Exome sequencing and
disease-network analysis
Yaniv Erlich
Whitehead Institute for Biomedical Research
4/20/11
DNA Sudoku
Yaniv Erlich
Two projects with neurological diseases
1. Joubert syndrome in Ashkenazi Jews
2. Hereditary Spastic Paraparesis in Palestinians
yaniv@wi.mit.edu
Our approach
Intro.
Results
Conclusion
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:
Normal
Joubert
Thick and elongated
superior cerebellar
peduncles
Large
Interpeduncular
fossa
Cerebellum
Parisi, 2007
Exome sequencing and disease network
yaniv@wi.mit.edu
Our approach
Intro.
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
Exome sequencing and disease network
yaniv@wi.mit.edu
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.
Chr 11
Hundreds of exons…
Exome sequencing and disease network
yaniv@wi.mit.edu
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.
Exome sequencing and disease network
yaniv@wi.mit.edu
Intro.
Our approach
Results
Conclusion
Technical details
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
Exome sequencing and disease network
yaniv@wi.mit.edu
Intro.
Our approach
Conclusion
Results
Analysis of variations
SNPs in:
#
Mother
49,515
Child
48,142
That are shard…
23,986
And are heterozygous in mother and
homozygous in child…
And not in dbSNP
And are not synonymous
And are in the mapped locus
Exome sequencing and disease network
2,541
105
39
1
yaniv@wi.mit.edu
Intro.
Our approach
Results
Conclusion
And the winner is… TMEM216
Chromosome 11
Amino-acid change: Arg -> Leu
(CGC>CTC)
Exome sequencing and disease network
yaniv@wi.mit.edu
Intro.
Our approach
Results
Conclusion
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
Intro.
Our approach
Results
Conclusion
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
Intro.
Our approach
Results
Conclusion
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
Intro.
Our approach
Results
Conclusion
Two projects with neurological diseases
1. Joubert syndrome in Ashkenazi Jews
2. Hereditary Spastic Paraparesis in Palestinians
yaniv@wi.mit.edu
Intro.
The problem
Our approach
Results
Conclusion
Hereditary Spastic Paraperesis
-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
4/20/11
Exome sequencing and disease network
(Weber J,2003)
Yaniv Erlich
Intro.
Our approach
The problem
Results
Conclusion
Thoughts on the Joubert rejection process
Currently, the rejection process is based on two classes of arguments:
1. Genetic arguments
2. Loss of function arguments
SNPs in:
#
Mother
49,515
Child
48,142
That are shard…
Genetic
arguments
Loss-offunction
arguments
23,986
And are heterozygous in mother and
homozygous in child…
And not in dbSNP
2,541
105
And are not synonymous
And are in the mapped locus
39
1
Genetic arguments have weaker power with smaller families
We need a new class of arguments
Exome sequencing and disease network
yaniv@wi.mit.edu
Intro.
The problem
Our approach
Results
Conclusion
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
Intro.
The problem
Our approach
Results
Conclusion
Data gathering stage
Whole exome seq.
with Illumina
Whole genome
genotyping with
Affy 250K array
4/20/11
Exome sequencing and disease network
Yaniv Erlich
Intro.
The problem
Our approach
Results
Conclusion
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
4/20/11
Exome sequencing and disease network
Yaniv Erlich
Intro.
The problem
Our approach
Results
Conclusion
Exclusion process
Total
Genes
Position
Sequenced
Genes
Position
Now what?
4/20/11
Exome sequencing and disease network
Yaniv Erlich
Intro.
The problem
Our approach
Results
Conclusion
Disease network analysis rank KIF1A as the top candidate
4/20/11
Exome sequencing and disease network
Yaniv Erlich
Intro.
The problem
Our approach
Results
Conclusion
Validation by loss of function analysis
Total
Genes
4/20/11
Position
Exome sequencing and disease network
Sequenced
Genes
Position
Yaniv Erlich
Intro.
The problem
Our approach
Results
Conclusion
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
4/20/11
Exome sequencing and disease network
Yaniv Erlich
Intro.
The problem
Our approach
Results
Conclusion
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
Erlich Lab
Whitehead Institute
Positions available (yaniv@wi.mit.edu)
yaniv@wi.mit.edu