We found an unexpectedly high proportion of

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We found an unexpectedly high proportion of literature-annotated disease mutations that were incorrect, incomplete, or common poly- morphisms. Differentiation of common polymorphisms from disease mutations requires genotyping a large number of unaffected individ- uals. Severe, orphan disease mutations should be uncommon (<1% incidence) and should not be found in the homozygous state in un- affected individuals. Unexpectedly, we found that 74% of disease mu- tation calls were accounted for by substitutions with incidences of 5%, of which almost one-half were homozygous in samples unaffected by the corresponding disease. Also unexpected was the finding that 14 of 113 literature-annotated disease mutations were incorrect. Thus, for many recessive diseases, HGMD, dbSNP, OMIM, and the literature are insufficient arbiters of whether variants are disease mutations. We have shown NGS of samples from affected individuals to be a powerful method for error correction: More than three-quarters of errors in mu- tation identification were Sanger sequencing interpretation errors or incorrect imputation of genomic mutations from cDNA sequencing. Key advantages of NGS are clonal derivation (facilitating unambiguous detection of heterozygous and indel variants), maintenance of phase information (allowing haplotype derivation for adjacent variants), and highly redundant coverage (resulting in extremely low consen- sus error rates). Thus, although we have shown that it is technically feasible to undertake comprehensive analysis of recessive gene se- quences, sequencing of many unaffected and affected samples will be required to establish an authoritative disease mutation database. Specifically, current reference resources contain common poly- morphisms that are annotated as disease mutations and erroneous disease mutations. Without reference database improvements, the clinical utility of comprehensive carrier testing will be limited. Aside from nonsense mutations and premature stop codons in known dis- ease genes and the study of affected individuals, additional bioinfor- matic approaches will be needed to distinguish rare benign variants from pathogenic variants: Amino acid substitution characteristics such as physicochemical and evolutionary conservation and location (where tertiary structure is known) are useful but not definitive. For many rare variants, functional assays will need to be developed to assess pathogenicity rigorously. Establishment of an authoritative database of disease mutations is clearly needed and represents a nas- cent bottleneck in progress toward prevention, diagnosis, and treat- ment of recessive diseases. In the interim, clinical interpretation of the functional importance or pathogenicity of variants will be challenging for many recessive diseases.
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  • Spring '11
  • Dr.Biology
  • DNA, Mutation, Genetic disorder, NGS, disease mutations

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