Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

Tomberg, Kärt; Westrick, Randal J.; Kotnik, Emilee N.; Cleuren, Audrey C. A.; Siemieniak, David; Zhu, Guojing; Saunders, Thomas L.; Ginsburg, David

doi:10.1371/journal.pgen.1007658

Cited by 6 publications

(3 citation statements)

References 53 publications

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“…In the screen reported here, most lines showed evidence for complex trait inheritance, predicting that more than one modifier segregated. In a recent published modifier screen for thrombosis, very few candidate genes were identified, likely because of the lack of power in finding linkages using standard quantitative trait mapping strategies (Tomberg et al 2018). Here, statistical tests that assessed association instead of linkage were used to identify multiple interacting loci in six lines, three of which had an effect only in the presence of other modifiers.…”

Section: Discussionmentioning

confidence: 99%

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

et al. 2020

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Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

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Section: Discussionmentioning

confidence: 99%

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

et al. 2020

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“…STOCK Crb1 rd8 mice were backcrossed to C57BL/6J (JAX stock number 000664) mice for seven generations to produce an incipient congenic strain, B6.Cg- Crb1 rd8 /Pjn (N 7 ), which was bred to homozygosity. Males from this incipient congenic colony were administered weekly intraperitoneal injections of ENU (Millipore Sigma, Burlington, MA, USA; N3385) for three consecutive weeks at a concentration of 80 mg/kg per treatment [ 109 , 110 ]. Following return to fertility, the mutagenized G 0 males were backcrossed to B6.Cg- Crb1 rd8 /Pjn females, producing G 1 mice which were subsequently outcrossed to unmutagenized B6.Cg- Crb1 rd8 /Pjn (N 7 ) and resulting female G 2 mice were backcrossed to G 1 sires to produce G 3 mice [ 46 ].…”

Section: Methodsmentioning

confidence: 99%

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model

et al. 2022

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Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.

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“…Colony management and ENU mutagenesis STOCK Crb1 rd8 was backcrossed to C57BL/6J (JAX stock number 000664) for seven generations to produce an incipient congenic strain, B6.Cg-Crb1 rd8 /Pjn (N 7 ), which was bred to homozygosity. Males from this incipient congenic colony were administered weekly intraperitoneal injections of N-ethyl-N-nitrosourea (ENU) for three consecutive weeks at a concentration of 80 mg/kg per treatment [94], [95]. Following return to fertility, the mutagenized G 0 males were backcrossed to B6.Cg-Crb1 rd8 /Pjn females, producing G 1 mice which were subsequently outcrossed to unmutagenized B6.Cg-Crb1 rd8 /Pjn (N 7 ) and resulting female G 2 mice were backcrossed to G 1 sires to produce G 3 mice [46].…”

Section: Methodsmentioning

confidence: 99%

Identification of Arhgef12 and Prkci as Genetic Modifiers of Retinal Dysplasia in the Crb1^rd8 Mouse Model

Weatherly

Collin

Charette

et al. 2021

Preprint

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Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. In both models at one month of age, Müller glia and microglia mislocalization at dysplastic lesions was similar to that in B6.Cg-Crb1rd8/Pjn mice, while photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms.

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Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

Cited by 6 publications

References 53 publications

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model

Identification of Arhgef12 and Prkci as Genetic Modifiers of Retinal Dysplasia in the Crb1^rd8 Mouse Model

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Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

Cited by 6 publications

References 53 publications

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model

Identification of Arhgef12 and Prkci as Genetic Modifiers of Retinal Dysplasia in the Crb1rd8 Mouse Model

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Identification of Arhgef12 and Prkci as Genetic Modifiers of Retinal Dysplasia in the Crb1^rd8 Mouse Model