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TITLE: Exome sequencing expedites gene discovery for rare diseases
AUTHOR NAMES:
Kun Huang (1T3)
MD candidate, University of Toronto
CORRESPONDING AUTHOR EMAIL ADDRESS:
Kun Huang
University of Toronto, Faculty of Medicine
1 King's College Circle,
Medical Sciences Building, Room 2109
Toronto, ON, M5S 1A8
Tel: (647) 863-2715 email: kun.huang@utoronto.ca
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Kun Huang
For the past several decades, genes underlying Mendelian disorders have been identified through traditional positional cloning strategies which often have reduced power due to marked locus heterogeneity, small kindred sizes, or substantially reproductive disadvantage. 1 2 The emergence of next generation sequencing techniques (ie. whole-genome, whole-exome and whole-transcriptome sequencing) allows substantial advances in identifying genetic alterations.
In theory, whole-genome sequencing of all human genes for discovery of genetic variants could potentially identify the gene underlying any given monogenic disease 3 . However the cost associated with sequencing whole genome is daunting. An alternative approach, exome sequencing, involves the targeted resequencing of all protein coding regions, which only requires ~5% as much sequencing as a whole human genome. 3-6 An increasing number of studies in the past two years have demonstrated whole-exome sequencing to be a powerful approach to identify causative genes underlying extremely rare Mendelian disorders. 7-14 #
In order to effectively process the massive sequencing data, some assumptions, albeit arbitrary, are made about causal mutations underlying Mendelian disease. 4 13 14 First, the disease is monogenic and caused by a single mutation. Secondly, the mutation has a significant effect on phenotypes; therefore it is most likely coding and highly penetrant, i.e. missense and nonsense substitutions, coding indels as well as splice acceptor and donor site changes.
Thirdly, the mutation would be rare or novel, and probably private to affected individuals. Where necessary, a further assumption is often made that the disease is genetically homogenous, i.e. unrelated affected individuals have mutations in the same gene, at least for the individuals whose DNA were sequenced for the study.
The proof-of-concept of exome sequencing was first demonstrated in 2009 in a rare
Mendelian disorder called Freeman –Sheldon syndrome to show the feasibility of this technique.
Through only four cases, this study was able to identify MYH3 as the single causal gene. 4
# Reprint from Huang K. Exome sequencing expedites disease gene discovery. Clin Genet 2011 and Huang K. De novo paradigm: the ultimate answer to the paradox in mental retardation? Clin Genet 2011 with permission by Wiley-Blackwell.
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Since then, more than 40 exome sequencing studies have applied various strategies to identify the causal variants for different disorders such as Miller syndrome 14 , Kabuki syndrome 13 ,
Fowler syndrome 15 , Perrault Syndrome 16 and Schinzel-Giedion syndrome 17 . Some studies also integrated exome sequencing data with traditionally used linkage and homozygosity analysis 10 18 .
However, the avalanche of data from exome sequencing provides a statistical and computational challenge: how to separate the causative alterations from the noise caused by normal variants. Based on the aforementioned assumptions, the primary filter used to identify potentially causal mutations is variant function, with the rationale that mutations which are disruptive to proteins and/or at more conserved sites are more likely to be pathogenic.
Therefore, non-coding and synonymous variants are often greatly down-weighted. Tools like
SIFT 19 , PolyPhen 20 21 , CDPred 22 , PhyloP 23 and GERP 24 25 are developed to rank the variants by potential effect on protein structure and function, and also by conservation scores. Although such strategy has been justified for many studies, this will most certainly not always be the case.
Shortcomings of this method include the inability to capture regulatory or evolutionary conserved sequences in non-coding regions. As more disorders are studied, there will be a growing need for functional annotation of non-coding regions. 26
Empirical analysis of published exomes estimates about 20 000 single-nucleotide variants in a given exome. 4 13 14 For rare mutations that give major effect and distinctive phenotype, they are not expected to be found in the population at large, and hence will not be seen in genome-wide scans for variants [e.g. the 1000 Genomes Project 27 ], nor in polymorphism repositories [e.g. dbSNP 28 ]. Exclusion from these data sets is typically an important criterion in defining a rare, novel or private variant. This simple assumption and filtering strategy offers an advantage to quickly sift through the exome data for promising causal variants. However, a caveat to note is that dbSNP has a considerable false-positive rate of 15 –
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17%. 29 It is possible that the recessive disease-causing mutations from a normal carrier are deposited in the database.
As an illustration, a recent study by Haack et al 30 provides an elegant example of how exome sequencing in combination with appropriate filtering strategies can be effective in the elucidation of a human respiratory chain disease, mitochondrial complex I deficiency (figure 1).
Discovering the molecular basis of this disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. Using whole exome sequencing followed by filtering with prioritization of mitochondrial protein (figure 1), Haack et al identified heterozygous mutations in ACAD9, a mitochondrial acyl-CoA dehydrogenase gene, from a single individual with severe, isolated complex I deficiency. The authors went on and screened 120 additional complex I defective index cases for ACAD9 mutations. Two additional unrelated cases and a total of five pathogenic ACAD9 alleles were identified, further supporting mutations in ACAD9 are associated with a mitochondrial disorder dominated by severe and generalized complex I deficiency. 2 #
Of particular excitement is that supplementation of riboflavin whose metabolite fosters
ACADs assembly and stability resulted in a significant increase of complex I activity in mutant cell cultures from the patient. 30 This is the first exome sequencing study that also obtained a promising clinical response. Follow-up clinical trial is needed to establish the efficacy of a supplementation with vitamins and cofactors in individuals with ACAD9 mutations. 2
Exome sequencing revolutionizes the way that the genetic bases of Mendelian disorders are studied. More studies now have applied exome and whole-genome sequencing to common and genetically complex diseases such as mental retardation. 31 32 Albeit an emerging new technique, exome sequencing has already expedited the disease gene discovery and is on the horizon to make personalized medicine a reality.
# Reprint from Huang K. Exome sequencing expedites disease gene discovery. Clin Genet 2011 with permission by Wiley-
Blackwell.
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Figure 1. Exome sequencing and filtering strategy (adapted from Huang, 2011 2 )
Kun Huang
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Reference
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