Download Identifying Mutations Responsible for Rare Disorders Using New

Identifying Mutations Responsible for
Rare Disorders Using New
Technologies
Jacek Majewski,
Department of Human Genetics, McGill University, Montreal, QC Canada
Mendelian Diseases
• Clear mode of inheritance – dominant of
recessive
• High penetrance – having the mutation
determines the phenotype with near certainty
• Clear phenotypic consequences
• Low environmental influence – lack of
phenocopies
• Examples – Tay-Sachs disease, Cystic Fibrosis…
Finding causes of Mendelian
Disorders
• Although many of those disorders are rare (1:2000 –
1:100,000 incidence), taken together they constitute
a substantial health burden
• It is estimated that over 90% of Mendelian disorders
are caused by mutations in the coding regions
(missense, nonsense, frameshifts, in/dels, splicing)
• Traditionally, the approach was via genetic linkage
analysis, followed by targeted sequencing of
candidate genes.
• In view of new technologies – can we do better?
1993
Homozygosity mapping Today
The American Journal of Human Genetics(2010)doi:10.1016/j.ajhg.2010.09.005
“Conventional” mutation mapping strategy for autosomal recessive disorders. Here we collected four
patients from consanguinous Bedouin families in Qatar with the Van den Ende-Gupta Syndrome
(VDEGS). Genotyping using SNP arrays identified a region of shared homozygosity – presumably inherited
from a common ancestor and containing the mutation - 2.4 mb in length, containing 44 genes.
Sanger Sequencing and Exome Capture
We concurrently conducted
conventional Sanger sequencing of
candidate genes and whole-exome
sequencing. In this case, the targeted
candidate approach was the winner,
identifying two distinct pathogenic
mutations in the SCARF2 gene. A few
days later, results from exome
sequencing confirmed this result and
helped to exclude all other candidate
genes in the region.
A. Presence
and
effect
c.1328_1329delTG mutation.
of
B. Presence and effect of c.773G>A
mutation.
C. Conservation of residues near 773G>A
(p.C258Y) mutation.
D. Exome capture and sequencing results
in the mother of patient 1.
High Throughput Sequencing
• E.g. Illumina HiSeq, or ABI Solid
• Main characteristics – typically produce
millions to hundreds of millions short (50100bp) sequencing reads per application
• Reads can be aligned to the reference
genome, and variants identified
• Currently, sequencing of the entire human
genome is still quite expensive - $9k today.
• Can we sequence a subset of the genome?
What is Exome Capture?
•
A method that uses specific probes to capture only the coding portions of all
annotated exons within the genome. This is followed by high throughput shotgun
sequencing. Our group uses the Agilent SureSelect Human All Exon Kit (in solution
beads), followed by Illumina GAIIx, 76 bp read sequencing (single lane).
Coverage Statistics
Exome coverage obtained by Agilent All
Exon in solution capture process, followed by
Illumina sequencing. The bars represent
coverage from 1, 2, and 3 lanes of 76 base
reads. E.g., two lanes of sequence produce
an average 52.3X coverage of the exome,
90% of the exome is covered at >10X level,
and 95% at > 5X. In our experience, 2 lanes
of sequencing provide excellent coverage at
a
reasonable
cost.
Further,
slight
improvement in coverage upon increasing
the number of lanes is not warranted.
Current bottom line – should be
able to get high quality 50X
exome for around $2k
Current Analysis Pipeline
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Exome Capture and Sequencing by Genome Quebec Platforms
Base calling, QC – standard Illumina Pipeline
Alignment to reference genome – BWA
Retain unique alignments only
SNV and in/del calls (SAMTools, GATK)
CNV calls (in house)
Annotate functional variants as (ANNOVAR)
Filtering
– Other exomes (~50) – Key!!!
– dbSNP
– 1000 Genomes
• Relax filtering criteria if necessary
END USER OUTPUT
Filtering The Data
Filtering The Data
Visualization, manual QC and
inspection
Visualization, manual QC and
inspection
ONGOING Projects
• Part of the FORGE consortium, Canada-wide
(GC/CIHR)
• Part of the IGNITE project, Dalhousie-Atlantic
Canada (GC)
• RaDiCAL (McGill/MUHC)
• Numerous collaborations with individual
researchers – McGill, Quebec, Canada, France,
Poland, Qatar, Lebanon…
Successes so far - new genes
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Fowler Syndrome
Mitochondrial Disease (2)
Vitamin B12 metabolism (2)
Nephrotic Syndrome
LCA
Hajdu-Cheney Syndrome
Hyper IgM (2)
Novel developmental dysmorphisms (2)
…
Two unrelated patients – total study time = 2 weeks from DNA sample to gene
Schematic of the FLVCR2 mutations found in two patients (F1 and F3).
A. The four mutations in FLVCR2 identified in F1 and F3 using whole exome sequencing as visualized using the Integrative
Genomics Viewer from the Broad Institute (http://www.broadinstitute.org/igv).
B Mutations in FLVCR2 are shown relative to the protein domains.
Mol Cell Biol. 2010 Sep 7. [Epub ahead of print]
The Fowler Syndrome associated protein FLVCR2 is an importer of heme.
Duffy SP, Shing J, Saraon P, Berger LC, Eiden MV, Wilde A, Tailor CS.
Program in Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada; Department of Molecular Genetics,
University of Toronto, Toronto, ON M5G, Canada; National Institute of Mental Health, Laboratory of Cellular & Molecular
Regulation, Bethesda, MD 20892 USA.
Hajdu-Cheney Syndrome
(with Mark Samuels and Jeremy Schwartzentruber)
• Rare dominant disease, characterized by bone
deterioration (our first dominant, FORGE project)
• < 100 patients known worldwide
• DNA from 3 affected family members, + 3 unrelated
individuals
• Exome sequencing
• Identification of variants
• 2 days after sequence data available – gene found
• All patients have truncating mutations in the last exon of
the gene Notch2
• Paper prepared for publication during the next week
• But...
Monday...
Summary
• Exome Sequencing is a fast and efficient way to
identify new disease genes
• In our hands, success rates vary from 100% (families)
to 33% (single individuals)
• Other applications:
- genomic molecular diagnosis
- genetic testing
Summary
• Exome sequencing is a rapid and increasingly affordable method of
identifying disease mutations
• CNVs (large structural variants) can be detected
• We have successfully found mutations in a number of additional recessive
disorders, using only a single individual
• In the next few months dozens of new disease genes/mutations will be
identified
• Next step - Genomic Molecular Diagnosis