Nicolas Macaisne1,2, Maria Sol Touzon3, Aleksander Rajkovic4, Judith L Yanowitz5,6,7,8,9. 1. Magee-Womens Research Institute, Pittsburgh, PA, 15217, USA. 2. Université de Paris, CNRS, Institut Jacques Monod, 75013, Paris, France. 3. Endocrinology Department, Research Unit Garrahan Consejo Nacional de Investigaciones Cientificas Y Tecnologicas, Hospital de Pediatr a Garrahan, 1245, Ciudad Autonoma de Buenos Aires, Argentina. 4. Department of Pathology, Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA, 94158, USA. 5. Magee-Womens Research Institute, Pittsburgh, PA, 15217, USA. yanowitzjl@mwri.magee.edu. 6. Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, 15213, USA. yanowitzjl@mwri.magee.edu. 7. Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA, 15213, USA. yanowitzjl@mwri.magee.edu. 8. Department of Microbiology and Molecular Genetics and the Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, 15213, USA. yanowitzjl@mwri.magee.edu. 9. Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA, 15213, USA. yanowitzjl@mwri.magee.edu.
Abstract
PURPOSE: In women under the age of 40, primary ovarian insufficiency (POI) is a devastating diagnosis with significant prevalence of 1-4% (Rajkovic and Pangas, Semin Reprod Med. 35(3):231-40, 2017). POI is characterized by amenorrhea with elevated levels of follicle stimulating hormone (FSH) and reduced estrogen levels, mimicking the menopausal state. Genetic determinants account for just over 10% of POI cases, yet determining whether particular single nucleotide polymorphisms (SNPs) are pathogenic is challenging. METHODS: We performed exome sequencing on a cohort of women with POI. CRISPR mutagenesis was employed to create a mutation in a conserved amino acid in the nematode protein. Functional relevance was assessed by analysis of bivalents and aberrant DNA morphologies in diakinesis nuclei. RESULTS: We identified a nonsynonymous c.C1051G; p.R351G variant, in a conserved region of the MSH5 protein. Mutation of this conserved amino acid in the C. elegans homolog, msh-5, revealed defective crossover outcomes in the homozygous and hemizygous states. CONCLUSIONS: These studies further implicate MSH5 as a POI gene and c.C1051G; p.R351G variant as likely playing a functional role in mammalian meiosis. This approach also highlights the ability of model organisms, such as C. elegans, to rapidly and inexpensively identify alleles of interest for further studies in mammalian models.
PURPOSE: In women under the age of 40, primary ovarian insufficiency (POI) is a devastating diagnosis with significant prevalence of 1-4% (Rajkovic and Pangas, Semin Reprod Med. 35(3):231-40, 2017). POI is characterized by amenorrhea with elevated levels of follicle stimulating hormone (FSH) and reduced estrogen levels, mimicking the menopausal state. Genetic determinants account for just over 10% of POI cases, yet determining whether particular single nucleotide polymorphisms (SNPs) are pathogenic is challenging. METHODS: We performed exome sequencing on a cohort of women with POI. CRISPR mutagenesis was employed to create a mutation in a conserved amino acid in the nematode protein. Functional relevance was assessed by analysis of bivalents and aberrant DNA morphologies in diakinesis nuclei. RESULTS: We identified a nonsynonymous c.C1051G; p.R351G variant, in a conserved region of the MSH5 protein. Mutation of this conserved amino acid in the C. elegans homolog, msh-5, revealed defective crossover outcomes in the homozygous and hemizygous states. CONCLUSIONS: These studies further implicate MSH5 as a POI gene and c.C1051G; p.R351G variant as likely playing a functional role in mammalian meiosis. This approach also highlights the ability of model organisms, such as C. elegans, to rapidly and inexpensively identify alleles of interest for further studies in mammalian models.
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