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Shotgun Approach to Functional Annotation of Genes

Shotgun Approach to Functional Annotation of Genes As clinical disease predisposition testing becomes more common, the problem of efficiently interpreting sequence variation will grow more acute. Following ACMG guidelines, many sequence variants in susceptibility genes can immediately be placed in clinically actionable pathogenic or not‐pathogenic categories. But other variants – primarily missense substitutions and splice junction variants, are initially reported by testing laboratories as Unclassified Variants (UVs). While several consortia aspire to integrate bioinformatic, human genetics, and functional assay data towards UV evaluation, the laboratory functional assay component tends to be laborious and low throughput.Working towards a generalizable strategy for increasing the throughput of functional assays, Ghosh et al. (Hum Mutat 36:260–269, 2015) created a demonstration shotgun‐mutant Syngeneic Variance Library (SyVal) for exon 27 of BRCA2 in a cell line that is hemizygous for this gene. As a proof of principle, they demonstrated a systematic modular approach to creation of libraries of syngeneic cell lines that harbor one or a few nucleotide substitutions in the selected target gene at its endogenous locus, paired with application of relatively high‐throughput assays to those libraries of cell lines, in order to evaluate dysfunctionality attributable to the introduced sequence variant(s). Upgraded with String‐based DNA fragment libraries and TALEN or CRISPR‐Cas9 technology to increase site‐specific integration, Ghosh et al. note that this approach could become an important strategy for functional evaluation of large numbers of variants in many different genes.The authors discuss some clear drawbacks. Despite the syngeneic nature of the cell line library, there were 2–3‐fold differences in drug sensitivity between a pair of clones that harbored the same nonsense substitution. That scale of variation could make it difficult to accurately distinguish between neutral and moderately damaging variants, or between moderately and severely damaging variants. A second issue involves assay selection. In large multi‐domain disease susceptibility proteins, different protein domains may have different functions that are relevant to the disease process. Especially for evaluating missense substitutions, it is indeed critical that appropriate assays are applied to each individual domain and that the applicability of the results be validated for the given disease susceptibility. For any given disease‐gene combination, these issues need to be considered carefully before high‐throughput functional assays can transition from research to clinical application. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Mutation Wiley

Shotgun Approach to Functional Annotation of Genes

Tavtigian, Sean V.
Human Mutation , Volume 36 (2) – Feb 1, 2015
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Publisher
Wiley
Copyright
Copyright © 2015 Wiley Periodicals, Inc.
ISSN
1059-7794
eISSN
1098-1004
DOI
10.1002/humu.22746
Publisher site
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Abstract

As clinical disease predisposition testing becomes more common, the problem of efficiently interpreting sequence variation will grow more acute. Following ACMG guidelines, many sequence variants in susceptibility genes can immediately be placed in clinically actionable pathogenic or not‐pathogenic categories. But other variants – primarily missense substitutions and splice junction variants, are initially reported by testing laboratories as Unclassified Variants (UVs). While several consortia aspire to integrate bioinformatic, human genetics, and functional assay data towards UV evaluation, the laboratory functional assay component tends to be laborious and low throughput.Working towards a generalizable strategy for increasing the throughput of functional assays, Ghosh et al. (Hum Mutat 36:260–269, 2015) created a demonstration shotgun‐mutant Syngeneic Variance Library (SyVal) for exon 27 of BRCA2 in a cell line that is hemizygous for this gene. As a proof of principle, they demonstrated a systematic modular approach to creation of libraries of syngeneic cell lines that harbor one or a few nucleotide substitutions in the selected target gene at its endogenous locus, paired with application of relatively high‐throughput assays to those libraries of cell lines, in order to evaluate dysfunctionality attributable to the introduced sequence variant(s). Upgraded with String‐based DNA fragment libraries and TALEN or CRISPR‐Cas9 technology to increase site‐specific integration, Ghosh et al. note that this approach could become an important strategy for functional evaluation of large numbers of variants in many different genes.The authors discuss some clear drawbacks. Despite the syngeneic nature of the cell line library, there were 2–3‐fold differences in drug sensitivity between a pair of clones that harbored the same nonsense substitution. That scale of variation could make it difficult to accurately distinguish between neutral and moderately damaging variants, or between moderately and severely damaging variants. A second issue involves assay selection. In large multi‐domain disease susceptibility proteins, different protein domains may have different functions that are relevant to the disease process. Especially for evaluating missense substitutions, it is indeed critical that appropriate assays are applied to each individual domain and that the applicability of the results be validated for the given disease susceptibility. For any given disease‐gene combination, these issues need to be considered carefully before high‐throughput functional assays can transition from research to clinical application.

Journal

Human Mutation – Wiley

Published: Feb 1, 2015

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