Whitehead Institute for Biomedical Research

1. Exome sequencing and disease-network analysis Whitehead Institute for Biomedical Research Yaniv Erlich Yaniv Erlich

2. Two projects with neurological diseases • Joubert syndrome in Ashkenazi Jews • Hereditary Spastic Paraparesis in Palestinians yaniv@wi.mit.edu

3. What is Joubert Syndrome? • Cerebello-oculo-renal phenotype: • - Hypoplasia of the cerabellar vermis • - Pyschomotor retardation and hyptonia • - Extra digits in upper and lower limbs (sometimes) • - Lazy eye • - Renal insufficiency • Molar Tooth Sign in MRI: Thick and elongated superior cerebellar peduncles Joubert Normal Large Interpeduncular fossa Cerebellum Parisi, 2007 Exome sequencing and disease network yaniv@wi.mit.edu

4. The cases • Dor-Yeshorim identifies 13 cases in Jewish Ashkenazi families • 8 families, where 3 are part of the same clan. • Autosomal recessive pattern • Founder effect Exome sequencing and disease network yaniv@wi.mit.edu

5. The starting point • 9 genes are known to cause Joubert syndrome in other populations • Sequencing those genes revealed normal results • Social implication… • Using autozygoustity mapping Hadassah found a strong signal from the centromeric region of chromosome 11. Chr 11 Hundreds of exons… Exome sequencing and disease network yaniv@wi.mit.edu

6. Finding the mutation – our approach • Sequence the entire exome of a healthy mother and an affected daughter. • Finding mutations that are: • - Heterozygous in the mother • - Homozygous in the child • - Not in dbSNP • - Causing a change in the coding region • Two lanes 36x2 per specimen • Hadassah sequenced the exons in the region according to a prioritized list of candidate exons. Exome sequencing and disease network yaniv@wi.mit.edu

7. Technical details Exome sequencing and disease network yaniv@wi.mit.edu

8. Analysis of variations Exome sequencing and disease network yaniv@wi.mit.edu

9. And the winner is… TMEM216 Chromosome 11 Amino-acid change: Arg -> Leu (CGC>CTC) Exome sequencing and disease network yaniv@wi.mit.edu

10. Who are you TMEM216? • Transmembrane protein • 88 amino-acid • Not a single paper on that gene • Conservation analysis: Exome sequencing and disease network yaniv@wi.mit.edu

11. Additional lines of evidence • PolyPhen predicated as damaging • Hadassah found the same mutation (double blind) • - Found in all 13 cases and parents found as carriers. From: Elpeleg Orly Sent: Sun 12/6/2009 11:56 PM To: Erlich, Yaniv Subject: RE: Preliminary analysis BINGO ________________________________________ From: Erlich, Yaniv [erlich@cshl.edu] Sent: Sun 12/6/2009 23:32 To: Elpeleg Orly Subject: Preliminary analysis Hi Orly, The only potential homozygous SNP mutation we found on chr11 between 59.5M-62M that is not in dbSNP and has an affect on the protein (missense, nonsense, splice) is TMEM216 Arg12->Leu (chr11:60918013). Are we right? Thanks, Yaniv Exome sequencing and disease network yaniv@wi.mit.edu

12. Summary • We found that TMEM216 is the causative mutation of Joubert syndrome in Ashkenazi Jews. • The project took 2 months end-to-end in our side. • We used only 4 lanes of Illumina GAII with paired-end 36nt reads. • Israel Ministry of Health added the mutation to their test panel. • Carrier rate in Ashkenazi Jews is 1:92 Exome sequencing and disease network yaniv@wi.mit.edu

13. Two projects with neurological diseases • Joubert syndrome in Ashkenazi Jews • Hereditary Spastic Paraparesis in Palestinians yaniv@wi.mit.edu

14. Hereditary Spastic Paraperesis • A single Palestinianfamily: • 3 brothers suffers from progressive weakness of the legs and abnormal gait • Phenotype is HSP – degradation of the pyramidal tract. • 20 genes have been documented (Weber J,2003) Yaniv Erlich

15. Thoughts on the Joubert rejection process • Currently, the rejection process is based on two classes of arguments: • Genetic arguments • Loss of function arguments Genetic arguments Loss-of-function arguments Genetic arguments have weaker power with smaller families We need a new class of arguments Exome sequencing and disease network yaniv@wi.mit.edu

16. Disease-network analysis Idea: Genes with a similar phenotype have a similar biological signature Zippi Brownstein, 2009 Method: Stratify variations according to their similarity to known disease genes. Exome sequencing and disease network yaniv@wi.mit.edu

17. Data gathering stage Whole exome seq. with Illumina Whole genome genotyping with Affy 250K array Yaniv Erlich

18. Results • 20 known genes that cause HSP were found intact • None of the 40,000 disease causing mutations in HGMD were found in the exome sequencing data. • X-linked mutation was ruled out. • Genotyping identified 4 regions of homozygosity: Conclusion: it is a new gene Yaniv Erlich

19. Exclusion process Total Sequenced Genes Genes Position Position Now what? Yaniv Erlich

20. Disease network analysis rank KIF1A as the top candidate Yaniv Erlich

21. Validation by loss of function analysis Total Sequenced Genes Genes Position Position Yaniv Erlich

22. Supporting evidence • Sanger sequencing confirmed the mutation in third affected child. 4 healthy brothers were not homozygous for the mutation • KIF1A is a kinesin. Phenotype is neuronal. • The region of KIF1A was suspected to cause HSP by a previous study with multiple Algerian family. • Conservation suggests a functional domain in KIF1A Yaniv Erlich

23. Summary • Identifying a new gene for spastic paraperesis. • Using only a single family and a new class of arguments • The rate of the mutation is 1:200 in Palestinians. Exome sequencing and disease network yaniv@wi.mit.edu

24. Erlich Lab Whitehead Institute Positions available (yaniv@wi.mit.edu) yaniv@wi.mit.edu