1. M Keramatipour1 Mohammad Keramatipour MD, PhD Keramatipour@tums.ac.ir Genetic Analysis in Polycystic Kidney Diseases (PKD): ADPKD, ARPKD

2. M Keramatipour Overview of the Lecture Introducing to Polycystic Kidney Diseases Introducing to Polycystic Kidney Diseases  ADPKD  ARPKD Genetic analysis: focus on new technologies Genetic analysis: focus on new technologies  Sanger sequencing  Next generation sequencing … 2

3. M Keramatipour Polycystic Kidney Diseases Genetic disorders characterized by development of multiple abnormal cysts, typically in both kidneys Genetic disorders characterized by development of multiple abnormal cysts, typically in both kidneys In human / some other animal In human / some other animal > 600 000 affected in US, among both sexes & all races > 600 000 affected in US, among both sexes & all races Cause of ~ 10% end-stage Cause of ~ 10% end-stage Renal disease Two main forms: Two main forms:  ADPKD  ARPKd 3

4. M Keramatipour Genetics of ARPKD Gene: PKHD1 – Ch: 6, 67 exons Gene: PKHD1 – Ch: 6, 67 exons An autosomal recessive disease An autosomal recessive disease 4

5. M Keramatipour Autosomal Dominant PKD Epidemiology: Epidemiology:  Frequency of 1/500 to 1/1000  3 rd cause of ESRD worldwide (4 th in Iran)  50% of patients require renal transplantation by the age 50 – 60 years Presentation: variable phenotype Presentation: variable phenotype  Abdominal pain  Hematuria  Hypertension  Cysts in other organs  ……. 5

6. M Keramatipour Pathophysiology of ADPKD A ciliopathy A ciliopathy Two proteins can be defective Two proteins can be defective  Polycystin 1  Polycystin 2 Major roles in: Major roles in:  Ion channels  Intracellular calcium homoeostasis  Cell to cell or cell to matrix interactions  Cell cycling & differentiation 6

7. M Keramatipour Genetics of ADPKD An autosomal dominant inherited diseases An autosomal dominant inherited diseases 7

8. M Keramatipour Causative Genes Two known genes Two known genes PKD1 gene encodes polycystin 1 PKD1 gene encodes polycystin 1  16p13.3 PKD2 encodes polycystin 2 PKD2 encodes polycystin 2  4q22.1 8

9. M Keramatipour PKD1 Gene Large gene: 46 exons, 12912 bp coding sequence Large gene: 46 exons, 12912 bp coding sequence Intra-chromosomal duplications Intra-chromosomal duplications 6 pseudogenes (PKD1P1-6) – 97 – 99% homology 6 pseudogenes (PKD1P1-6) – 97 – 99% homology 13 to 16 Mb proximal to the gene 13 to 16 Mb proximal to the gene High GC content High GC content No hot spot / mutations No hot spot / mutations Allelic heterogeneity Allelic heterogeneity Unclassified variants Unclassified variants 9

10. M Keramatipour PKD2 Gene 15 exons, 2904 bp coding sequences 15 exons, 2904 bp coding sequences 10

11. M Keramatipour Genetic Testing for ADPKD Two main approaches: Two main approaches:  Direct genetic analysis  Indirect genetic analysis  Followed by direct analysis Direct genetic analysis Direct genetic analysis  Standard PCR & Sanger sequencing: PKD2  Long range / nested PCR & Sanger sequencing: PKD1  Next generation sequencing: PKD1 & PKD2 11

12. M Keramatipour Sanger vs. NGS 12 PCR, Sanger sequencing Target region capture/exome + NGS

13. M Keramatipour Workflow of Genetic Analysis 13

14. M Keramatipour Indirect Genetic Analysis of ADPKD Using STR markers to test the linkage in the family Using STR markers to test the linkage in the family Multiplex PCR & Fragment analysis Multiplex PCR & Fragment analysis Assigning genotypes Assigning genotypes Linkage analysis Linkage analysis 14

15. M Keramatipour Sample Experiment 15

16. M Keramatipour Indirect Testing Linkage analysis: EasyLinkage Softeware Linkage analysis: EasyLinkage Softeware 16

17. M Keramatipour Analysis of PKD1 Gene Long-range PCR / Nested PCR & Sanger Sequencing Long-range PCR / Nested PCR & Sanger Sequencing 17

18. M Keramatipour 15 Families Analyzed 18

19. M Keramatipour Following Linkage Analysis 15 Families were investigated: 15 Families were investigated:  Confirmed linkage to PKD1 in 9 families (60%)  Confirmed linkage to PKD2 in 2 families (~ 14%)  Linkage to both loci was excluded in one family  Inconclusive results in 3 families (20%) Direct PCR and Sanger sequencing Direct PCR and Sanger sequencing Next generation sequencing: three families Next generation sequencing: three families 19

20. M Keramatipour 20 No ESRD until 82 years Youngest patient:15y c.2359-2373del23 p.E787Vfs E1 3- F E13 -R PKD-IR-05

21. M Keramatipour 21 PKD-IR-12 PKD2, c.180_181delGC Age of ESRD in the pedigree: ~70 Extra renal complications Age of onset: 45 Age of ESRD: 53 Blood pressure No other complication PKD- IR-07 PKD2, c.556C>T (p.R186X)

22. M Keramatipour 22 Predictive toolsp.Q61Gfs*30p.E787Vfs Mutation tasterDisease causing Cadd score2733 TypeNull allele SegregationYes Population Frequency No Variant Interpretatioin

23. M Keramatipour 23 PKD1: c.7702A>G, p. R2568G Age of ESRD in the pedigree: ~50 Youngest patient: 2Y NGS analysis PKD-IR-03

24. M Keramatipour Summary of the Results 24 Relative contributions in pathogenesis Reports:Ours: PKD1: 70-85% PKD1: 67% PKD2: 15-35% PKD2: 33% Pedigree specific mutation > 70% 100% Novel mutations ? ~up to 50% 66%

25. M Keramatipour Conclusion Different types of new mutations in PKD1 & PKD2 genes Different types of new mutations in PKD1 & PKD2 genes Novel mutations are very common in Iranian patients Novel mutations are very common in Iranian patients Long-range PCR and Sanger Sequencing is the choice method for analyzing PKD1 gene Long-range PCR and Sanger Sequencing is the choice method for analyzing PKD1 gene PKD2 gene can be analyzed by Sanger as well as NGS PKD2 gene can be analyzed by Sanger as well as NGS Choice method for analysis of PKHD1 gene (ARPKD) is NGS Choice method for analysis of PKHD1 gene (ARPKD) is NGS 25

26. M Keramatipour Acknowledgement Contributors Contributors  Dr M Entezam  Dr M Ayati  Dr M R Khatami  Dr Saddati  Dr Rozbeh And mainly: patients and their families And mainly: patients and their families Pishgam Biotech Company Pishgam Biotech Company 26

27. M Keramatipour Thank you for listening, any comments? 27