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 Structural Biology. The authors declare no 
conflict of interest.