A homozygous NOBOX truncating variant causes defective transcriptional activation and leads to primary ovarian insufficiency.

STUDY QUESTION:Does a novel homozygous NOBOX truncating variant, identified in whole exome sequencing (WES) of patients with primary ovarian insufficiency (POI), cause defective transcriptional activation of multiple oocyte-related genes? SUMMARY ANSWER:A novel homozygous truncating mutation of NOBOX was confirmed to exhibit a loss-of-function effect using well-defined molecular and functional analyses. WHAT IS KNOWN ALREADY:Several NOBOX mutations have been reported to be associated with POI but all of them are heterozygous mutations. STUDY DESIGN, SIZE, DURATION:This is a cross sectional study in 96 patients diagnosed with POI and 211 women not diagnosed with POI in China. PARTICIPANTS/MATERIALS, SETTING, METHODS:Blood samples collected from the participants were subjected to whole exome sequencing. Full-length transcript of NOBOX was cloned directly from human fetal ovary (FO). Functional analysis was performed for a NOBOX sequence variant associated with POI. MAIN RESULTS AND THE ROLE OF CHANCE:One novel homozygous truncating variant, chr7:144098161delC, in the NOBOX gene was found in a POI patient. The truncating variant showed a severe defect in transcriptional activation of GDF9 a well-known target NOBOX. Furthermore, using real-time quantitative PCR analysis, we found many oocyte-related genes were expressed at lower level in truncating variant cells than in control cells. In addition, we found that the truncated NOBOX lost its ability to induce the G2/M arrest.Notably, our results confirmed that the 1725 bp NOBOX transcript is expressed in human FO and is the only functional isoform in transcriptional activation assays. LIMITATIONS REASONS FOR CAUTION:Although the in vitro assays demonstrated the loss-of-function effect of truncating mutation on NOBOX transcriptional activation, further studies are needed to validate its long-term effects on folliculogenesis and POI. WIDER IMPLICATIONS OF THE FINDINGS:This is the first homozygous mutation of NOBOX associated with POI showing a loss-of-function effect using well-defined molecular and functional analyses. These results will aid both researchers and clinicians in understanding the molecular pathology of NOBOX and POI to develop diagnostic assays or therapeutic approaches. STUDY FUNDING/COMPETING INTERESTS:Research funding is provided by the Ministry of Science and Technology of China [2012CB944704; 2012CB966702], the National Natural Science Foundation of China [Grant number: 31171429] and Beijing Advanced Innovation Center for The authors declare no conflict of interest.

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