Genetics in Pediatric Nephrology. S Alexander J Fletcher Children s Hospital at Westmead National Kidney Transplant Institute

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1 Genetics in Pediatric Nephrology S Alexander J Fletcher Children s Hospital at Westmead National Kidney Transplant Institute

2 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 4 To understand the ways of investigating novel renal disease

3 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

4 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 coinheritance 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

5 What is a Chromosome

6 Genes mrna Protein

7 What is a Gene

8 What is a Mutation

9 Inheritance- Polygenic vs Monogenic

10 What is Linkage

11 How Do You Find The Right Gene (Muhle et. al. Paediatrics 2007)

12 X-linked Recessive Inheritance

13 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

14 Genetic Renal Disease (%) A Genetic Diagnosis As The Underlying Aetiology For Paediatric Renal Transplantation

15 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 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 1976 AD 8q13.3 Juvenile Nephronophthisis 1975 AR NPHP1 2q13 (2001-4) NPHP2 9q31.1 NPHP3 3q22.1 NPHP4 1p36.1 NPHP5 3q13.3

16 Disease Category Disease Year Disease Inheritance Gene Loci HUS D(-) HUS 1975 AR 1q32 (1997) Obstructive Nephropathy PUV Triad Syndrome Unknown Sporadic > Familial Vascular Thrombosis Renal Artery Stenosis Polygenic Polygenic 1q23 (Factor V ) 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 AD AD AD AR 16p13.3 (1994) 4q21-23 (1996)? 6p21.1-p12 (1994) Systemic Disease SLE IDDM Polygenic Polygenic SLEN1-3 10q22.3; 2q34-35;11p15.6 (2002) 17q23 Pyelonephritis 2001 Polygenic Toll-like receptor (9q32-q33 TLR4) IL-8 Deficiency

17 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

18 Minimal Change NS CAKUT Alports Syndrome Lupus Nephritis FSGS ADPKD Gitelman Syndrome Juvenile NPHP VACTERL SRNS ARPKD Cystinosis HUS (Atypical) Bartter Syndrome TRIAD Congenital Nephrotic BOR Syndrome Renal Coloboma Denys-Drash Nail-Patella Syndrome Dent Disease Number Genetic Renal Disease in Paediatrics (ANZPNA) Australia

19 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

20 Genetic Renal Disease Large number of the diseases we treat are familial Renal disease: AD AR ADPCKD Renal Coloboma Syndrome Congenital Nephrotic Syndrome Nephronopthisis X Linked Alports Disease

21 Genetics Monozygous-single gene AR AD X linked Polygenic-multiple genes Lupus Modifier Genes

22 Important Genetic Disorders PKD Nephrotic Syndrome Alports Nephronophthisis Tubular Disorders

23 AD 1:800-1:1000 PKD1 85% PKD2 15% Bimodal Early Onset childhood Adult ADPKD

24 Presentation Hypertension Renal Mass Renal Failure Occasionally hematuria Family History Associated Features Biliary Cysts Pancreatic Cysts Hernias Male Infertility Mitral Valve prolapse, inguinal hernias ADPKD

25 ARPKD

26 ARPKD Incidence 1:20000 PKHD1 Pathology Fusiform Dilatation of the CDs Biliary dysgenesis and fibrosis

27 ARPKD

28 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

29 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 Mutation Types (Mutation Number) Extrarenal Protein Name Splice site, frame-shifts (insertions and deletions), substitutions; nonsense and missense (PKD1-266)(PKD2-63) Hepatic, Cerebral Aneurysms, Cardiac valve AbN, HT Polycystin-1 Polycystin-2 Frameshift, non-sense, splicing alterations; compound heterozygote (PKHD1 111) Hepatic (Biliary, Congenital Hepatic fibrosis, PHT); SHT 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)

30 Proteins Involved in PKD (Igarashi and Somlo JASN 2002)

31 Models of Polycystin-1 and Polycystin-2 Signaling ORPK Mouse - Polaris (intraflagellar transport) CPK recessive Mouse Cystin (primary apical cilia) PKD 1 and PKD 2 Polycystin 1 and 2 (Igarashi and Somlo JASN 2002)

32 Gene Exons Sequence 23 Exons Full gene 67 exons Diagnosis 85% Testing ARPKD

33 Telomere D6S272 D6S465 D6S1714 D6S2010 D6S1344 D6S1623 Centromere PKHD1 Gene D6S436 A B A B A B A A A A A B A B A B A B A A A A A B 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

34 Imaging in PKD

35 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

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40 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

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42 GBM in Alport syndrome

43 Type IV collagen a1(iv) a 6(IV) chains 3 networks - (a1) 2 a2 and a3a4a5 and (a5) 2 a6 COL4A1 COL4A6 at 13q, 2q and Xq

44 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

45 Transepithelial Salt Resorption in a Cell of the Thick Ascending Limb of the Loop of Henle. NEJM :1281

46 46 Chromosomes

47 Chromosomes 1-22 Genome

48 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

49 Single X Men

50 In the coding sequence SNPs Coding is in triplets correspond to an AA or a stop codon Human genome mutation every 300 bases on average Single nucleotide polymorphisms SNPs

51 SNPs

52 Linkage Dysequilibrium

53 Genome Wide Association Studies Large numbers of affected patients and looking for linkage to genes

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55 In AR Disease Homozygosity Mapping 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

56 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.

57 Comparative Genomic Hybridisation

58 Whole Genome Sequencing Whole Genome Sequencing Exome Sequencing 1000 Genome Project

59 Human Genome Project Sequenced the Human Genome Available in UCSC Browser Information about genes syndromes OMIM On Line Mendelian Inheritance in Man

60 Gene Phenotype Syndrome OMIM Data on the gene, clinical features, genetics, animal models NIH Regularly updated

61 Sample Genome Viewer Image, TP53 Region base position UCSC genes RefSeq genes MGC clones mrnas & ESTs 28 species compared single species compared SNPs repeats

62 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