Post on 03-Nov-2020
Genetics in Pediatric Nephrology
S Alexander J Fletcher
Children’s Hospital at Westmead
National Kidney Transplant Institute
OBJECTIVES
To understand the basis of inheritance of genetic
diseases and the different underlying mutations
To recognise common genetic conditions found in
nephrology including PKD, nephrotic syndrome, Alports and tubular disorders
To be aware of the common databases of genetic
information
To understand the ways of investigating novel renal
disease
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2
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Genetic Definitions
Genome the entire complement of genetic material in the full set of chromosomes
Gene a unit of hereditary material of ordered sequence that encodes a product
Exon the coding sequence of a gene
Allele any one of the alternative forms of a given gene
Heterozygous
Homozygous
Definitions Continued
Sequence the order of nucleotide bases in a DNA molecule
Mutation a structural alteration in the DNA that is permanent often leading to abnormal/absent product
Haploinsufficiency a single copy of the normal gene is incapable of providing sufficient protein production as to assure normal function
Single Nucleotide Polymorphism a variation in which a single nucleotide (A,C,T,G) in the sequence is altered from the base in its usual position.
Microsatellite repetitive segments of DNA with enough variation between individuals that its inheritance and co-inheritance with alleles of a given gene can be traced
Linkage Analysis Using polymorphisms (normal variants) or microsatellites that are near or within a gene of interest to track within a family the inheritance of a disease-causing mutation in that gene
What is a Chromosome
Genes
mRNA
Protein
What is a Gene
What is a Mutation
Inheritance- Polygenic vs Monogenic
What is Linkage
How Do You Find The “Right” Gene
(Muhle et. al. Paediatrics 2007)
X-linked Recessive Inheritance
Genetic Diversity in Renal Disease
Mendelian ARPKD ADPKD Nephronophthisis Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)
Chromosomal Associated abnormalities of the Kidney Trisomy 13, 18 Turners Syndrome 45XO – 60% renal malformation
Polygenic SLE IDDM
Mitochondrial Renal Syndromes in Mitochondrial cytopathies
Single Nucleotide Polymorphisms - Association studies Cytokine single nucleotide polymorphims and allograft outcomes ACE and ACE-R polymorphisms and hypertension/CRF
A Genetic Diagnosis As The Underlying
Aetiology For Paediatric Renal Transplantation
0
20
40
60
80
100
Gen
eti
c R
en
al
Dis
ease (
%)
2
4552
86
1963-1972 1973-1982 1983-1992 1993-2004
Renal Disease Categories, Disease Inheritance and Gene Loci Linked to Each Disease
Disease Category
Disease Year Disease
Inheritance
Gene Loci
Glomerulonephritis Idiopathic NS FSGS 1
FSGS 2
FSGS 3
IgA Nephropathy
1995
1995
1999
2003
2000
AR 1q25-q31 (1995)
19q13 (1998)
11q21-q22 (1999)
Chr 6 (2003)
1q31-q42; 6q22-q23 (2000)
Reflux Nephropathy VUR 1973/75 ?AD
Polygenic
1p13 (2000)
Renal Dysplasia BOR
Juvenile Nephronophthisis
1976
1975
AD
AR
8q13.3
NPHP1 – 2q13 (2001-4)
NPHP2 – 9q31.1
NPHP3 – 3q22.1
NPHP4 – 1p36.1
NPHP5 – 3q13.3
Disease Category
Disease Year Disease
Inheritance
Gene Loci
HUS D(-) HUS 1975 AR 1q32 (1997)
Obstructive Nephropathy
PUV
Triad Syndrome
1983
2000
-
-
Unknown
Sporadic > Familial
Vascular Thrombosis
Renal Artery Stenosis
2000
1999
Polygenic
Polygenic
1q23 (Factor V - 1988)
17q23 (ACE Polymorphism)
Metabolic Cystinosis 1952 AR 17p13 (1995)
Drug Toxicity CypP450 (3A5, 3A4) 2004 Polygenic 7q22.1 (1994)
Polycystic Kidney Disease
PKD1
PKD2
PKD3
ARPKD
1961
1996
1997
1994
AD
AD
AD
AR
16p13.3 (1994)
4q21-23 (1996)
?
6p21.1-p12 (1994)
Systemic Disease SLE
IDDM
2002
1996
Polygenic
Polygenic
SLEN1-3 10q22.3; 2q34-35;11p15.6 (2002)
17q23
Pyelonephritis 2001 Polygenic Toll-like receptor
(9q32-q33 –TLR4)
IL-8 Deficiency
Genetic Factors in ESRD
Linkage at several loci Satko et al. KI (2005)
Ch 3q (Type 1 and 2 DN);
Chr 10q (DN and Non-Diabetic Nephropathy);
Chr 18q (Type 2 DN)
3-9 increase risk of ESRD if relatives have ESRD
FHx ESRD more predictive for development of CRI then level of BP
and glucose control (Freedman JASN 1997; Petteitt Diabetilogica 1990)
Familial clustering
Population based screening in US and Singapore
Lebanon (Barbari et al Mol. Immunol 2003)
26% HD pts were offspring of consanguineous marriages (CM)
35% pts CM early onset ESRD vs 21% non CM
Genetic Renal Disease in Paediatrics
(ANZPNA) Australia
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50
100
150
200
250
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2122242528
58121517
Num
ber
IDENTIFICATION OF GENETIC DISEASE
Known Genes
Directly (small genes)
Specific disease causing mutations
Indirectly (large genes)
Linkage with DNA Microsatellites
Linkage with SNP’s
Unknown Genes
Large families
Linkage
Genetic Renal Disease
• Large number of the diseases we treat are
familial
• Renal disease:
• AD ADPCKD
Renal Coloboma Syndrome
• AR Congenital Nephrotic Syndrome
Nephronopthisis
• X Linked Alports Disease
Genetics
• Monozygous-single gene
• AR
• AD
• X linked
• Polygenic-multiple genes
• Lupus
• Modifier Genes
Important Genetic Disorders
PKD
Nephrotic Syndrome
Alports
Nephronophthisis
Tubular Disorders
ADPKD
• AD
• 1:800-1:1000
• PKD1 85%
• PKD2 15%
• Bimodal
• Early Onset childhood
• Adult
ADPKD
• Presentation
• Hypertension
• Renal Mass
• Renal Failure
• Occasionally hematuria
• Family History
• Associated Features
• Biliary Cysts Pancreatic
Cysts Hernias
• Male Infertility
• Mitral Valve prolapse ,
inguinal hernias
ARPKD
ARPKD
• Incidence 1:20000
• PKHD1
• Pathology Fusiform Dilatation of the CDs
• Biliary dysgenesis and fibrosis
ARPKD
Cystic Disease in Childhood
• Many conditions where cysts are part of a larger
spectrum of disease
• When isolated to the kidney
• Large Kidneys ARPKD
• Echogenic ARPKD over ADPKD
• Cysts AD>AR
• Stones AR>AD
• Pelvicalyectasis AR>AD
• Liver Large Echogenic AR>AD
Adeeva 2006
Polycystic Kidney Disease
ADPKD ARPKD
Inheritance AD – Variable phenotype AR
Incidence 1/500 – 1/1000 1/6000 – 1/40000
Age of Onset ESRD PKD 1 -53yr; PKD 2 – 69yr Infancy
Location of Cysts All nephron segments Collecting ducts
Gene PKD 1 (Chr 16p13.3) – 85%
PKD2 (Chr 4q21-23) – 15%
PKHD1 (Chr 6p21.1)
Exon Number PKD 1 – 46; PKD 2 - 15 86
Mutation Types
(Mutation Number)
Splice site, frame-shifts (insertions and
deletions), substitutions; nonsense and
missense
(PKD1 - 266)(PKD2 - 63)
Frameshift, non-sense, splicing alterations;
compound heterozygote
(PKHD1 – 111)
Extrarenal Hepatic, Cerebral Aneurysms,
Cardiac valve AbN, HT
Hepatic (Biliary, Congenital Hepatic
fibrosis, PHT); SHT
Protein Name Polycystin-1
Polycystin-2
Fibrocystin/Polyductin
?cell surface receptor, secreted protein
Protein Size P1 – 4302aa; P2 – 968aa 4074 aa
Protein Function Ion Transport, Cell Adhesion, Modulating Ca
Entry, Mediates mechanosensation in
Primary Cilia
Unknown
(Igarashi and Somlo JASN 2002)
Proteins Involved in PKD
(Igarashi and Somlo JASN 2002)
Models of Polycystin-1 and Polycystin-2
Signaling
(Igarashi and Somlo JASN 2002)
ORPK Mouse - Polaris (intraflagellar transport)
CPK recessive Mouse – Cystin (primary apical cilia)
PKD 1 and PKD 2 – Polycystin 1 and 2
Testing ARPKD
• Gene
• Exons
• Sequence 23 Exons
• Full gene 67 exons
• Diagnosis 85%
Telomere Centromere
D6S272 D6S465 D6S1714 D6S2010 D6S1344 D6S1623
PKHD1 Gene
26/09/2005
D6S 272
D6S 465
D6S1714
D6S2010
D6S1344
D6S1623
A B
A B
A B
A A
A A
A B
A B
A B
A B
A A
A A
A B
A B
A B
A B
A A
A A
A B
A B
A B
A B
A A
A A
A B
A B
A B
A B
A A
A A
A B
D6S436
Imaging in PKD
Congenital Nephrotic Syndrome
• Rare
• 50% associated with gene mutations
• Commonest
• AR Finnish Nephrotic Syndrome Nephrin
• AR podocin
• AR Pierson’s Sd associated eye changes
• AD Dennys Drash Syndrome WT-1 defects
Alport syndrome
One in 50,000
XL 85%, AR 15%, AD – rare
Haematuria, renal failure, hearing loss,
lenticonus, retinopathy
Lamellated GBM
Due to mutations in COL4A5 (XL) or
COL4A3/COL4A4 (AR, AD)
GBM lacks a3(IV), a4(IV), a5(IV) chains
GBM in Alport syndrome
Type IV collagen
a1(IV) – a 6(IV) chains
3 networks - (a1)2a2 and a3a4a5 and (a5)2a6
COL4A1 – COL4A6 at 13q, 2q and Xq
Tubular disorders: Bartters and Gitelmanns
Bartter’s AR
Chloride wasting disorder of TAL Henle
Present with systemic alkalosis
hypercalciuria and nephrocalcinosis
Severe neonatal form
Gitelmans AR
Milder
Hypokalemia poor growth and cramps
Hypomagnesemia
Defect is in the distal tubule
Transepithelial Salt Resorption in a Cell of the
Thick Ascending Limb of the Loop of Henle.
NEJM 2004 350:1281
46 Chromosomes
Genome
• Chromosomes 1-22
Women
• X1
• X2
• 1 Is inactivated in each cell on a random basis
• Some women having a higher number of affected cells
• Lyonisation
• Alports Disease 10% women affected
Men
• Single X
SNPs
• In the coding sequence
• Coding is in triplets correspond to an AA or a
stop codon
• Human genome mutation every 300 bases on
average
• Single nucleotide polymorphisms
• SNPs
SNPs
Linkage Dysequilibrium
Genome Wide Association Studies
Large numbers of affected patients and looking for linkage to genes
Homozygosity Mapping
• In AR Disease
• Affected patients in consanguineous families will
inherit the same DNA at the site of the mutation
• Markers along the genome the same pair will be
inherited at sites of mutation in affected
individuals
• Parents and unaffected members will not have
pairs at this site
Copy Number Variation
A copy number variant (CNV) is a segment of DNA in which differences of copy-number (number of copies of a molecule or portions of it) have been found by comparison of two or more genomes.
Size one kilobase to several megabases in size.
Humans (being diploid) ordinarily have two copies of each autosomal region, one per chromosome.
Altered due to deletion or duplication.
Comparative Genomic Hybridisation
Whole Genome Sequencing
Whole Genome
Sequencing
Exome
Sequencing
1000 Genome
Project
Human Genome Project
• Sequenced the Human Genome
• Available in UCSC Browser
• Information about genes syndromes
• OMIM On Line Mendelian Inheritance in Man
OMIM
Gene
Phenotype
Syndrome
Data on the gene, clinical features, genetics,
animal models
NIH
Regularly updated
http://www.ncbi.nlm.nih.gov/sites/entrez
Sample Genome Viewer Image, TP53 Region
base position
UCSC genes
RefSeq genes
mRNAs & ESTs
repeats
28 species compared
SNPs
single species compared
MGC clones
Summary
• Increasing number of renal diseases particularly in the
young are genetic
• Key to take a family history
• Look for non-renal manifestations which suggest an
associated syndrome; eye examination
• Within known genes there can be significant variability
• Unknown diseases with a reasonable number of affected
individuals it is possible to identify the cause
• Increasingly this will affect outcomes and treatment