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Premature ovarian insufficiency (POI) is a severe clinical syndrome defined by ovarian dysfunction in women less than 40 years old who generally manifest with infertility, menstrual disturbance, elevated gonadotrophins, and low estradiol levels. STAG3 is considered a genetic aetiology of POI, which facilitates entry of REC8 into the nucleus of a cell and plays an essential role in gametogenesis. At present, only six truncated variants associated with POI have been reported; there have been no reports of an in-frame variant of STAG3 causing POI. In this study, two novel homozygous in-frame variants (c.877_885del, p.293_295del; c.891_893dupTGA, p.297_298insAsp) in STAG3 were identified in two sisters with POI from a five-generation consanguineous Han Chinese family. To evaluate the effects of these two variants, we performed fluorescence localization and co-immunoprecipitation analyses using in vitro cell model. The two variants were shown to be pathogenic, as neither STAG3 nor REC8 entered nuclei, and interactions between mutant STAG3 and REC8 or SMC1A were absent. To the best of our knowledge, this is the first report on in-frame variants of STAG3 that cause POI. This finding extends the spectrum of variants in STAG3 and sheds new light on the genetic origins of POI.

A novel glycoside hydrolases family 57 gene (gh-57) was found from a metagenomic fosmid library constructed from a black smoker chimney sample 4143-1 from the Mothra hydrothermal vent at the Juan de Fuca Ridge. Sequence and homology analysis using BLAST revealed that it had high similarity to gh-57 family. Conserved domain research revealed that the novel gh-57 contained a Glyco-hydro-57 domain and five conserved regions, including two putative catalytic residues Glu¹⁵⁴ and Asp²⁶³. The three-dimensional features of the protein and its homologue from Pyrococcus horikoshii OT3 known as α-amylase were generated by homology modeling. The gh-57 gene was cloned, expressed, and purified in Escherichia coli using pQE system. Enzyme activity revealed that the recombinant protein could hydrolyze soluble starch and demonstrated amylase activity. It showed an optimal pH of 7.5, an optimal temperature of 90 °C, and its thermostability at 90 °C could remain over 50% enzyme activity for 4 h. The enzyme activity could be increased by DTT and Mg²⁺ while an inhibitory effect was observed with EDTA, ATP, and Ca²⁺. These results showed that the gh-57 gene was a novel thermostable amylase from oceanic microorganisms.

Background: Premature ovarian insufficiency (POI) is one major cause of female infertility, minichromosome maintenance complex component 8 (MCM8) has been reported to be responsible for POI. Methods: Whole-exome sequencing was performed to identify the genetic variants of women with POI. Sanger sequencing was used to validate the variants in all the family members. Various bioinformatic software was used for the pathogenicity assessment. Reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative PCR, and a chromosomal instability study induced by mitomycin C were performed to analyze the functional effects of the variant. Results: A novel homozygous frameshift mutation (NM_032485.4:c.351_354 delAAAG) of MCM8 gene was identified in the patients, segregated with POI in this family. This mutation is predicted to produce truncated MCM8 protein and to be pathogenic. Reverse transcription polymerase chain reaction revealed that the frameshift mutation led to a remarkably reduced level of MCM8 transcript products, and chromosomal instability study showed that the ability of mutant MCM8 to repair DNA breaks was impaired. Conclusion: We identified a novel homozygous frameshift mutation in the MCM8 gene in two affected sisters with POI, and functional analysis revealed that this mutation is pathogenic. Our findings enrich the MCM8 mutation spectrum and might help clinicians to make a precise diagnosis, thereby allowing better family planning and genetic counseling. K E Y W O R D Sloss-of-function, MCM8 gene, premature ovarian insufficiency, whole-exome sequencing