Modeling primary ovarian insufficiency-associated loci in C. elegans identifies novel pathogenic allele of MSH5

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.

Keywords: C. elegans; Crossover; Infertility; MSH5; Meiosis; POI.

Fig. 1

Identification of putative pathogenic allele in POI patient. A Pedigree of POI patient. B CLUSTAL multi-sequence alignment of the C. elegans, human, and mouse MSH5 proteins showing the conserved arginine that is mutated in the human patient and the CRISPR-edited worms (grey highlight)

Fig. 2

msh-5(ea36) is impaired in crossover formation. Shown are the numbers of DAPI⁺ bodies in diakinesis oocytes for the respective genotypes. Control dpy-1 animals had only the expected 6 DAPI⁺ bodies. msh-5 heterozygous worms similarly had predominantly 6 DAPI bodies, whereas the msh-5(me23) null worms had 12 univalents in almost all nuclei. msh-5(ea36) mutant animals have increased numbers of univalents and chromosome fusions.