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Designing a Comprehensive Stepwise Diagnostic Approach to Iranian Patients with Mental

Retardation, by Determining the Molecular Profile of DNA Copy Number Variations

Tehran University of Medical SciencesDepartment of Medical Genetics

Presented for PhD Degree

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Presented by:Maryam Rafati

Supervised by:SR Ghaffari MSc MD PhD

Tehran University of Medical SciencesDepartment of Medical Genetics

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Advisors:ZT Keihanidoust MD, MR Eshraghian PhD

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Main Goal

Determining the genetic causes of “Familial MR”

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Main Questions

Is there any differences between “Sporadic” and “Familial” MR regarding their underlying genetic causes?

Should a different diagnostic approach be adopted for “Familial” rather than “Sporadic” MR?

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Outline

Introduction Materials and method Results Discussion

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Introduction

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Higher Cerebral Development Dysfunction

Mental retardation

Cerebral palsy Abnormal motor actions and postural mechanisms Non-progressive abnormalities of the developing brain limited, stereotypic, and uncoordinated voluntary movements

Autism A behaviorally defined syndrome characterized by

▪ Atypical social interaction▪ Disordered verbal and nonverbal communication▪ Restricted areas of interest▪ Limited imaginative play▪ A need for sameness

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Mental retardation

Mental retardation is a serious and lifelong disability that places heavy demands on society and the health system

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American Association on Mental Retardation, 1992

“ mental retardation is not something you have, like blue eyes or a bad heart, nor is it something you are, like short or thin. It is not a medical disorder or a mental disorder… mental retardation reflects the “ fit “ between the capabilities of individuals and the structure and expectations of their environment. “

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Definition

MR is accepted as having three components:

1) Significantly abnormal intellectual performance, generally determined by a test of intelligence

2) Onset during development before the age of 183) Impairment of the ability to adapt to the environment

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Global developmental delay

Reserved for children five years of age or younger

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Global developmental delay

Global developmental delay (DD) describes significant delay in two or more of the following areas:

Cognition Speech/language Gross/fine motor skills Social/personal skills Daily living

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Prevalence

Prevalence: 1% - 3%

Mild MR occurring 7-10 times more frequently than moderate or severe MR. Mild MR: 29.8/1000 Mod-severe MR: 3.8/1000

In Iranian population: 1.8 – 2.7%

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Why diagnosis?

Estimating the recurrence risk in future pregnancies Prenatal diagnosis Minimizing the number of diagnostic procedures Short-term and long-term prognosis Treatment options

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Recurrence risk

Variable depending on the etiology

From very low ( the same as normal population) to 50% and even in rare situations to 75 -100%

Irrespective of etiology, empiric risk: 8.4%

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Recurrence Risks for Severe MR

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Study Brothers Sisters All SibsMale index caseHerbst and Baird (1982)

1 in 12 1 in 33 1 in 18

Bundey et al. (1985)

1 in 10 1 in 20 1 in 13

Female index caseHerbst and Baird (1982)

1 in 22 1 in 17 1 in 19

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Classification

Severity:

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• (IQ : 50-70)Mild

• (IQ : 35-50)Moderate

• (IQ : 20-35) Severe

• (IQ < 20)Profound

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Classification

Pedigree analysis:

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Sporadic

Familial

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Etiology

Genetic

Non-genetic

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Prenatal and perinatal events

Infections

Environmental factors

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Genetic causes of sporadic MR

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Genetic causes of sporadic MR

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33%

3%9%

1%

55%

Chromosomal abnormalitiesFragile-X syndromeX-linked MRInborn errors of metabolismDe novo dominant mutations

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Genetic causes of sporadic MR

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9%

24%

3%9%1%

55%

Microscopic aberrationsSubmicroscopic aberrationsFragile-X syndromeX-linked MRInborn errors of metabolismDe novo dominant mutations

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Submicroscopic Cromosomal Abnormalities

Subtelomeric Rearrangements• 0.5-15%• Unselected patients: 5%

Common Microdeletion and Microduplication (CMMSs) Syndromes• 5.8-9.5%

Genomic Copy Number Variations (CNVs)• 10-17%

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Diagnostic approach to sporadic MR

Guidelines based on the assessment of1. Chromosomal abnormalities

▪ Microscopic▪ Submicroscopic

2. Fragile-X syndrome

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Diagnostic approach to MR

2005 (Van Karnebeek et al.) For all the patients:

1. Family history, neurologic and dysmorphologic exam2. Karyotype3. Assessment of Fragile-X syndrome

For highly selected patients:1. Investigation of Subtelomeric aberrations (FISH) 2. Investigation of common microdeletion and

microduplication syndromes (CMMSs)

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Diagnostic approach to MR

Many studies during 2005-2010

1. Karyotype2. Assessment of Fragile-X syndrome3. Assessment of DNA copy number differences

(Array-CGH, MLPA, …)

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Diagnostic approach to MR

2009 (Hochstenbach et al.), retrospective investigation of 36325 patients karyotype

2010 (Miller et al.), analysis of array testing on 21698 patients

Conclusion:chromosomal microarray is a first-tier clinical

diagnostic test

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Diagnostic Methods

Karyotype Assessment of fragile-X syndrome FISH MLPA Array-based techniques

Array-CGH SNP Array

Exome sequencing Next-generation sequencing

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Karyotype

The first technique for studying chromosomal abnormalities

Advantages: Genomic Detection of balanced abnormalities

Disadvantages: Low resolution (3-5 Mb) Labor intensive

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FISH

The first molecular cytogenetic technique

Advantages: Higher resolution

Disadvantages: Limited tergets

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MLPA

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MLPA

Advantages: Investigation of 40-50 genomic regions in a single reaction Quantitative results (carrier detection) Detection of duplications High-throughput Universal primers Relatively inexpensive

Disadvantages: Inability to detect

▪ Balanced abnormalities▪ Low-level mosaicism▪ Novel DNA copy number differences

Limited genomic targets

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Array-CGH

BAC-array CGH Oligonucleotide

array-CGH

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SNP array

Advantages: Very high resolution

(>1000000 probes) Detection of LOH

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Next-generation sequencing

Promising technique in detecting novel genetic changes (CNVs, single gene disorders)

Technique of choice in near future

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Materials and Methods

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Overview of the study design

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Sporadic Patients

Clinical screening

Laboratory investigati

ons

Familial patients

Clinical screening

QuestionnaireScoring

Laboratory investigatio

ns of selected families

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Sporadic patients

Inclusion criteria: Developmental delay (DD)or Mental retardation (MR)or Multiple congenital anomalies (MCA)

Exclusion criteria: Known inborn errors of metabolism Documented non-genetic causes of DD/MR

▪ Neonatal uncontrolled icter▪ Perinatal events

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Familial patients

Inclusion criteria: The presence of at least two individuals in the first

degree relatives affected with DD/MR/MCA

Exclusion criteria: Families with known genetic causes

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UNIQUE SAMPLE SET

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Sampling

Place: Imam Khomeini Hospital

Duration: 1386 (Aban) -1390 (Mehr)

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Screening phase

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Completing the questionnaire

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Data extraction

Drawing pedigree based on the questionnaires data Data entry to the Progeny

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ScoringPhase 1

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Scoring criteria

Number of affected individuals in siblings(example: 4 affected persons 1+2+3+4=10)

Affected parents: each of them 2 scores

Prenatal onset (sonographic positive findings)

Multiple organ involvements: each one 1

Non-consanguinity of parents: 1

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Laboratory phase

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Genetic counseling

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Drawing pedigreePast medical history

PrenatalPerinatalpostnatal

Recording past medical history in detail

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Pedigree drawn real time

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Pedigree drawn by progeny

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Informed consent signed before sampling by patients and their guardians

Informed concent

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Physical examination

Measurement of height and head circumference

Neurologic exam Dysmorphologic exam

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Photography

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Example: accessory nipples in 3 affected individuals in a family

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Sampling

Heparinized Blood EDTA Blood

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Designing patients database using Progeny software

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Stepwise approach to MR

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Karyotype

Assessment of Fragile-X syndrome

Assessment of subtelomeric rearrangements (MLPA)

Assessment of CMMSs (MLPA)

Scoring

Array-CGH

SNP Array Rafati M

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Karyotype

G-Banding: Lymphocyte culture Harvest Slide preparation Giemsa staining

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Fragile-X syndrome

PCR screening: Determining CGG repeat expansion of FMR1 gene

Triplet-primed PCR: Determining pre-mutations and full mutations

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MLPA optimization

Step 1: Detection of common chromosomal aneuploidies

Step 2: Assessment of deletions and duplications of F8 gene in

severe hamophilia A patients

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Subtelomeric rearrangements

Techniques: MLPA

▪ Using two alternative probe mixes▪ Family study to rule out polymorphisms

FISH (in case of positive findings)

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CMMSs

Technique:

MLPA Using 1 screening and 2 confirmatory probe mixes

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Investigated Syndromes:

Cri du Chat syndrome MECP2 / Xq28 duplication

1p36 deletion syndrome

DiGeorge syndrome 22q Rubinstein-Taybi syndrome

2p16 microdeletion

DiGeorge region 2 Smith-Magenis syndrome 3q29 microdeletion

Langer-Giedion syndrome Sotos syndrome 5q35.3 9q22.3 microdeletion

Miller-Dieker syndrome WAGR syndrome 15q24 deletion syndrome

NF1 microdeletion syndrome

Williams syndrome 17q21 microdeletion

Prader-Willi / Angelman Wolf-Hirschhorn 4p16.3 22q13 / Phelan-Mcdermid

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Scoring Phase 2

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Scoring criteria

Number of affected individuals1+2+3+…

Non-consanguinity +3

Recurrent abortion +2

Pedigree analysis (Dominant) +3

Multiple congenital anomalies +3

Severity of MR Moderate +2Severe

+3

Affected parents (each of them) +2

Dysmorphism+2

Negative scores

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Number of normal siblings -1-2-3…

Consanguinity-3

Pedigree analysis (Recessive) -3

Pedigree analysis (X-linked) -3

•Positive scores

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Selection

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array-CGHRafati M

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Array-based techniques

Array-CGH Agilent 44K EmArray Cyto6000

SNP Array Illumina humanOmni 1-Quad 1134514 markers Resolution: 6 kbp

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Results

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Screening phase

Number of families Number of patients

Sporadic patients 279 279

Familial patients

• First step 551 1260

• Second step (questionnaire) 355 647

Total 830 1539

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Laboratory phase

Number of families Number of patients

Sporadic 55 55

Familial 116 371

Total 171 426

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Screening phasesporadic patients

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Screening phasesporadic patients

Diagnosis Number of patients Diagnostic method

Chromosomal abnormalities Numerical 23 karyotype

Structural 9 karyotype

• Mosaicism 4 karyotype

Syndromes DiGeorge Syndrome 2 FISH

Miller-Dieker Syndrome 1 Clinical and paraclinical findings

Rubinstein-Taybi Syndrome 1 Clinical and paraclinical findings

Kabuki Syndrome 1 Clinical and paraclinical findings

Hallermann-Streiff Syndrome 1 Clinical and paraclinical findings

Laurence-Moon-Bardet-Biedl Syndrome

1 Clinical and paraclinical findings

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Continued

Single gene disorders Phenylketonuria (PKU) 3 Measurement of Phenylalanine level

Methylmalonic Acidemia (MMA) 1 MS/MS

Aminoacids HPLC Maple Syrup Urine Disease

(MSUD)2

GM2 Gangliosidosis (Tay-Sachs Disease)

2 Enzyme activity assessment of Hexosaminidase A

GM2 Gangliosidosis (Sandhoff Disease)

1 assessment of Hexosaminidase A and B Enzyme activity

Niemenn-Pick Disease 1 Mucopolysaccharidosis type I

(MPS1)1

Krabbe Disease 1 Whole gene sequencing

Non-ketotic Hyperglycinemia (Glycin Encephalopathy)

1 Whole gene sequencing

Metachromatic Leukodystrophy 1 assessment of arylsulfatase Enzyme activity

Saposine-B Deficiency 1 Whole gene sequencing

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Screening PhaseFamilial Patients

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Number of affected individuals in the first degree relatives

2 3 4 5 6 8 12

49.40%

30.20%

10.60%

4.70%

1.70%0.40%

0.00%

36.30% 36.30%

17.50%

5.00%

2.00%2.00%

0.98%

Screening phase

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Laboratory Phase Findings

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MLPA optimization

STEP 1: Prenatal diagnosis of chromosomal aneuploidies As a stand-alone test:

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74 amniotic fluid samples

5 Trisomy 21

1 XXY

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MLPA optimization

STEP 2:

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347 Previously Investigated Severe

Hemophilia A Patients

10 Patients No PCR amplification in

some exons

Confirmation of deletionsDetermining the deletion

extensionCarrier detection

1 patient with no mutation detected

Duplication of exon

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MLPA

Optimization

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Sporadic MRGenetic test Abnormal finding Number (%)Karyotype Trisomy 21 4 (7.2%)

Partial deletion of 9p telomeric region

1 (1.8%)

Fragile-X syndrome CGG repeat expansion 1 (1.8%)Subtelomeric rearrangements

Duplication of 2p telomeric region

1 (1.8%)

CMMSs Williams syndrome 1 (1.8%)Cat Eye syndrome 1 (1.8%)

Cytogenetically visible chromosomal abnormalities

5 (9%)

Cryptic chromosomal abnormalities

3 (5.4%)

Fragile-X syndrome 1 (1.8%)Total 9 (16.3%)/55

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Sporadic Williams syndrome

Summary of clinical findings: Global Developmental delay Mental retardation Facial dysmorphic features

▪ Micrognathia ▪ Midface hypoplasia▪ Wide mouth▪ Prominent upper and lower lips▪ Short philtrum▪ Full nose tip▪ Periorbital fullness

Non-facial dysmorphic features▪ Additional skin creases of the fingers

Personality▪ Outgoing overfriendly personality

Investigations with normal results Karyotype Visual status

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Dysmorphic features

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MLPA analysis

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Cat Eye syndrome

Summary of clinical findings: Developmental delay Hypotonia Facial dysmorphic features

▪ Dolichocephaly ▪ Cleft palate▪ Low-set ears▪ External ears deformity

Failure to thrive (FTT) Congenital Heart Disease (CHD)

Large ASD with left to right shunt Bilateral inguinal hernia

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MLPA analysis

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Patient resultsInvestigated genes using 4

MLPA probemixes Genes Genomic regions

Gain P070 IL17RA

Cat eye syndrome region

- SLC25A18Gain P036 BID

- MICAL3- USP18- LCR22-A

Gain P064 CLTCL1

commonly-

deleted DiGeorge region

DiGeorge region

- HIRAGain P064 CDC45Gain P245 / P064 CLDN5Gain P245 GP1BB

- TBX1- TXNRD2- DGCR8

Gain P064 ARVCFLCR22-B

- ZNF74Gain P064 KLHL22

- MED15LCR22-C

Normal P245 / P064 SNAP29

Not Investigated

- LZTR1- LCR22-D- HIC2- PPIL2- TOP3B

LCR22-E- RTDR1- GNAZ- RTDR1- RAB36

LCR22-F- SMARCB1

LCR22-G- SNRPD3

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Laboratory PhaseFamilial Patients

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Karyotype

All normal Inversion of chromosome 2 (normal variation)

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Fragile-X Syndrome

Description Number (%)

Diagnosed families 8 (6.9%)

Diagnosed patients 23

Males 21 (91.3%)

Females 2 (8.7%)

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Fragile-X syndrome

Control (unaffected) Control (affected) Contrlo (unaffected) MR-63 (No Band) MR-35 (No Band) Control (unaffected) Control negative (No DNA) Size marker (100-1000bp)

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1 2 3 4 5 6 7 8 1 2 3 4 5 6

•Control (affected)•MR-69-MO (Mother)•MR-69-S2M (Normal male)•MR-69-P2M (Affected male)•Size marker (100-1000bp)•Control negative (No DNA)

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Pedigree

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Subtelomeric Rearrangements

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Family ID MLPA probemix Results Type of aberrations

MR-92P036

P070

Partial monosomy of 13qter and partial trisomy of 9pter

Clinically significant pathogenic changes

MR-51P036

P070Xpter and Xqter Gain

MR-130P036

P0701pter Gain

Aberrations with unknown clinical significance

MR-130P036

P07012qter Gain

MR-1 P070 3pter loss

Genomic variants

MR-1 P070 4qter Gain

MR-61 P070 4qter Gain

MR-105 P070 4qter Gain

MR-109 P036 21q11.2 Gain

MR-116 P070 4qter loss (homozygous)Rafati M

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Unbalanced Subtelomeric Rearrangement

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Pedigree

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Family history

Non-consanguineous healthy parents History of one abortion at 12 weeks of gestation Two infantile deaths due to Multiple Congenital

Anomalies (MCA) One affected child with developmental delay/mental

retardation (DD/MR)

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Clinical findings

Second pregnancy:

Cleft lip/cleft palate Intestinal atresia Death at 2 months of age Failure to thrive (FTT)

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Clinical findings

Third pregnancy: (A 12-year old girl )

At birth: Wt: 2850 gr HC: 33 cm Microcephaly Poor sucking (feeding difficulties) Facial Dysmorphism:

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Facial and non-facial dysmorphic features

▪ Hypertelorism ▪ Ptosis ▪ Epicanthal fold▪ Low-set ears▪ Broad nasal bridge▪ Long forehead

▪ Broad distance between 1st and 2nd toes

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Clinical findings

Developmental delay/mental retardation Intractable seizure (onset: 3.5 y) Behavioral disorder Autistic features

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Previous genetic tests

Karyotype: 46,XX

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Family history

Fourth pregnancy: Prenatal:

▪ IUGR▪ Choanal atresia▪ Polyhydramnios▪ Intestinal obstruction

Prenatal diagnosis (Cordocentesis): karyotype: normal

Postnatal: Club foot Duodenal atresia Death at 3 months of age

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Assessment of subtelomeric rearrangements

Technique: MLPA Result:

• Deletion of 13q telomeric region

• Duplication of 9p telomeric region

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FISH analysis

Confirmatory test: FISH Result:

• Partial monosomy of 13qter

• Partial trisomy of 9pter

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9pter

13qter

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Investigation of the Parents

Technique: Metaphase FISH

Mother: normal

Father: balanced translocation of 13q and 9p telomeric regions

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9pter

13qter

Chr 9

Chr 13

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Assessment of CMMSs

A familiy with hereditary Williams syndrome Others: no deletion or duplication detected

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Hereditary Williams Syndrome

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Patient 1 (the proband)

Clinical findings: ▪ Developmental delay (most prominently in walking)▪ Mental retardation▪ Growth retardation

Dysmorphic features: ▪ Microcephaly▪ Micrognathia▪ Midface hypoplasia▪ Wide mouth▪ Prominent lower lip▪ Long philtrum▪ Strabismus

Personality: outgiong overfriendly personality

Echocardigraphy Pulmonary stenosis 2/15/2012

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Patient 2 (the father)

Summary of clinical and paraclinical findings At age of 37-year:

Ht: 165.5 cm (<5th centile) HC: 53.5 cm (<5th centile)

Dysmorphic features: Micrognathia Strabismus Periorbital fullness Long philtrum

No hypertension (blood pressure: 125/80 mmHg) Echocardiography:

Mild aortic stenosis Tricuspid valva regurgitation Mitral valave regurgitation Mitral valve prolapse

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MLPA analysis

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Williams syndrome critical region

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Hereditary Williams syndrome

Reference Number of Reported Families (Cases)

Method of WBS Ascertainment

Molecular Confirmation

TechniqueMorris et al. (1993) 3 (6) Clinical manifestations Quantitative southern

analysis (Ewart et al (1993) confirmed the deletion in two of the

families]Sadler et al. (1993) 1 (2) Clinical manifestations -

Ounap et al. (1999) 1 (2) Clinical manifestations FISH1 (just the son was investigated, the mother

was not available)

Pankau et al. (2001) 2 (4) Clinical manifestations FISH (both of the patients)

Metcalfe et al. (2005) 1 (2) Clinical manifestations FISH (both of the patients)

The present study 1(2) Screening for cryptic chromosomal abnormalities

MLPA2 ((both of the patients))

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Findings of array-based techniques

Clinically significant findings: Related to familial MR: 0 Unrelated to familial MR: 1

▪ Charcot-Marie-Tooth

Benign copy number changes: 3 families

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Familial MR and CMT

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Array Result

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Array result

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case a CGH ISCN nomenclature # probesSize

loss(bp) Size gaine (bp) Clinical target

MR-82 arr chg 6P25.3 (204528-284802)X1 6 80274  Benign CNC

  arr chg 8P23.1 (7040596-8117330)X1 6 1076762  Benign CNC

  arr chg 10q26.3 (135104029-135227522)X3 6  123,494<500Kb, CYP2E1 gene, SYCE1 gene

  arr chg 16p11.2-11.1 (34339543-34584850)X3 5  245,308Benign CNC

 

arr chg 17p12 (14052497-15382791)X3 38 1,330,295

CNC with significance pathogenic effect,

     

COX10, HS3ST3B1, PMP22 Genes

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Family study

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Loss of heterozygosity (LOH)

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1 p31.3 p31.1 q12 q32.1 q41 q43 q44

Base Position 25,624,620 50,471,950 75,319,280 100,166,610 125,013,940 149,861,270 174,708,600 199,555,930 224,403,260 249,250,590

Cytogenetic Band p3 p3p3p3p3 p… p p32.p3 p31.1 p2p2pp21pp21p13 p1 p p1 q12 q21q2q21 q q23 q2q2 q…q31q3q31q32.1 q3q3 q41 q q43 q44Sequence (+) No sequence data file found for this chromosome.

LOC643837LOC643837LOC643837LOC643837LOC643837LOC643837AK091100FAM41C KIAA1922

IGSF21

BC040588BC131526CAMK2N1CAMK2N1CAMK2N1MUL1 ARCH

ZBTB8OSC1orf94

RRAGCRRAGCRRAGCRRAGCMYCBP

AKR1A1

ELAVL4DMRTA2

TMEM48

YIPF1YIPF1YIPF1YIPF1

NFIA

AK3L1

SGIP1SGIP1SGIP1SGIP1

SGIP1

PIN1LBC041341

IFI44L

KIAA0786LPHN2LPHN2LPHN2

LPHN2

U80773HFM1

CDC7

PTBP2PTBLPPTBP2

PTBP2

DPYD

LPPR4

BC043293

PRMT6

BC053344RBM15RBM15RBM15

BC069739

SLC16A1 CR936796FLJ00310DQ579288DQ586768BC053679BC071797BC029473DQ596563

TAGLN2

CS1SLAMF7SLAMF7

F11R

F11RF11R

FCER1G

FMO3

TNFSF18

TNFSF18TNFSF4

RFWD2

PAPPA2PAPPA2SEC16B

RGL1

FAM5C

FAM5CDBCCR1L

RGS18

RGS21RGS21

KCNT2

KCNT2

LRRN2

LRRN2SLC26A9

FAM72AFAM72A

CD46sytXIV

C1orf133

TGFB2

TGFB2SLC30A10

DUSP10DUSP10

DQ584993

TLR5

PARP1

SIPA1L2

SIPA1L2

BC016972RYR2RYR2

LOC339535

FMN2

FHOR2M7

OR2M7

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Pedigree

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Discussion

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Outline

Summary of results

Comparison of “Familial” MR with “Sporadic” MR

Comparison of the current study with the previous investigations of “Familial” MR

Diagnostic algorithms

Suggestions

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Sporadic MR

0.00%4.00%8.00%

12.00%16.00%

Sporadic MR (current study)Sporadic MR (previous stud-ies)

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Summary of results (familial MR)

0%1%2%3%4%5%6%7%

Familial MR (current study)

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“Familial” versus “Sporadic” MR

371 Patients from 116 Families

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131

Karyotype

Sporadic MR: 9.5% Present study: 0% (371

patients from 116 families)

Sporadic MR

Familial MR

0.00%

2.00%

4.00%

6.00%

8.00%

10.00%

Cytogenetically vis-ible abnormalities

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Fragile-X syndrome

Sporadic MR: 2-5% (mean: 3%)

Present study: 7% (371 patients from 116 families)

Sporadic MR

Familial MR0%1%2%3%4%5%6%7%

Fragile-X syndrome

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Subtelomeric aberrations

Sporadic MR: 0.5-9% (mean: 5%)

Present study: 0.9% (248 patients from 108 families)

Sporadic MR

Familial MR0%

1%

2%

3%

4%

5%

Subtelomeric Rearrangements

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Previous subtelomeric investigations

Reports in support of the importance of “positive family history” (most of the studies)

Reports with conflicting data

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Positive family history, as a predicting factor?

Positive family history is the main single predicting factor of subtelomeric rearrangements

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Conflicting data in the previous studies

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The current study

Our results could be compared with the prevalence of hereditary subtelomeric aberrations (0-2%)

The present study finding:Severity of MR, dysmorphism and multiple congenital

anomalies are more important than positive family history by alone

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CMMSs

Sporadic MR: 5.8-9% Present study: 0.9% (245

patients from 107 families)

More coverage of common syndromes in this study (18 CMMSs)

Sporadic MR

Familial MR0%

1%

2%

3%

4%

5%

CMMSs

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CMMSs

Mechanisms: De novo: misalignment during recombination due to LCRs Hereditary:

▪ Balanced translocation in the parents▪ Autosomal dominant transmissions

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Array-based techniques

Sporadic MR: 10-17%

The findings were not explanatory for “familial” mental retardation

Sporadic MR

Familial MR

0%2%4%6%8%

10%12%14%16%18%

Genomic CNVs

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144

Interpretation

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“Familial” versus “Sporadic” MR

Karyo

type

Fragil

e-X Sy

ndrom

e

Subte

lomeri

c cha

nges

CMMSs

Other g

enom

ic CNCs

048

1216

Familial MRSporadic MR

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Familial versus Sporadic MR

Sporadic MR

Familial MR0.00%5.00%

10.00%15.00%20.00%25.00%30.00%35.00%40.00%

Chromosomal Abnormali-tiesFragile-X syndrome

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Interpretation

2011, Jehee et al.

Karyotype + MLPA investigations of subtelomeric rearrangements and CMMSs = detection of 76.5% of all chromosomal abnormalities detected by whole genomic array screening

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148

Conclusion

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No pathogenic

CNV is expected in

the remained families

More coverage on CMMSs

KaryotypeMLPA,

subtelomeric aberrations

MLPA, CMMSs75.6% No pathogenic

change in array-CGH study of highly selected

patients

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Low contribution of chromosomal abnormalities to “Familial” MR

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Autosomal dominant single gene disorders

2009-2011, Hamdan et al. Investigation of 197 synaptic genes (glutamate receptor, …)

in 95 patients: 11 new mutations found

2011, Nature, Vissere et al. Exome sequencing of 10 patients with sporadic MR: 6

pathogenic mutations found (60%) More than all of the previous investigations

New paradigm of de novo dominant mutations in MR

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New classification

Sporadic MR Familial

MR

0.00%10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%

Chromosomal Abnormali-tiesFragile-X syndromeSingle gene disorders

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Comparison with the previous studies

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Previous investigations of Familial MR

Few published studies Studies on Iranian population

Autosomal recessive MR (2011, Najmabadi et al.) X-linked MR

A. R. Pouya et al. Fragile X syndrome screening of families with consanguineous and non-consanguineous parents in the Iranian population. European Journal of Medical Genetics (2009), doi:10.1016/j.ejmg.2009.03.014

Najmabadi H et al. Homozygosity mapping in consanguineous families reveals extreme heterogeneity of non-syndromic autosomal recessive mental retardation and identifies 8 novel gene loci. Hum Genet. 2007 Mar;121(1):43-8. Epub 2006 Nov 21

Kahrizi K et al. An autosomal recessive syndrome of severe mental retardation, cataract, coloboma and kyphosis maps to the pericentromeric region of chromosome 4. Eur J Hum Genet. 2009 Jan;17(1):125-8. Epub 2008 Sep 10.2/15/2012

154

Parents Relationship

55%

15%

6%

24%

First CousinSecond CousinFar relatedNon-con-san-guineous

76%

24%

Parents Rela-tionship

ConsanguineousNon-consanguineous

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Pedigree analysis

78%

5%17%

Pedigree analysisAutosomal Recessive Established X-linkedPutative X-linked

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Sample Set Characteristics

40%

60%

Selected families

ConsanguineousNon-consanguineous

39%

49%

6%2% 4%

Screening phaseNon-consanguinousFirst CousinFirst Cousin, Once removedSecond CousinFar related

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Pedigree analysisthe present study

66%

24%

10%

Chromosomal or Autosomal Dominant

Autosomal Recessive

X-linked

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Comparison of studied sample sets

Autosomal Recessive X-Linked

Autosomal Dominant

0%10%20%30%40%50%60%70%80%

The present studyNajmabadi et al.

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Diagnostic Algorithms

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Sporadic MR

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Karyotype

Numerical or Structural Chromosomal Abnormality Normal

Assessment of Fragile-X syndrome

Fragile-X syndrome No CGG repeat expansion

Availability of array-based techniques + cost

Aavailable

array-CGH orSNP array

Pathogenic Copy Number

Variation

No pathogenic

change

Exome Sequencing (de novo dominant mutations)

New Suggestion

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Karyotype

Numerical or Structural Chromosomal Abnormality Normal

Assessment of Fragile-X syndrome

Fragile-X syndrome No CGG repeat expansion

Availability of array-based techniques + cost

Aavailable

array-CGH orSNP array

Pathogenic Copy Number

Variation

No pathogenic

change

Exome Sequencing (de novo dominant mutations)

Not Available

MLPA (Subtelomeric Rearrangements + CMMSs)

Subtelomeric Aberration

CMMSs

No pathogenic

change

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Familial MR

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Pedigree

Analysis

Autosomal Recessive

Assessment of Known

single gene disorders

Mutation Detected

No Mutation Detected

Exome Sequencing

Next-generation Sequencing

X-linked

Assessment of Fragile-X Syndrome

Fragile-X

Syndrome

No CGG repeat

Expansion

Assessment of Known XLMR

Mutation Detected

No Mutation Detected

Exome Sequencing

Next-generation Sequencing

Autosomal Dominant

DysmorphismMultiple Congenital

AnomaliesRecurrent Abortion/Infertility

Yes

Karyotype

Numerical or Structural

AbnormalitiesNormal

Availability of array-based techniques +

cost

Aavailable

array-CGH or

SNP array

Pathogenic Copy Number

VariationNo pathogenic

change

Exome Sequencing (de novo dominant

mutations)

Not Available

MLPA (Subtelomeric Rearrangeme

nts + CMMSs)

Subtelomeric Aberration

CMMSs

No pathogenic change

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Pedigree

Analysis

Autosomal Recessive

Assessment of Known single gene disorders

Mutation Detected

No Mutation Detected

Exome Sequencing

Next-generation Sequencing

X-linked

Assessment of Fragile-X Syndrome

Fragile-X

Syndrome

No CGG repeat

Expansion

Assessment of Known XLMR

Mutation Detected

No Mutation Detected

Exome Sequencing

Next-generation Sequencing

Autosomal Dominant

DysmorphismMultiple Congenital

AnomaliesRecurrent Abortion/Infertility

Yes

Karyotype

Numerical or Structural

AbnormalitiesNormal

Availability of array-based techniques +

cost

Aavailable

array-CGH or

SNP array

Pathogenic Copy Number

VariationNo pathogenic

change

Exome Sequencing (de novo dominant

mutations)

Not Available

MLPA (Subtelomeric Rearrangeme

nts + CMMSs)

Subtelomeric Aberration

CMMSs

No pathogenic change

No

Exome Sequencing

Next-generation sequencing

Unknown

Assessment of Fragile-X

syndrome

Fragile-X

syndrome

No CGG repeat

expansion

New Suggestion

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List

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Acknowledgements

Comprehensive Genetic Center, Imam Khomeini Hospital

Department of Medical Genetics, TUMS Hope Generation Foundation Tehran Welfare Organization Gene clinic

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Thanks for Your Attention

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