POI-associated EIF4ENIF1 mutations exhibit impaired translation regulation abilities.

Various genetic variants have been found to be associated with the clinical onset of premature ovarian insufficiency (POI). However, when measured in vitro, the functional influence of the variants can be difficult to determine. By whole-exome sequencing (WES) of 93 patients with sporadic POI, we found a missense variant c.623G > A;p.R208H in the EIF4ENIF1 gene. In silico prediction of the variant using different algorithms suggested it might be a damaging variant. We compared the property of EIF4ENIF1 R208H and Q842P, a POI-related mutant that we reported previously, with wildtype (WT) protein using 293FT cells in vitro. Surprisingly, a change in subcellular distribution and granule forming ability (Q842P) and nuclear import capacity (R208H) was not observed, despite domain prediction evidences. Since EIF4ENIF1 was reported to inhibit translation, we employed T&T-seq, a translation-transcription dual-omics sequencing method, to profile gene expression upon overexpression of EIF4ENIF1 WT and mutants. EIF4ENIF1 WT overexpression group exhibited significantly (P < 0.0001) lower translation efficiency (TE) than empty vector or GFP overexpression control group. Surprisingly, EIF4ENIF1 Q842P overexpression failed to repress global translation, showing an overall TE significantly higher than WT group. Overexpression R208H significantly (P < 0.0001) lowered the overall TE, whereas exhibiting a reduced translation inhibitory effect on high-TE genes (TE > 2 in GFP control group). Several fertility-associated genes, such as AMH in Q842P group and SERPINE1 and THBS1 in R208H group, was translationally up-regulated in mutant groups versus WT control, suggesting a potential mechanism of mutated EIF4ENIF1 causing POI via impaired translation repression. It is further proposed that T&T-seq can be a sensitive evaluation tool for the measurement of functional alteration by variants in many other translational regulator genes, not only EIF4ENIF1, helping to eliminate misinterpretation of clinical significance of genetic variants.

TP63 truncating mutation causes increased cell apoptosis and premature ovarian insufficiency by enhanced transcriptional activation of CLCA2.

BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear. METHODS: Ninety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI. RESULTS: By WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression. CONCLUSION: Our findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Regulation and control of wenshen yangxue granule combined with clomifene citrate on INH-ACT-FS system in patients with follicular maldevelopment infertility / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the clinical curative effect of Wenshen Yangxue Granule (WSYXG) combined with clomifene citrate (CC) in treating follicular maldevelopment (FM) infertility, and to explore its possible action channels.</p><p><b>METHODS</b>Ninety patients with FM of Shen-deficiency blood stasis syndrome were randomly assigned to 3 groups, i.e., the Chinese medicine group (CMG, treated with WXYXG), the Western medicine group (WMG, treated with CC), and the combination group of Chinese medicine and Western medicine (CG, treated with both WSYXG and CC), 30 cases in each group. Three menstrual cycles were totally observed. Serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2 ), inhibin B (INHB), activin A (ACTA), and follistatin (FS) were tested before and after treatment, and the ovulation was monitored and their basic body temperature measured.</p><p><b>RESULTS</b>There was no statistical difference in clinical efficacy among the three groups (P> 0.05). Better effects on the Chinese medicine syndrome efficacy, the ovulation rate, and the endometrium thickness on the ovulation day were shown in CMG and CG than in WMG, showing statistical difference (P < 0.05). The E2 level increased on the third day of the first menstrual cycle in CG when compared with before treatment. On the 10th day of the 1st menstrual cycle, the INHB and FS increased and the ACTA decreased, showing statistical difference (P < 0.05). On the 10th day of the 3rd menstrual cycle the serum LH level decreased more obviously in CG than in WMG, showing statistical difference (P < 0.05). On the 3rd day of the 3rd menstrual cycle in CG, the INHB was negatively correlated with FSH (r = -0.492,P < 0.01), and INHB on the 10th day was positively correlated with E2 and FS (r = 0.682, 0.772, P < 0.01), and negatively correlated with ACTA on the 10th day (r = -0.635, P < 0.01).</p><p><b>CONCLUSION</b>WSYXG combined with CC could improve Chinese medicine syndrome, regulate the expressions of FM patients' ovary local factors INHB, ACTA and FS, improve the condition of ovary functions, and control the follicle development.</p>