN-associated
nuclease activity, thus any mutation in this region will render the MRN and RBBP8
complex ineffective and cause the impaired production of single stranded DNA that
results in the inactivation of ATR, which ultimately results in defective apoptopic
activity in patients due to hypersensitivity to DNA damage.
NRXN1
In a Dutch family (D1) a 607 kb deletion was identified, which encompasses the
upstream promoter that is used to generate the α-neurexin of NRXN1. In addition in
the family MRQ12 besides the RBBP8 mutation a 455 kb deletion was identified
which encompasses the downstream promoter, which is used to generate β-neurexin
of NRXN1. Heterozygous variations and a deletion affecting NRXN1 have been
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previously reported to be involved in autism (Feng et al., 2006; Yan et al., 2008;),
schizophrenia (Gauthier et al., 2011), and many other psychiatric and psychotic
disorders (Ching et al., 2010). A number of studies have shown that Neurexins have
an essential role in the development of the synapse, not in its initial adhesion but in
the recruitment of molecular components and its maturation (Zhang et al., 2005).
There are three Neurexin genes in mammals, which have the capacity to generate a
stunning variety of distinct transcripts by using alternate promoters, splice sites and
exons (Rowen et al., 2002; Tabuchi and Sudhof, 2002). For NRXN1, most transcripts
use the upstream promoter and encode alpha-neurexin isoform; while fewer
transcripts are produced from the downstream promoter and encode beta-neurexin
isoform. α-Neurexins contain epidermal growth factor-like (EGF-like) sequences and
laminin G domains, and they interact with neurexophilins. β-Neurexins lack EGF-like
sequences and contain fewer laminin G domains than α-neurexins (Kirov et al., 2008).
Interestingly, knockout of the three α-neurexins, leaving the three β-neurexins intact,
leads to perinatal lethality due to the loss of presynaptic Ca2+
channel function
(Missler et al., 2003). The studies conducted till now on knockout mice lacking all
three α-neurexins but with normal expression of β-neurexins have shown that α-
neurexins have a unique function that is not covered by β-neurexins. Moreover, a
patient with mild ID and autistic features reported by Zahir et al. (2008), was shown
to have heterozygous deletion that specifically affected NRXN1-, leaving NRXN1-
intact. The heterozygous loss of exon 13 to exon 19 containing some coding exons for
α-neurexins, as well as ablation of the promoter for β-neurexin in family MRQ12, has
resulted in a mild ID of the mother and might be the white matter abnormalities of the
brain in the two affected sons. This result thus also suggests an important role of α-
neurexins as well as β-neurexin. The white matter disease of the MRQ12 patients is
also consistent with the findings of multiple studies, where neurexin-1α has been
reported to interact with postsynaptic neuroligins (NLGNs) mediating GABAergic
and glutamatergic synapse function. Different studies have reported neurexin-1α to
bind to leucine-rich repeat transmembrane protein (LRRTM2), instructing presynaptic
and mediating postsynaptic differentiation of glutametergic synapses and ultimately
reduced expression of NRXN1, which may lead to the white matter alterations (de Wit
et al., 2009; Ko et al., 2009; Siddiqui et al., 2010). In a study conducted on a family
with complex phenotype, as in the current family MRQ12, the authors found a
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compound heterozygous NRXN1 mutation; a 451 kb deletion including the upstream
promoter and the first introns as well as point mutation (c.2880-IG>A, IVS14-IG>A;
50,723,234 Hg19) (Duong et al., 2012).
In family D1 the female proband had gonadal dysgenesis apart from ID, this
combined phenotype may be explained by the NRXN1 deletion because it has been
previously shown that the NRXN1-α has a role not only in calcium dependent release
of neurotransmitters but also calcium induced release of secretory granules from the
endocrine cells including those of the pituitary glands (Dudanova et al., 2007).
Furthermore NRXN1 is also expressed in the hypothalamus of prepubertal females of
primates (Mungenast and Ojeda, 2005). Phenotype variations may reflect the highly
pleiotropic effects associated with NRXN1, which is also reflected by a few patients
with complex structural malformations reported in different studies (Zahir et al.,
2008; Ching et al., 2010; Gregor et al., 2011). The loss of the 455 kb in the family D1
female proband occurred de novo, in addition the two brothers in MRQ12 inherited
NRXN1 deletions from their mother but both of them were severely affected and their
phenotype was of Seckle and Jawad Diseases, which is proposed to be due to RBBP8
donor splice site mutation, while the mother was ostensibly less affected than her
children and in addition she had diabetes mellitus type 1, the mutation in NRXN1 can
possibly explain her variable phenotype. This suggests that the change in RBBP8
together with deletion in NRXN1 gene may be fully penetrant, and that genetic
background effects and/or environmental factors cause clinical variability and thus in
MRQ12 the digenic model of inheritance is proposed. Diabetes mellitus has not been
reported previously to be associated with NRXN1 mutations and therefore it cannot be
concluded whether this feature in the mother of MRQ12 is due to this deletion or
another genetic predisposition. However, it is of note that the Neurexins are expressed
in β-cells of the pancreas and any abberations in the protein could potentially lead to a
decrease in function of the pancreas (Suckow et al., 2008).
The current findings are consistent with the findings of Jawad et al. (2003) and Qvist
et al. (2011), that the changes in the C-terminal region of RBBP8 are involved in
severe phenotype such as those of Seckle, Jawad syndrome and the current MRQ12
syndrome. Additionally as reported in other studies also the current work suggests
that deletions in NRXN1 is involved in various neurological disorders, however, the
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current findings are novel in showing that the strong phenotype could also result from
heterozygous deletion of NRXN1 alone like in the patient D1. Further efforts to
investigate such variable phenotypes associated with this unstable genomic region
will provide insight into the role of RBBP8 and NRXN1 in the development of
dysmorphic features such as microcepahly, language delays, autism and
morphological abnormalities of the brain and ID.
Genotyping of families MRQ11, MRQ14, MRQ15 and MRQ20 revealed homozygous
regions in some families such as MRQ11 and MRQ14 but no homozygosity was
found in MRQ15 and MRQ20. The phenotypic features of MRQ11 were of non-
syndromic ID, in addition they did not have any structural anomalies in their organs
and brain, except that their IQ was in the range of moderate ID (IQ: 36-51).
ZNF589
By exome sequencing and variant testing a substitution at p.L319H was identified in
gene ZNF589 segregating with the disease in the family in a recessive manner. The in
silico analysis revealed that the substitution results in a missense change in the amino
acid Leucine to Histidine, the two amino acids differ from each other with a
Grantham distance of 99, which depicts a moderate physiochemical difference. The
phyloP score for the missense change is 2.965, which is suggestive of a pathogenic
role of this particular variant. The variation was heterozygous in the mother and
unaffected sibling but it was homozygous in the two affected children. ZNF589 is
localized at the cytogenetic band 3p21.31, it is a 41189Da protein and it is 364aa long,
it belongs to the kruppel C2H2-type zinc-finger protein family. ZNF589 consists of a
conserved KRAB domain at the aminoterminus and also four zinc fingers of the
C2H2 type at the carboxy terminus. Upon alternative splicing of ZNF589 two
products are obtained that encode a protein of 361 and 421 amino acids, which differ
from each other at the carboxy terminus (Liu et al., 1999). In the current study the
missense change c.956T>A is located in the domain C2H2- type3, Polyphen and SIFT
score revealed this particular defect in the gene to be pathogenic. The functional data
from the study of Liu et al. (1999), has shown that ZNF589 is involved in
hematopoiesis, because of its localization in the bone marrow derived stem cells.
Gene card identifies its expression in all parts of the brain in adults as well as fetal
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brain. The gene ZNF589 has never been reported previously to function in the
pathogenesis of ID but the data published by Najamabadi et al. (2011), have reported
a missense change in another zinc finger gene ZNF526, which is kruppel C2H2-type
zinc-finger protein, in a consanguineous family of nonsyndromic ID, the gene was
reported to be the probable activator of mRNA translation. In another study Shoichet
et al. (2003), reported four substitutions (I125R, Q262Q, D315E and Q486Q) in
ZNF41 in 200 X-Linked ID probands, this gene encodes another kruppel C2H2-type
zinc-finger protein. Lugtenberg et al. (2006), have also reported the involvement of a
zinc finger gene in a patient with ID, retinal dystrophy and dwarfness. They therefore
further tested ZNF673 and ZNF674 in 28 families with nonsyndromic X-linked ID,
which revealed a nonsense mutation, p.E118X, in the coding sequence of ZNF674 in
one family (Lugtenberg et al., 2006). This mutation is predicted to result in a
truncated protein containing the Kruppel-associated box domains but lacking the zinc-
finger domains, which are crucial for DNA binding. They further screened an
additional 306 patients with X-linked ID, and found two amino acid substitutions,
p.T343M and p.P412L, that were not found in unaffected individuals (Lugtenberg et
al., 2006). In agreement with the results published by these previous studies, in the
current study the pathogenic role of ZNF589 in the family MRQ11 is supported. The
zinc finger genes are housekeeping genes such as ZNF589 that has been localized in
bone marrow stem cell and has been reported to be involved in DNA dependent
transcription repression, the missense change identified in the current study was found
in the C2H2 effector domain that is a highly conserved motif in the ZNF589 protein.
The C2H2 domains are located in the amino-terminal region of the protein where they
have different roles in transcriptional regulation by interacting with different cellular
molecules (Ding et al., 2009). Although no functional assay was performed to detect
the effect of the particular change in ZNF589 at the protein level but as the change is
affecting the conserved domain of the ZNF589 it can be predicted that this mutation
will disturb the function of the protein at the cellular level. By the support of data
published on other Kruppel-associated box domain zinc finger genes the pathogenic
role of the mutation p.L319H in MRQ11 is proposed. The identification of ZNF589
in non syndromic ID among the affected members in MRQ11 may indicate a critical
role of the zinc-finger genes suggesting them to be crucial for human cognitive
functioning.
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MLL4
The family MRQ14 had three affected members all of whom were found to be
homozygous carriers of the pathogenic variant p.P819H, c.2456C>T. The phenotype
of the family was very severe with IQ < 20 and the disease was also associated with
other features such as muscle spasticity and dimorphic facial features. The phenotype
of the three affected members resembled Kleefstra syndrome (MIM 610253), which is
characterized by facial dysmorphism, hypotonia and mild to severe ID, the syndrome
is rare with unknown prevalence. The family MRQ14 with Kleefstra syndrome
features is being reported for the first time from Pakistan. As the family had mostly
males affected, so the X chromosome variants obtained from whole exome
sequencing data were analyzed manually to detect for any pathogenic variant, only
one variant TXLNGF was found, which did not segregate with the disease in the
family. Exome sequencing identified multiple homozygous and compound
heterozygous variants in different genes but the only variant, which segregated was
the missense substitution in MLL4. The variant has a high phyloP score of 4.429 and
the Grantham distance depicting the physiochemical difference between Proline and
Leucine was moderate i.e. 98. The variant was not found to be present in any of the
domains but in silico analysis by SIFT reported it not to be tolerated. The variant
segregated in the family in a recessive manner. Although till date dominant mutations
for EHMT1 and MLL3 have been reported for Kleefstra syndrome (Kleefstra et al.,
2012), but no recessive or homozygous mutations has been reported in any of those
previously identified genes.
MLL4 belongs to the MLL (mixed-lineage leukemia) family, it is found to be
ubiquitously expressed in adult tissues and also found to be expressed in solid tumor
lines, which suggests its involvement in human cancer. The protein encoded by this
gene has multiple domains such as CXXC zinc finger, three PHD zinc fingers, SET
(suppressor of variegation, enhancer of zeste, and trithorax) and two FY domains. Of
all the domains, the SET domain is the most conserved domain of MLL4 protein,
which actually characterizes the MLL family genes (http://genome.ucsc.edu/cgi-
bin/hgGene?hgg_gene). The Gene ontology database (GO) reports a mouse model
with abnormal levels of MLL4 to suffer from abnormal somite development
(MP:0001688), open neural tube (MP:0000929), increased apoptosis (MP:0006042),
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kinked neural tube (MP:0003400), embryonic lethality during organogenesis
(MP:0006207), abnormal embryonic growth retardation (MP:0003984), embryonic
growth arrest (MP:0001730), reduced female fertility (MP:0001923), abnormal DNA
methylation (MP:0008877) and reduced female fertility (MP:0001923). Kleefstra et
al. (2012), reported that only 25% of patients with Kleefstra syndrome from their
cohort were found to be associated with haploinsufficiency in EHMT1 gene while
other patients had de novo mutations in MBD5, SMARCB1, NR1I3 and MLL3. The
EHMT1 negative patients actually had mutations in genes that are biologically related
to the EHMT1. As in the current study no dominant mutation was found but the de
novo change reported in MLL3 is supportive of the current findings as both MLL3 and
MLL4 belong to the same family. In addition in one recent study on a cohort of 86
patients of Kabuki syndrome (MIM 147920), which is characterized by dysmorphic
facial features, bone deformities, hypotonia, congenital heart defects, ID, urinary tract
and respiratory tract infections, 41 likely pathogenic mutations were identified in
MLL2 gene, which also belongs to the MLL family (Makrythanasis et al., 2013).
MLL genes MLL1-MLL4 along with their drosophila orthologs such as trithorax trx
and trithorax related trr express protein products, which are involved in methylating
histone H3 on lysine 4. MLL4 belongs to the trr ortholog and it has been shown to be
involved in cell proliferation by its evident role in human cancer but its mechanism of
action is not yet known. It has also been reported in a study by Kanda et al. (2013),
that the mutants of trr in the drosophila model result in restricted tissue growth, which
explains the growth retardation in the mouse model with low levels of MLL4 and also
in the current family where the affected members had growth delays. MLL4 like
MLL3 is a part of ASCOM (activating signal cointegrator-2) coactivator complex,
which has an important role in epigenetic regulation together with the nuclear-
receptor transactivation and forms a connection between the two complexes (Kleefstra
et al., 2012). Kim et al. (2009), have previously reported in their study that ASCOM-
MLL4 play an essential role in Farnesoid X Receptor transactivation through their
H3K4 trimethylation activity, hence it can be proposed on the basis of these studies
that MLL4 could also be a part of the chromatin modification module purposed by
Kleefstra et al. (2012), along with MLL3, SMARCB1 and NR1I3 and that MLL4 can
also be functionally related to EHMT1 gene haploinsufficiency, which leads to
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Kleefstra syndrome. EHMT1 encodes a histone methyltransferase, which is capable of
histone 3 lysine 9 dimethylation (H3K9me2) in euchromatic regions of the genome
(Tachibana et al., 2005). No functional assay was performed in the current study to
determine the exact function of MLL4, thus based upon the previous studies it is
purposed that the change in MLL4 can be responsible for epigenetic modifications by
interacting with the genes of chromatin modification module leading to the severe
phenotype in family MRQ14. It can also be proposed that not only genes that are
widely expressed in the brain and synaptic plasticity pathways but genes of other
pathways such as the genes of epigenetic regulatory mechanism can also be
responsible for the pathogenic phenotype of ID.
HHAT
The family MRQ15 in the current study did not have any consanguinity in the parents,
although their ethnic group was the same. Exome sequencing of the proband in this
family revealed very few variants with good pathogenicity score and only one variant
was found to be common (c.1158G>C, p.W386C) in the gene HHAT (Hedgehog
Acyltransferase) in the two affected members. The change has a high phyloP score
and Grantham distance, which points to the high physiochemical differences between
the wild type and variant amino acid. The particular variant gene has no domains and
the substitution was not found in any of the unaffected members while it was present
in the two affected children as a heterozygous change. It is thus purposed that this
variation among the affected members of MRQ15 has occurred as a result of a de
novo change. The phenotype of the affected offsprings was very severe with IQ < 20
and they both had speech impairment and facial deformities but no brain structural
defect was noted. The current results are in agreement with the previous results in
which de novo changes have been shown to occur in two sisters with Noonan
syndrome, as a germline HRAS mutation (G12A) in one sister and as another
germline KRAS mutation (F156L) in the other (Sovik et al., 2007). Similar to the
current findings de novo heterozygous c.1568T>C substitution in exon 13 of TP63 has
been reported previously in two siblings, which resulted in a p.L523P change in the
SAM domain of TP63 protein (Barbaro et al., 2012). In that study the two siblings
were found to suffer from Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC)
syndrome, while genetic analysis of the unaffected parents revealed no defect in
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TP63, the authors thus proposed that germ line mosaicisim could be the explanation
of the occurrence of the same de novo change in TP63 in both the offsprings although
no change was present in the parents. They further suggested that the phenomena of
mosaicism sometimes include only very few number of cells (very low grade somatic
mosaicism) or there could be the phenomena of maternal gonadal mosaicism, which
could be responsible for the de novo mutation in the two affected siblings. The
particular de novo change in HHAT among the two siblings in MRQ15 is in
agreement with the suggested phenomena of germline mosaicisim reported by
Barbaro et al. (2012).
HHAT, which is also referred to as Skinny hedgehog' actually is the precursor of an
enzyme that acts within the secretory pathway to catalyze amino-terminal
palmitoylation of 'hedgehog. HHAT is included in the Hedgehog family of gene; it
encodes a glycoprotein that undergoes autoproteolytic cleavage post-translational
modification to generate its active form. The modification, which involves the lipid
modification, is actually required for multimerization activity and distribution of
hedgehog proteins. The gene ontology database suggests that low levels of HHAT in
mouse phenotype leads to disproportionate dwarfism (MP:0002427), oligodactyly
(MP:0000565), neonatal lethality (MP:0002058), decreased motor neuron number
(MP:0000939), decreased embryo size (MP:0001698), absent floor plate
(MP:0000926), chondrodystrophy (MP:0002657), holoprosencephaly (MP:0005157),
and abnormal spinal cord interneuron morphology (MP:0004100). The database
further describes its role in multicelleular organ development
(www.geneontology.org)
Jennifer et al. (2012), have previously shown that HHAT insertional mutation was
responsible for the holoprosencephaly in a mouse model, together with acrania and
agnathia. Holoprosencephaly is the severe brain structural disorder, which inhibits the
brain to bifurcate into left and right hemispheres during perinatal development
(Milner et al., 2012). Although the condition is very rare but still 1 in 20,000 live
births have holoprosencephaly in humans. Jennifer et al. (2012), further proposed that
HHAT was a strong candidate gene for the congenital human disorder because they
found by different functional assays that HHAT actually palmitoylates hedgehog
proteins and were important for maintaining of the long range signaling processes
126
during emrbryogenesis. They also proposed that HHAT is required for
posttranslational palmitoylation of Hedgehog proteins hence their absence could
diminish secretions of hedgehog proteins, this could lead to abnormal patterning and
extensive apoptosis within the craniofacial primordial which leads to the structural
defects in holoprosencephaly (Milner et al., 2012). This could be the possible
explanation of the current results in which only the heterozygous de novo change in
HHAT was identified among the two affected sibs, the other allele was found to be
normal hence the effect of the mutation with a predicted strong pathogenicity, did not
result in any structural malformation in both the sibs but the brain cognitive function
was highly affected, which also resulted in speech impairment in patients.
TPO
Family MRQ18 had only one affected member, the parents had consanguineous
marriage. The affected son had congenital hypothyroidism and learning difficulties
and his IQ was below average. The gene TPO thyroid peroxidase was sequenced, as a
number of defects in this gene have already been established to be causative for ID
associated with thyroid disorders (Medeiros-Neto et al., 1993). In silico analysis
revealed the identified variant g.1418194C>G, p.Ala5Gly in exon 2 not to be
pathogenic as the phyloP score was -1.17 and the variant amino acid was present in
other species as well, hence the underlying genetic cause remains unclear in the
family MRQ18.
Benefits to the patients
The results of the solved families can help in the genetic screening and counseling of
the family members which could possibly eliminate the disease in the subsequent
generations.
Unsolved Families
The families PKMR71, MRQ16 and PKMR176, which were suspected to be X linked
families due to their pedigree structure did not reveal any aberration in the X
chromosome and thus seem to be of autosomal recessive ID, so the next step to
unravel the genetic cause in these families could be whole exome sequencing. While
in the other families among which exome sequencing did not reveal any pathogenic
127
mutation, there might be some deep intronic change that could be involved in the
manifestation of the disease pathology, to solve these families, upcoming powerful
tools such as parallel DNA sequencing and single molecule sequencing could be the
solution. The families in which a number of homozygous regions were found and also
among which only one polymorphic region was identified with heterozygous deletion,
the genetic cause responsible for pathogenecity remains unclear and exome
sequencing could be the solution as no definitive homozygous region was identified in
these families. The family for which the coding exons of RELN were sequenced and
no pathogenic defect was found, there might be some other gene in the identified
homozygous region working in the pathology, which needs to be elucidated further.
128
Conclusion
During the current study multiple families with genetic cause of ID were identified,
pathogenicity in these families could be due to the high inbreeding in them. In
addition spontaneous mutations could also be an intricate cause because of excessive
usage of pharmaceutical preparations and exposure to radioactive materials.
Techniques such as Microarray, Exome sequencing was found to be valuable tool in
the elucidation of ID in these families and hence establishment of such techniques in
diagnostic facilities could be helpful in prenatal diagnosis of such disorders, which
can result in reducing the disease burden in the country.
Chapter 5
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130
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Appendix
158
Appendix 1. List of primers for all coding exons of TPO.
Exon # Direction Sequence 5' - 3'
Product
size
Exon 2
Forward CTCAGCAGGGAGACAAGGAC
286bp Reverse ACAGGGGCCTAAAGTGACTC
Exon 3
Forward GGCTTGAGGAACAAAGCAAC
237bp Reverse CACGTGTGTGGATGTCAGG
Exon 4
Forward TGTCACATTGTCTGGGACAC
408bp Reverse GTCCCATCCTGCTTGGTC
Exon 5
Forward CCCCAGTTACATATGAATCCC
344bp Reverse CCAGTGGTGGTAGAAGTTCC
Exon 6
Forward TTCTCCCTGAGAATGGTGTC
343bp Reverse GGATCTGAAGGGATTGCAC
Exon 7
Forward AAGAACCACACCAGGAAGTG
441bp Reverse GCACCAGATATTCACAACTGC
Exon 8-1
Forward CTACAGAGGACTGGAGGGG
530bp Reverse GTGTGCAGTGCCGTCAG
Exon 8-2
Forward GGCCCTAGAGAGGCAGC
439bp Reverse TGCAAGTACCTGGGAGAGAG
Exon 9
Forward AAGATGCTCTTCCACACTGC
441bp Reverse TTGTGCACATGTACCCTAGAAC
Exon 10
Forward
GCAACAGAAAGAATGAGACTC
C
395bp Reverse AAGGCCTCAGATTTGTGATTG
Exon 11
Forward ACCATGGCATGAGTGAGATG
408bp Reverse GAATGTGAATGCAGGTCTGG
Exon 12
Forward AGGCTGGCAGCACACAG
431bp Reverse GTAAGGCAGGGCGTTGAC
Exon 13
Forward GGAAGAGGCCGTGTGTG
464bp Reverse TGCTCCTGGGAAGATAACAG
Exon 14
Forward ACCTCCCCAGAGAGAAGC
358bp Reverse GTGTCTTATTCATCTCCGCC
Exon 15
Forward AGGACAGGGTATGGCCC
318bp Reverse TCAATGCCTTTGAGATACCAG
Exon 16
Forward AATGAAAGTGGGTTGGAGTG
363bp Reverse AACAGAAATATGCAGGGAGC
Exon 17
Forward ATGACAAGCAAGAAGGATGG
314bp Reverse GTGTTGGGAAAACAGTCGTC
159
Appendix 2. List of primers for exons of NTF3.
Exon # Direction Sequence 5' - 3'
Product
size
Exon 1
Forward TCCCCTGCTGGGTAGTG
293bp Reverse ACTTTGGGGAAGACACAAGC
Exon 2-1
Forward GGTACCCTCTCTCGTGCC
571bp Reverse CTCGGTGACTCTTATGCTCC
Exon 2-2
Forward AGAGACGCTACAACTCACCG
627bp Reverse
AGCTTATATACTGTAGGGTTGC
TG
160
Appendix 3. List of primers for all coding exon of NRXN1.
Exon # Direction Sequence 5' - 3'
Product
size
Exon2_1
Forward CCTTTCTGTCTCTCGGGACC
553 Reverse GCTGAACACCGTCATGTCC
Exon2_2
Forward CAGCTTCTCCATCTTCTGCG
595 Reverse CAGCTTCTCCATCTTCTGCG
Exon3
Forward CTGGGTAGTGGAGGCCAAG
301 Reverse GTGTTGATTGCCTTGCTTTG
Exon 4
Forward GATGTGTTTGGTGCCTTGC
249 Reverse CCAGAAACCAACAAATGTTCAG
Exon 5
Forward AGGAAAAGAGATGTCTGTCACTG
333 Reverse TAATTTGCAAGCATTTGCCC
Exon 6-7
Forward CATGTCATTCATGGAATGTTTC
304 Reverse AGCAGAAGAGTACCAACCGC
Exon8
Forward ATGGGTTCCGTCATAGGTC
440 Reverse GTGCCGTTTGACTCTGGAAC
Exon 9
Forward TTTGCCTGAGAAATGTTGCC
746 Reverse TTCATGGTGTGAAACAGAAGC
Exon 10
Forward GCCTCCAAGGAGTTAACCATC
610 Reverse
GCTTGCAAACAGAGTTTACTTCA
G
Exon 11
Forward CTCTTTCCAGCTCACAGTTTTG
464 Reverse TTCCATGGCAAACAGGCTAC
Exon 12
Forward CCCGGAAGGGTGGACTG
265 Reverse ACAACCCAGATGATGGACTG
Exon 13
Forward TTGAAAATTGGTCCAGCCC
427 Reverse
TGGATTGTGTGAATACATTTCAA
G
Exon 14
Forward GTACCAAGGAGGGATTTGGC
622 Reverse GTCCTCCACCTGCTCCG
Exon 15
Forward
GACTTCTAGTAAGGATGGAACCA
C
402 Reverse GCAGAAGGTACAAACACACCC
Exon 16
Forward AGCACTTTGGGGAAAACAGC
413 Reverse CCAGTTATCAGAATTTTGCTGG
Exon 17
Forward CATATCATCTGTGGTGTTGGC
344 Reverse GGTTATGACAGTTCGACTGACTC
Exon 18
Forward GATTTACTTTGCCATAGTTTTGTG
418 Reverse AACAAAGTACTGGTTTCTGGGG
Exon 19
Forward CAAAGCACATTTTAAAACAATAC
359 Reverse ACCACAATTTGGTGAAACGGGG
161
Appendix 3.Contin.
Exon Direction Sequence
Product
size
Reverse TCATTTTGGGTTAATGCTTGC 456
Exon 21
Forward
TGAATTATGAGTCAAATGCCTCT
C
581 Reverse TCAAAGGAAGCTGTAGTGCC
Exon 22
Forward GTTGGCTGTCACCATTTCTG
559 Reverse AAGCCCTGTGTGCTATACCC
Exon 23-24
Forward CACAGCAACAAATCTCCCAG
738 Reverse TTAGGGAATTTATTGGCCCC
162
Appendix 4. List of primers for all coding exon of RBBP8.
Exon # Direction Sequence 5' - 3'
Product
size
Exon 2
Forward ATTTGACCTGTCCAAAGACG
319bp Reverse CCCGGAACCTAAAAGCTG
Exon 3
Forward CGTAAGGGGCATATACTGTAAAC
283bp Reverse AAGGTCAAATTGGACATCAG
Exon 4
Forward TTCCAACCTGACCTTTTCAG
330bp Reverse GGAAAGATCACTTTCAATCCC
Exon 5
Forward TTCACAGTATTTGCCAAGTCAG
439bp Reverse CCAATGTGGACAGTATTTGC
Exon 6
Forward GGCACGGTGTGAGATGTAG
468bp Reverse TGTTGACACTTTCTTCAAGCAC
Exon 7
Forward TGGAACATTAGATGCAAGGG
496bp Reverse CGTATCTATACATAACACGGGC
Exon 8
Forward GCAATTTGGGTTAGAAGAAGC
648bp Reverse TGCCTGGTATACAACAGATGC
Exon 9
Forward TGTGAGCCTTTTCCTTCATC
322bp Reverse GTTTCAGGCCTTTACCCAAG
Exon 10
Forward ATTATGTGGCCTTTGTCTGG
345bp Reverse ATATGTTGAAGGAGAGAAATGG
Exon 11-1
Forward TGAAGAGAAGCCAAAAGCTG
627bp Reverse ATCCATTGGAAAAGGGAAAG
Exon 11-2
Forward AGGCCGAACATCCAAAAG
639bp Reverse GATGCAAGTTCACATCCTCC
Exon 12
Forward TGTTTTAGATGACATAAAGGTTTG
363bp Reverse TCGTCCTATAAATACAGACACCTG
Exon 13
Forward CATCTCGTGGTTTTGGTTTG
702bp
Reverse TCTCTCTTCGACCTCCCAG
Exon 14
Forward GCAGTAAAACCTAAATCCTTACC
Reverse AAACCTGCTTTATGGTGAAGAG
399bp
Exon 15
Forward GTCATGGGAATTTGTTGCAC
Reverse TTGGGTCAAAGGTACAGGAG 565bp
Exon 16
Forward TTAAATGAGTTGCTCAGAGGC
600bp Reverse TCTGACAAAGACAGCTCGAC
Exon 17
Forward GAATTTGCAGGTCACTTTGG
450bp Reverse TTACGCCTGGCTCAAATAAG
Exon 18
Forward TCATCAATGAGGATTAAGTTTCC
364bp Reverse TGTTGGGATTATAGGCGTGAG
Exon 19
Forward AGATCAATCATCAGCATCACAC
402bp Reverse GGTGCAAAAGCAAAATATCAC
163
Appendix 5. List of Primers for all coding exons of HPRT.
Exon# Direction Sequence 5' - 3'
Product
size
Exon 1
Forward GAACCTCTCGGCTTTCCC
302bp Reverse GTGACGTAAAGCCGAACCC
Exon 2
Forward TGTAATGCTCTCATTGAAACAGC
274bp Reverse TGGCCTAGTTTATGTTCAAATAGC
Exon 3
Forward TATTGCCCAGGTGGTGTG
377bp Reverse AAATGAAAGCAAGTATGGTTTGC
Exon 4
Forward CTTGATTGAAGATGGGTGG
647bp Reverse AGAGTCCCACAGAGGCAGAC
Exon 5
Forward TTTGGATAATTCCTTAGGGTTG
208bp Reverse GAGGAATTTCTCTCCCTGGC
Exon 6
Forward TTTTGGTGAGAATTACTGTGCTG
308bp Reverse TTTTCGAAGATAAAATGACAGTTG
Exon 7-8
Forward GCACGGATGAAATGAAACAG
502bp Reverse CATCAGTCTGTTCAAATTATGAGG
Exon 9
Forward TGCTATTCTTGCCTTTCATTTC
208bp Reverse CTAAGCAGATGGCCACAGAAC
164
Appendix 6. List of primers for all coding exons of BBS10, which was sequenced in 7
fragments (1 for Exon 1, and 6 to cover the large Exon 2).
Exon # Direction Sequence 5' - 3'
Product
size
Exon 01 Forward
TGTAAAACGACGGCCAGTTTCTGCCTTCGC
GTACAAC
535bp Exon 01 Reverse
CAGGAAACAGCTATGACCGGGACAAGAGCT
CCACAGAG
Exon 02_01 Forward
TGTAAAACGACGGCCAGTTGGTTTTAAGAT
GTGGGAAGC
536bp Exon 02_01 Reverse
CAGGAAACAGCTATGACCCAAATACACCAA
TCCCACTTTT
Exon 02_02 Forward
CAGGAAACAGCTATGACCGCGAAAAGGCCT
GTGGTG
635bp Exon 02_02 Reverse
TGTAAAACGACGGCCAGTTGCTAAAGAGAG
AACATTGTGTAGG
Exon 02_03 Forward
TGTAAAACGACGGCCAGTTTCCACTTCTGG
ATCAGAGTTT
636bp Exon 02_03 Reverse
CAGGAAACAGCTATGACCGGCCTTTGTATT
GAGCCATT
Exon 02_04 Forward
TGTAAAACGACGGCCAGTACCAGTGCATGG
TCTCATTG
632bp Exon 02_04 Reverse
CAGGAAACAGCTATGACCAAACACAACCAG
CTGGCATA
Exon 02_05 Forward
TGTAAAACGACGGCCAGTCCAAACAGTTGA
AACGCTGA
532bp Exon 02_05 Reverse
CAGGAAACAGCTATGACCTTCCAAACCTGT
CTGACTGC
Exon 02_06 Forward
TGTAAAACGACGGCCAGTTGCCATCAATCA
GAAGAAACC
500bp Exon 02_06 Reverse
CAGGAAACAGCTATGACCGGTCTGGTGACC
TTAGTGTGC
165
Appendix 7. List of primers for all coding exons of .
Exon # Direction Sequence 5'-3'
Product
size
Exon1
Forward AGTGTCCCTGCACTTCTTGC
347bp Reverse ACAAGGTGCTCGCCGAC
Exon2
Forward TGACTGAAAGTTGTCCATGC
611bp Reverse GAGTGTCACCAAGCGATTAGC
Exon3-4
Forward CCAGTTAATGTGTGAGGCCC
666bp Reverse GGAGAAGCTTACACTTCTGTCAAC
Exon5
Forward AGGTTGTCAATCTTGGGTGG
307bp Reverse AAAACCACCTGGGTTTGGAG
Exon6-7
Forward CCACTAATGGATTGGAAGCG
534bp Reverse AACATTGCCAAGGAACGAAC
Exon8-9
Forward TGAGAAGTTGTAGGGCTTCATAG
732bp Reverse ACCCTGGCAATGACACTCTC
Exon10
Forward AAGGGTATTTTCCCACAGGG
689bp Reverse GCTCGGATGATGGTGTCTG
Exon11
Forward ACCTGTGCCATCTTGGAGTC
573bp Reverse AATGGTTTCTCAGGCCCC
Exon12
Forward TTAACCCTCAAGTGTTTTGGG
435bp Reverse TACAGGTTACAAGCCACCCC
Exon13
Forward CCCTCAAGAATGGCTGTACC
328bp Reverse CCACATGAGAAATCTATGCCC
Exon14
Forward TGCTGGAAATAATGCAGTTTG
520bp Reverse CATGGTGCTCAGGAGCTAAAC
Exon15
Forward TTGGTATAAGCGAACAGGGG
393bp Reverse CATAGAGGACATGCATTTTCAAC
Exon16
Forward TCAAAGCAAACTAGCAGTTACTCG
427bp Reverse CAGCTCTGGAGTGTGAAAGG
Exon17
Forward TTCCCTCCTGGTTTTATGTTG
314bp Reverse TTATTTTCATTCTTAGCACCCG
166
Appendix 8. List of primers for all coding exons of RELN.
Exon # Direction Sequence 5' - 3'
Product
size
Exon1
Forward CTTCTTCTCGCCTTCTCTCC
435bp Reverse ATGGTTCTTGTTTCCAAGGC
Exon2
Forward TTCCGAGCTTCTTTATTCTCC
448bp Reverse CAATTCACCCAACATCAAGC
Exon3
Forward CAATGATTCCAGCAGGTCAC
569bp Reverse TCCAATTTAGACCTTTGGAGG
Exon 4
Forward CGCACATCTGCTGTTTTCAG
677bp Reverse CATTTTCAACAAAGCTGACAGG
Exon5
Forward GGCAGCATGAATGTTTCCTC
442bp Reverse ATTTAACCAATAAGTGTTGGCAC
Exon6
Forward CCTTAGTAGTTTGAGGGACAAAGC
385p Reverse AAGCCCAGTTCCATAGCTTAAC
Exon7
Forward CAAAATGGTGATTTGAGCTGAG
374bp Reverse AGGCCATAGAATCACCCCTC
Exon8
Forward AATACCATTCACTGCTGGAGAG
460bp Reverse CCCACAGGGGAAGAAGAAAC
Exon9
Forward GAAGAATGGGGAAGTTACCAGAC
384bp Reverse CAGGGCTGGTCATAATATGTG
Exon10
Forward TTTTCCATGTAAACCTTTTGTAGG
720bp Reverse TTCAGCCCAGTACACAGCAC
Exon11
Forward GCTCTTTTGATTTGAGAGTCCATC
374bp Reverse GCTTTCGCCATTTAACAATATG
Exon12
Forward AAATTCATCCCTCTACAGGAAAAC
529bp Reverse GGCTTCTGCTCTGTCTGGAG
Exon13
Forward GCCTGTCAGAACAGGGATG
349bp Reverse GGTTATTCTTAAGTGACATCTGGG
Exon14
Forward CCTGGTGCTTAATAAATGGCAG
475bp Reverse TTTTCATTTGGCAAAAGTGTG
Exon15
Forward TGCCCTTTGAAAGCTGGTC
490bp Reverse TTGCATCCAGTTTACCAAAGATAC
Exon16
Forward GTGCTTTTCCAAGCACACAG
319bp Reverse
GCAAGACCATGACTTAGAAATGT
G
Exon17
Forward CCATATGTCACCCTGGGTTC
418bp Reverse ATGAGGAATGCTAAATTTGTCC
Exon18
Forward ACCATTCAATCAAATCCTAGTCTC
474bp Reverse GTAGGCTCCCTCCAACCC
167
Appendix .8 cont.
Exon # Direction Sequence 5' - 3'
Product
size
Exon19
Forward AACATTTTGTAAAAGAGGGGTGAG
399bp Reverse ATCTTTGTGCGCTCCATCTG
Exon20
Forward TGGAAACAGACAGGAAACTGG
586bp Reverse TGTAGACAAACAAGTAGGGTTGTG
Exon21
Forward GGCAAGGTTTAAACAAGTAAAATG
421bp Reverse TCTTTTCAACCAAACCTAAACTTC
Exon22
Forward AATACTCAATTTCTTTATGGCACC
586bp Reverse GCTAGGACAGTTTGGAAACCAC
Exon23
Forward GCCAGCTAAGCTCAAGTTCC
375bp Reverse TGGCTATGTCATTATTTATCGGTC
Exon24
Forward AACGTGGGGAACAATAGAACC
390bp Reverse CTGAGCAAAAGTAAGACATGGG
Exon25
Forward ACTAAATGGCATGACTGCCC
558bp Reverse AAATTCAAACATGAATACAAATGC
Exon26
Forward TCCAAACTTACGGGCTTTTG
525bp Reverse TGCTTGATGATTTAACCAGACTTC
Exon27
Forward GTACATGGCCCAGCATGTC
423bp Reverse GCTTTCGTACAGGTGAGTCCC
Exon28
Forward GAGCTTTGGCAAGGAAACAG
1210bp Reverse TTTCTCAGTAAAGGGAGAGACATC
Exon29
Forward GGTTAAGTGCAGTCAAGCAGAC
443bp Reverse TCATGTGCCTGGGTTGTG
Exon30
Forward ATGGACATTCTTCCTTTGCC
473bp Reverse AAACCACCTATTTTATTCCAAAGC
Exon31
Forward TCTCATATGCCCTTTGAGTTTG
357bp Reverse AGCCACCGATTCTACAAAGC
Exon32
Forward GTTACACAAATGTGCCCTCG
473bp Reverse GCAATCAGATGAATAAAGGTGG
Exon33
Forward CACCTTGCACTGTGTGTTTC
429bp Reverse ATGGGTAGTTAGGCACACGG
Exon34
Forward GAAAAGTCAAAGCACATAGGGG
503bp Reverse ATAATAGAATCCCCAGGCCG
Exon35-36
Forward AGAGAGAACTGGACCCTGGAG
645bp Reverse TCATTTGAAGAGATTCAGAAATCC
Exon37
Forward AAGCCATTAAGAGAGGGGAAAC
358bp Reverse AACTTATGGGCCTTAAATCCG
Exon38 Forward AAGCATTTGGACTATAAAGCTTCC 409bp
Reverse TCAGTTGACAAACACTTCCTCC 409bp
168
Appendix 8 cont.
Exon39-40
Forward TGATGCCAAAATCTCTTACATCC
603bp Reverse TGAGTTGCTTTTGTCCTTTGC
Exon41
Forward AGTTCCCAGGAAAATGGACC
462bp Reverse TGATCTGCTTGAGAACAGAGG
Exon42
Forward TCCTGGAGATTGAGGAATGG
577bp Reverse TCACTCACTGACCATTGTTTCC
Exon43
Forward AAGCTGAAATGTTGAAAAGGC
403bp Reverse CTTGAAATCGCTCTGCTTCC
Exon44
Forward CTTTCGTTCCCCTGCATTAC
485bp Reverse TTGGGCAAGTCACTCAACTG
Exon45
Forward CCCTCACATTCTGCTGGC
490bp Reverse CAGACCTATCAGAGAGGCAGAG
Exon46
Forward AGGAAGCATTTCAAAGCAGG
451bp Reverse TCATACATGGCAATACCATTAGC
Exon47
Forward TGTGTTACACTTCTCCATGCAC
334bp Reverse AGGAAGCATGGGAGTGATTC
Exon48
Forward GCAGGTGTATTCTGGCAAGC
415bp Reverse CTTGATTTGCCCATTAAACTTC
Exon49
Forward GGTGACAGAGCAAGACCCTG
400bp Reverse GGCATAAGTCTGAGGTCCCC
Exon50
Forward TATGTCCTTCCCTCACCCAC
486bp Reverse CCACTGGAACATCTGAAAAGG
Exon52
Forward TCTGAAAATACAGGATGGATGTG
451bp Reverse AAAGCACTTTACCCACAAATACA
Exon53
Forward AAACCAGCTTGAAAGTTCCATTAC
488bp Reverse AAGGATTTTCTTTCCTGGGG
Exon54-55
Forward TGAAAAGCATTTGAAATCAGCTC
657bp Reverse CAAACTGTGAAGTCAGCAAAGC
Exon56
Forward CAGGAGTATGGGCTAGGCAC
466bp Reverse TGGCTGAAAATTTCTTTGGG
Exon57
Forward GGTCTTTGCAAAAGATGACCTC
430bp Reverse CAACAATAAATCGGTCAGTTCAAG
Exon58
Forward CCCACCATTGCACTCCAG
395bp Reverse GAATGTATTCTTGGTATCCCAGC
Exon59
Forward GAACATGCAGTTGCCATTTG
600bp Reverse GAATCCACCAGAATTCATTGC
169
Appendix 8. cont.
Exon # Direction Sequence 5' - 3'
Product
size
Exon60
Forward ATCCCTCAGGCTGGAGTTTC
384bp Reverse ACATTTGGGAAGCCCTTTTC
Exon61
Forward TCACTGGGCTTGTGACTAATTC
298bp Reverse TGTGACTGCTCCTTGTCCTC
Exon62
Forward AGTATAAAGATGATTTCCCAGCC
684bp Reverse AGTCTTCGTTCTGCCACCAC
Exon63
Forward AAGCAAATCTGAATTCCATGC
365bp Reverse TGTAAGCCAAAGTGCTCCTG
Exon64-65
Forward GGCTTTCAGGTAATCACCAAG
359bp Reverse ATTTCTTCAAGGCCAAAGGC
Exon51
Forward
TGAAAATTTGCTTCAAGTATTTTT
G
281bp Reverse
ATCTTGATTATATAAACAACCAT
ACATTAGC
170
Appendix 9. List of primers of NRXN1 copy number detection by qPCR.
Exon # Direction Sequence 5' - 3'
Product
size
Exon 2
Forward GTGAAGCTGGACGATGAGC
100bp Reverse AGCACACACCTCCGTTGAG
Exon 5
Forward CTGAAAGCAGAGTGGAAAAGG
111bp Reverse CATGCTCCCTTGTTTATTGC
Exon 7
Forward CTCAAAACCCCATTCAAAGC
85bp Reverse CCAGTGTGAAGCATCAGTCC
Exon 13
Forward TATGGCATTCTGATGGCAAC
87bp Reverse GACCGTCAGTTTCACACGTC
Exon 16
Forward AAAATCACAACGCAAATCACC
111bp Reverse TTTCCATGTGAAGGGAGACC
Exon 19
Forward CCCAGTACACGAGCAGACAG
96bp Reverse GTCACCCAAGCCTGAAGAAC
Exon 22
Forward CCATGCAATCAGAGATGTCC
109bp Reverse CTCACCTGGCTAATGGGTTC
Exon 24
Forward AGGCTCAGCAGAAGTGATCC
92bp Reverse GGAGGATAAGGATGCACAGG
171
Appendix 10. Selected homozygous and compound heterozygous variants for the
family MRQ14, primer sequences, product sizes and annealing temperatures.
Gene Exon Sequence 5’ – 3’ Product
size
NME7 3 F-TGTAAGGACTACTTTGCTGAAATG
167bp R-TGTGCTAAGTGCCACAGGAG
PPP1R9A 2 F-TCGGAAATTGTTGGATTGC
188bp R-TCTTGAATCCCTTACAAAAACTCC
DYM 11 F-AGGTTTTACCAATTCTTGAGATTC
181bp R-GGGACATGTCTGTTCTGTTAACTC
DLG1 8 F-TCTGAGCTGCTTTTCTTTTCC
212bp R-GCCAATTTTATGAACTGGGAAC
KMT2B
(MLL4) 3
F-TCTGGGGTAGAGGAGAAGATG 197bp
R-GCTGGAAAGTGTCCAAGGAG
EHMT2 11 F-CCTCTTTTCCTCCATCCAAG
171bp R-ACCTCTCCGTCCACACTCTC
SMEK1 15 F-AGCCCAAGTTTCAAGCTTTC
195bp R-GCAAAATGGGAGTCAAGAATG
PLCD4 13 F-GCTGTGGCTATGTGCTGAAG
203bp R-GCTATCCGGCCTCTACATTC
VAV2 29 F-GGTACAGAGCAGGGTTCTGG
394bp R-TGCAGAGGGTGTCTGGAAC
ZNF227 6 F-CAGAGAATTCACCCAGGAGAG
207bp R-TTCCTCGCATTTATAGGGTTTC
UGT8 2 F-GGAGATTGACAGCAATCGAAC
221bp R-ACCCACTTCAGCAGGATACC
NAA15 17 F-AAGGAGGCCTCTTATCCAATG
210bp R-ATACCGTCACCATCAACCAC
DNAH17 78 F-GTGGGCTCTTTGCAGTGAC
243bp R-TGACCCATGTTTACCTTCAGC
DNAH17 76 F-CTACCCGCTGTCATCCTTC
255bp R-GAGGCACGAGCCTTCATTAC
SACS 10 F-TTGAATCATTTGATGTCCCAAG
229bp R-CAGGTACACAACCAATCACCTC
SACS 10 F-GACCCAGCTGCTCTCTTTG
213bp R-GTCCTGGATTTCTGACAGC
172
Appendix 10. cont.
TEP1 24 F-CGCCTTCTTCAGGACACAG
162bp R-TGTGCCTTACCTCCTTAGCC
TEP1 12 F-TCATACAGCATTGCCCTTTC
170bp R-TCAGCTTCTCCCTCTTGAGC
F, forward primer; R, reverse primer
173
Appendix 11. Family MRQ11 selected homozygous and compound heterozygous
variants, primer sequences, product sizes and annealing temperatures.
Gene Exon Sequence 5' - 3' Product
size
SMOX 7 F-GTGCTGTTTTCCGGTGAG
177bp R-GGCTGGAGGTCACGAGTTAG
TAS1R2 3 F-TGCGCCAGAACTTCACTG
261bp R-TGGTGTTGAAGGACAAGGTG
ATP11A 2 F-TGGGCAAGAGCTTTTGATG
204bp R-CAGACCCAAGCCAAGTTACC
ADORA2B 2 F-CTGCTGCCTTGTGAAGTGTC
291bp R-GCTGGCTGGAAAAGAGTGAC
ZNF589 14 F-TATGTCTGCGGAGAGTGTGG
168bp R-CGATTTCTCCCTTGTGTGTG
ZNF502 4 F-TGGGAAAACATTTCGATGTC
179bp R-ATGCCTTCCCACACCTATTG
ADHFE1 10 F-AGGAATTGCCAATGTTGATG
174bp R-AAATGAAATCTCAGACTTGCTCAC
ADHFE1 13 F-GACTGAACTCCACCCAGAGC
161bp R-GGCCATCATCAACATCCAG
CMYA5 2 F-GAAGGTGCTGGCAGAAAAAC
206bp R-CACCTTTCTCCTTTTCAGAA
CMYA5 2 F-AGAAACACCGCCATATTTGC
208bp R-CTCCAAGTGGTGCTTTGAAA
DCHS1 6 F-AGGCCATGTACGGCTTATG
216bp R-ACCTGAGTTCCAGCAGTGG
DCHS1 2 F-CATCTTTCTCAGTGCAGATGG
245bp R-CTGTGGCTGTAACCCTCAAG
DPAGT1 7 F-TTCAAATAGTGGCCCAGTCA
219bp R-CCCCTTGGATCTGATGTAGG
DPAGT1 1 F-CTTGCCCGTTACCTGAAGAG
190bp R-TTGAGGTCCTGACCACAGAG
DENND2C 7 F-ACAGAGCAAGACCCCATCTC
238bp R-TTCGTGTTTGCAACTTCACC
F, forward primers; R, reverse primers
174
Appendix 12. Selected homozygous and compound heterozygous variants of family
MRQ15, primer sequences, product sizes and annealing temperatures.
Gene Exon Sequence5' - 3' Product
size
SF3B3 3 F-CTTAGCCTCCCGAGTAGCTG
273bp R-GCCTAAAGGCCATGAGTGAC
LARGE 3 F-AAGCCCGTGTCTCTGTCAC
231bp R-AAGGTTCTCGCTGTCTCCAG
HHAT 9 F-GCAGCCCTCTGTATGTCTCC
259bp R-AAGGATCCTGCTCACCAGAC
NEDD4 1 F-CCGGAGATGACTTGGATAGC
330bp R-TCAATTCATTATCGACCACAGC
PLCH1 IVS13+1C>T
F-AAAGCCCAGCTCATTCTCTG 157bp
R-CCCTCCTTGCCAGTACTCTG
PDS5B IVS4+1C>T
F-AGTCATTGTGGTGAACTGTTGG 214bp
R-ATGTGCCAAAAGGTGAATTG
WASF1 IVS25-1G>A
F-GTGCAATATTCAGTTTTATTTTGG 258bp
R-TGCTTCCTCTTTTCCTTCCTC
DENND2A 1 F-GAGTCAGGCTTCAGAAGGAGTC
231bp R-TTTCTTCCCTTCCCATTCTG
HMCN1 46 F-AGGCAACTAATATTTCCATTCC
182bp R-AGGCCACCCATCTTTGAAC
HMCN1 85 F-TGCAATCCTGAATTGTGAGG
192bp R-ACAGAGGGGTATTGGCTGTG
MED13L 10 F-GGCTTAAATGGGACGCTAAC
164bp R-ACTGCCACAGGGAAATCATC
MED13L 6 F-TGTAGTGAGCATTTGTCCTGTG
150bp R-TTGAAATGGTGCAGGTGAAG
ZNF772 5 F-CATATGTGGGAAAGTTTTTAAT
227bp R-GAACACTCCAATGTTTAATGAG
ZNF772 5 F-GCAACACCAAGTGTGAGGAG
235bp R-TTCCCACATATGCCACACTC
F, forward primer; R, reverse primer
175
Appendix 13. Selected homozygous and compound heterozygous variants of family
MRQ20, primer sequences, product sizes and annealing temperatures.
Gene
c.DNA
change Sequence 5' - 3'
Prod
size
(bp)
CEP97 811G>A
F-TAGTCAAGGCAAGGGGAGAG
236 R-AGCACTTGCCTCAGGCTTAC
FLII 2983T>A
F-AAGAAGGCGAGGAAGCAAC
240 R-CTAACCTTCGCTGGGTCTG
P2RY11 440G>T
F-TCTTCATCACCTGCATCAGC
231 R-CTGTCCCCAGACACTTGATG
ZBTB46 920A>T
F-CTCAGAGCAGAGTGCTGGTG
284 R-GAAAGCCACCAGTGTGAGC
IKBKAP 2026C>T
F-ATGGAGAGTGGCTTGTTGTG
229 R-GCTTGGTACTTGGCTGAATG
GCSH 103C>G
F-CTGGCCAATCAGACCCTG
550 R-GTCCTCCCGCTACTTAAGGG
ADAMT
S18 3290C>T
F-
AGGAAGAGGGAGATGAAGTGC
248bp R-AGAACGCCTCATTTTTCGAC
ADAMT
S18 2483G>C
F-GAAGCATCGAAATCCAGGAG
281bp R-TGGCTCAAATTGCAGTCAAC
INTS7 2025C>G
F-
TGGAATGACAGTGAAAAGATGG
479bp R-GCTTTGGATGCTCTTTCTGG
INTS7 1347C>T
F-TTTTCTTTTTGGCGTGCAG
244bp R-CTTGTCTGTGGCTGATCGTC
KIF13B 5462C>T
F-TAAGAATGACGGTTCCATCG
295bp R-CACCCCCAGAAAAGTTCG
KIF13B 1226A>G
F-GGCAATGAAATCTCCAGAGC
165bp R-ATTGTGCAAACTGCCATGAG
176
Appendix 14. List of primers for variants of the family PKMR71.
Gene
c.DNA
change Sequence 5' - 3'
Product
size
Cxorf58 c.766 G>A
F-CCTGGCCAACTTAATCGTTC
342bp R-TGACCTATCTGGCAGAATGTG
DGAT2L6 c.338 T>A
F-GAGTCCCACATCAGCCTATGGC
325bp R-CTCCCCTACCTTGGTTCTC
IGBP1
c.acceptor
T>A
F-GCTTCAGAGCCCACAGAAC
245bp R-GACAAAACCGGAAGAAGCAC
TCEANC c.382 T>A
F-AACTTGCTCTCTGGGACCAC
345bp R-GTTACCAGCACACCCACTTG
SLC10A3 c.478 C>T
F-CCTTCTCAGGACAACCCAAC
309bp R-TCACAGAGCCATCTCCAATG
177
Appendix 15. List of primers for the variants of the family PKMR176.
Gene
c.DNA
change Sequence 5'-3'
Product
size
CCDC22 c.628A>G
F-gggggTgACTTgTCTgTATACTC
289bp R-CTTCTCTgAgTgCgTgAAgC
MID2 c.A>G716
F-ggTTATgAAACTgAgAAAgTTgg
320bp R-gAgACAAgggTCCAgAAATCC
TMEM164 c.G>A298
F-TAACATTgTCTCACTCTgTCTgg
243bp R-CCTTTgggAAgTgCAAAgAC
178
Appendix 16. Homozygous regions obtained among the affected members of family
MRQ14 and MRQ11.
Family MRQ14
Chromosome Flanking SNP Boundaries (MBs) Size (MBs) Ranking
Chromosome 19 rs16966190;rs41334244 32,19-45,45 13.2 1
Chromosome 18 rs17573901;rs2155957 36,34-48,69 12.3 2
Chromosome 1 rs643927;rs4617427 91,66-99,18 7.5 3
Chromosome 4 rs1397933;rs11733857 40,18-40,93 0.7 4
Family MRQ11
Chromosome 13 rs973907;rs7322187 91,72-105,1 13.3 1
Chromosome 3 rs3774389;rs2236975 42,26-50,48 8.2 2
Chromosome 2 rs10185886;rs6755276 26,07-33,94 7.8 3
Chromosome 3 rs3774589;rs7636357 53,81-60,71 6.3 4
SNP, single nucleotide polymorphism; MBs, mega bases
179
Appendix 17. List of families their homozygous regions obtained from Homozygosity
Mapper and candidate genes screened among them.
Family name Homozygous region Gene sequenced
MRQ1 Multiple regions homozygous No gene sequenced
MRQ2 No region > 1MB No gene sequenced
MRQ5 Chr12: 45,045-47,106 NTF3
MRQ11
Chr2: 26,077-33,941
Chr3: 53,817-60,714
Chr3:42,257-50,483
Chr13: 91,725-10,5116
Chr17: 79,002-32,232
No gene sequenced
MRQ12 Chr18:13,829-34,299 RBBP8, NRXN1
MRQ14
Chr1:16,673-99,180
Chr18:36,340-48,697
Chr19:32,196-45,458
No gene sequenced
MRQ15 No region>1MB No gene sequenced
MRQ16 No array performed
HPRT1 was sequenced (due to
phenotype match with Lesch Nyhan
Syndrome)
MRQ17 Chr7:95,492-10,398 RELN
MRQ18 No array performed
TPO was sequenced (due to
phenotype match with
hypothyroidism)
MRQ19
Chr12: 75,812-78,662
Chr12 :85,203-92,855
Chr16: 56,846-70,993
BBS2, BBS10, BBS14
MRQ20 No region>1MB No gene sequenced
180
Published and submitted Data
181
1. Agha, Z., Iqbal, Z., Azam, M., Hoefsloot, LH., van Bokhoven, H., Qamar, R.
(2013). A novel homozygous 10 nucleotide deletion in BBS10 causes Bardet–Biedl
syndrome in a Pakistani family. Gene. 519:171-181.
2. Agha Z, Iqbal Z, Azam M, Zweier C, Siddique M, de Leeuw N, Qamar R, van
Bokhoven H. (2014). A complex microcephaly syndrome in a Pakistani family
associated with a novel missense mutation in RBBP8 and a heterozygous deletion in
the NRXN1 gene. Gene. doi.org10.1016/J.gene.2014.01.027.
3.Agha, Z., Iqbal, Z., Azam, A., Ayub, H., Vissers, L., Gilissen, C., Benish, S.H.,
Riaz, M., Veltman, J., Pfundt, R., van Bokhoven, H. and Qamar, R. (2014) Exome
sequencing identifies three novel candidate genes implicated in intellectual
disability.(Submitted European Journal of Human Genetics).
4. Agha, Z., Iqbal, Z., Yntema, HG., Kleefstra, T., Zweier, C., Leeuw, N., Qamar,
R., van Bokhoven H., Willemsen, MH. A de novo microdeletion in NRXN1
responsible for Microcephaly syndrome in Dutch patient. (under preparation ).
