High throughput sequencing approaches to mutation discovery in the mouse

Simon, Michelle; Mallon, Ann‐Marie; Howell, Gareth R.; Reinholdt, Laura G.

doi:10.1007/s00335-012-9424-0

Cited by 5 publications

(6 citation statements)

References 101 publications

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“…Single‐nucleotide variant (SNV) cells were made using a customized version of The Genome Analysis Toolkit (GATK) with default parameters. Several triaging steps were made to reduce false positives . The 17 Mouse Genome data set was used to filter inbred SNP sites from the RCALC1 SNV data set, and common sites were removed from further investigation.…”

Section: Methodsmentioning

confidence: 99%

“…Several triaging steps were made to reduce false positives. (33) The 17 Mouse Genome data set (34) was used to filter inbred SNP sites from the RCALC1 SNV data set, and common sites were removed from further investigation. The remaining SNVs were further filtered by removing sites with an allele frequency <35% and >80%, a read depth <3, and a quality score <200.…”

Section: Exome Sequence Analysismentioning

confidence: 99%

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Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis

Gorvin

Loh

Stechman

et al. 2019

Journal of Bone and Mineral Research

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Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12‐month‐old progeny from a male mouse that had been treated with the chemical mutagen N‐ethyl‐N‐nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm‐Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome‐wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3‐B3 and whole‐exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain‐containing protein 4 (BRD4). The mutant heterozygous (Brd4+/M149T) mice, when compared with wild‐type (Brd4+/+) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4+/M149T and Brd4+/+ mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4+/M149T mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.

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Section: Methodsmentioning

confidence: 99%

Section: Exome Sequence Analysismentioning

confidence: 99%

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis

Gorvin

Loh

Stechman

et al. 2019

Journal of Bone and Mineral Research

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“…Single‐nucleotide variant (SNV) calls were made using a customized version of The Genome Analysis Toolkit (GATK) with default parameters. Several triaging steps were made to reduce false positives . The 17 Mouse Genome dataset was used to filter inbred SNP sites from the RCALC2 SNV dataset, and common sites were removed from further investigation.…”

Section: Methodsmentioning

confidence: 99%

“…Several triaging steps were made to reduce false positives. (26) The 17 Mouse Genome dataset (27) was used to filter inbred SNP sites from the RCALC2 SNV dataset, and common sites were removed from further investigation. The remaining SNVs were further filtered by removing sites with an allele frequency <35% and >80%, a read depth <3 and a quality score >200.…”

Section: Exome Sequence Analysismentioning

confidence: 99%

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice

Gorvin

Ahmad

Stechman

et al. 2018

Journal of Bone and Mineral Research

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Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X‐rays to identify renal opacities in N‐ethyl‐N‐nitrosourea (ENU)‐mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16‐Mbp region on chromosome 11D‐E2 and whole‐exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma‐2, accessory subunit ( Polg2 ) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co‐segregated with RCALC2. Kidneys of mutant mice ( Polg2 + / Y265X ) had lower POLG2 mRNA and protein expression, compared to wild‐type littermates ( Polg2 +/+ ). The Polg2 +/Y265X and Polg2 + / + mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2 + / Y265X kidneys was reduced compared to Polg2 +/+ mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2 + / Y265X mice, compared to Polg2 + / + littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

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“…Over the years a number of typical steps have been employed to remove the false positives. These steps include one or more of the following: a read depth threshold where variants found in less than the allotted number of reads are ignored, a quality threshold where variants in poorly mapped reads are ignored and inbred SNP identification where variants overlapping background SNV sites are ignored (Simon et al 2012 ). This prioritisation and filtering of SNVs is a crucial step in the NGS pipeline as false discovery of erroneous SNVs masquerading as real ENU variants can result in incorrect candidate genes, whereas over-filtering can result in the exclusion of the real causal mutation, resulting in the failure of the experiment.…”

Section: Next Generation Sequencingmentioning

confidence: 99%

Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing

et al. 2015

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Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to ‘finding a needle in a haystack’. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.

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High throughput sequencing approaches to mutation discovery in the mouse

Cited by 5 publications

References 101 publications

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice

Current strategies for mutation detection in phenotype-driven screens utilising next generation sequencing

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