whitehead institute for biomedical research
DESCRIPTION
Exome sequencing and disease-network analysis. Whitehead Institute for Biomedical Research. Yaniv Erlich. Two projects with neurological diseases. Joubert syndrome in Ashkenazi Jews Hereditary Spastic Paraparesis in Palestinians. [email protected]. What is Joubert Syndrome?. - PowerPoint PPT PresentationTRANSCRIPT
4/20/11 Yaniv ErlichDNA Sudoku
Yaniv Erlich
Exome sequencing and disease-network analysis
Whitehead Institute for Biomedical ResearchWhitehead Institute for Biomedical Research
Two projects with neurological diseases
1. Joubert syndrome in Ashkenazi Jews
2. Hereditary Spastic Paraparesis in Palestinians
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:
[email protected] sequencing and disease network
Parisi, 2007
Normal Joubert
Cerebellum
Thick and elongated superior cerebellar
peduncles
Large Interpeduncular
fossa
Intro. Our approach Results Conclusion
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
[email protected] sequencing and disease network
Intro. Our approach Results Conclusion
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.
[email protected] sequencing and disease network
Chr 11
Hundreds of exons…
Intro. Our approach Results Conclusion
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.
[email protected] sequencing and disease network
Intro. Our approach Results Conclusion
Technical details
[email protected] sequencing and disease network
Step Tool Comments
Sequencing Illumina Mother: ~67,400,000 single end-reads
Child: ~73,600,000 single end-reads
Alignment Bowtie - Default parameters -> poor results. (only 1% of reads were mapped).
- Iterative mapping
Total unique mapper:
Mother – 51 million (76%)
Child – 54 million (74%)
SNP calling SOAPsnp Only regions with x5 coverage or more
Intro. Our approach Results Conclusion
Analysis of variations
[email protected] sequencing and disease network
SNPs in: #
Mother 49,515
Child 48,142
That are shard… 23,986
And are heterozygous in mother and
homozygous in child… 2,541
And not in dbSNP 105
And are not synonymous 39
And are in the mapped locus 1
Intro. Our approach Results Conclusion
And the winner is… TMEM216
[email protected] sequencing and disease network
Amino-acid change: Arg -> Leu
(CGC>CTC)
Chromosome 11
Intro. Our approach Results Conclusion
Who are you TMEM216?
[email protected] sequencing and disease network
• Transmembrane protein
• 88 amino-acid
• Not a single paper on that gene
• Conservation analysis:
Intro. Our approach Results Conclusion
Additional lines of evidence
[email protected] sequencing and disease network
• PolyPhen predicated as damaging
•Hadassah found the same mutation (double blind)
- Found in all 13 cases and parents found as carriers.
Intro. Our approach Results Conclusion
From: Elpeleg OrlySent: Sun 12/6/2009 11:56 PMTo: Erlich, YanivSubject: RE: Preliminary analysis
BINGO________________________________________From: Erlich, Yaniv [[email protected]] Sent: Sun 12/6/2009 23:32To: Elpeleg OrlySubject: 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) isTMEM216 Arg12->Leu (chr11:60918013).
Are we right?Thanks,Yaniv
Summary
[email protected] sequencing and disease network
• 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
Intro. Our approach Results Conclusion
Two projects with neurological diseases
Intro. Our approach Results Conclusion
1. Joubert syndrome in Ashkenazi Jews
2. Hereditary Spastic Paraparesis in Palestinians
Hereditary Spastic Paraperesis
4/20/11 Yaniv ErlichExome sequencing and disease network
-A single Palestinian family:
-
- 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)
Intro. The problem Our approach Results Conclusion
Thoughts on the Joubert rejection process
[email protected] sequencing and disease network
SNPs in: #
Mother 49,515
Child 48,142
That are shard… 23,986
And are heterozygous in mother and
homozygous in child… 2,541
And not in dbSNP 105
And are not synonymous 39
And are in the mapped locus 1
Currently, the rejection process is based on two classes of arguments:1.Genetic arguments2.Loss of function arguments
Genetic arguments
Loss-of-function arguments
Genetic arguments have weaker power with smaller familiesWe need a new class of arguments
Intro. The problem Our approach Results Conclusion
Disease-network analysis
[email protected] sequencing and disease network
Idea: Genes with a similar phenotype have a similar biological signature
Method: Stratify variations according to their similarity to known disease genes.
Zippi Brownstein, 2009
Intro. The problem Our approach Results Conclusion
Data gathering stage
4/20/11 Yaniv ErlichExome sequencing and disease network
Intro. The problem Our approach Results Conclusion
Whole exome seq. with Illumina
Whole genome genotyping with Affy 250K array
Results
4/20/11 Yaniv ErlichExome sequencing and disease network
Intro. The problem Our approach Results Conclusion
• 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
Exclusion process
4/20/11 Yaniv ErlichExome sequencing and disease network
Intro. The problem Our approach Results Conclusion
TotalGenes Position
SequencedGenes Position
Now what?
Disease network analysis rank KIF1A as the top candidate
4/20/11 Yaniv ErlichExome sequencing and disease network
Intro. The problem Our approach Results Conclusion
Validation by loss of function analysis
4/20/11 Yaniv ErlichExome sequencing and disease network
Intro. The problem Our approach Results Conclusion
TotalGenes Position
SequencedGenes Position
Supporting evidence
4/20/11 Yaniv ErlichExome sequencing and disease network
Intro. The problem Our approach Results Conclusion
• 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
Summary
[email protected] sequencing and disease network
• 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.
Intro. The problem Our approach Results Conclusion
