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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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

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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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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