Premature ovarian insufficiency(POI)is a severe female repro-ductive disorder that affects 1％of women in general populations(European Society for Human Reproduction and Embryology[ESHRE]Guideline Group on POI et al.,2016).An increasing preva-lence up to 3.7％has been reported in a recent meta-analysis(Golezar et al.,2019).POI can lead to infertility or subfertility,as well as a range of complex complications suffering multi-organ sys-tems,seriously threatening women's health and reducing the life quality.By contrast,POI is a representative heterogeneous disease with multiple etiologies.While more than 70 causative POI genes have been identified,the etiology of more than half of the POI patients is still ambiguous(Jiao et al.,2018).Unreported POI causative genes,therefore,remain to be identified.

AbstractBackgroundPremature ovarian insufficiency (POI) is a highly heterogeneous disease, and up to 25% of cases can be explained by genetic causes. The transcription factor WT1 has long been reported to play a crucial role in ovary function. Wt1‐mutated female mice exhibited POI‐like phenotypes.Methods and ResultsIn this study, whole exome sequencing (WES) was applied to find the cause of POI in Han Chinese women. A nonsense variant in the WT1 gene: NM_024426.6:c.1387C>T(p.R463*) was identified in a non‐syndromic POI woman. The variant is a heterozygous de novo mutation that is very rare in the human population. The son of the patient inherited the mutation and developed Wilms’ tumor and urethral malformation at the age of 7. According to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, the novel variant is categorized as pathogenic. Western blot analysis further demonstrated that the WT1 variant could produce a truncated WT1 isoform in vitro.ConclusionsA rare heterozygous nonsense WT1 mutant is associated with non‐syndromic POI and Wilms’ tumor. Our finding characterized another pathogenic WT1 variant, providing insight into genetic counseling.

BackgroundPremature ovarian insufficiency (POI) is a common disease in women that leads to a reduced reproductive lifespan. The aetiology of POI is genetically heterogeneous, with certain double-strand break (DSB) repair genes being implicated in POI. Although non-homologous end joining (NHEJ) is an efficient DSB repair pathway, the functional relationship between this pathway and POI remains unknown.Methods and resultsWe conducted whole-exome sequencing in a Chinese family and identified a rare heterozygous loss-of-function variant in non-homologous end joining factor 1 (NHEJ1): c.532C>T (p.R178*), which co-segregated with POI and irregular menstruation. The amount of NHEJ1 protein in the proband was half of the normal level, indicating a link between NHEJ1 haploinsufficiency and POI. Furthermore, another rare heterozygous NHEJ1 variant c.500A>G (p.Y167C) was identified in one of 100 sporadic POI cases. Both variants were predicted to be deleterious by multiple in silico tools. In vitro assays showed that knock-down of NHEJ1 in human KGN ovarian cells impaired DNA repair capacity. We also generated a knock-in mouse model with a heterozygous Nhej1 variant equivalent to NHEJ1 p.R178* in familial patients. Compared with wild-type mice, heterozygous Nhej1-mutated female mice required a longer time to first birth, and displayed reduced numbers of primordial and growing follicles. Moreover, these mice exhibited higher sensitivity to DSB-inducing drugs. All these phenotypes are analogous to the progressive loss of ovarian function observed in POI.ConclusionsOur observations in both humans and mice suggest that NHEJ1 haploinsufficiency is associated with non-syndromic POI, providing novel insights into genetic counselling and clinical prevention of POI.

Premature ovarian insufficiency (POI) is a severe female reproductive disorder that affects 1%−2% of women in general populations.1 The analysis of familial POI implies that genetic aberrations strongly influence the onset of POI.2 However, a large proportion of POI cases remain idiopathic, suggesting that novel causative or susceptible factors are yet to be discovered.3 Here, we identify HSD17B12 dosage insufficiency as a novel mechanism underlying human POI. A nonconsanguineous Han Chinese pedigree with two daughters exhibiting POI was investigated (Table S1). The proband (II-1, Figure 1A) presented primary amenorrhea. Ultrasound examination revealed the infantile uterus (size: 19 × 14 × 18 mm) and invisible ovaries. She was treated with hormone replacement therapy at age 19 to induce menarche and maintain sexual development and cyclical bleeding. Her younger sister (II-2) experienced normal first menarche at 12 years old. But she was diagnosed with early-onset POI at 13 years old when occurring amenorrhea. Both sisters have normal 46,XX karyotypes. Their FMR1 CGG repeat lengths are in the regular polymorphic range. Both parents were reported as healthy with normal high-resolution karyotypes. The mother had normal pubertal development and continued to have regular menstrual periods at her age of 49. No history of associated endocrinopathies or autoimmune disorders was found in this family. We conducted whole-exome sequencing and subsequent array-based comparative genomic hybridization (aCGH) and found that both index POI sisters harbored compound heterozygous variants in HSD17B12 (Figure 1A), including a novel missense variant (M1: c.610G > C, p.A204P) and a novel deletion (M2). The maternally inherited missense variant (Figure 1B) was confirmed by Sanger sequencing (Table S2) and predicated to be pathogenic by bioinformatic tools (Table 1). Additionally, long-range polymerase chain reaction (PCR) confirmed a paternally inherited deletion affecting HSD17B12 exons 5 and 6 (Figure 1C). HSD17B12 (hydroxysteroid 17-beta dehydrogenase 12) encodes a major enzyme responsible for the estrone to estradiol (E2) conversion.4 Importantly, the A204 position of HSD17B12 is conserved according to phyloP and phastCons programs (Table 1), and the p.A204P variant could disturb the structural stability of HSD17B12 by missing hydrogenbonds formed between A204 and other amino acids (Figure S1). Additionally, both HSD17B12 missense and deletion variants carried by both index sisters are located in the crucial short-chain dehydrogenase/reductase domain (Figure 1D) that is conserved during evolution.5 Dramatically, no obvious HSD17B12 was detected in the blood samples of index sisters (Figure S2), indicating deleterious effects of HSD17B12 variants. To further investigate the pathogenesis of bi-allelic HSD17B12 variants in POI, we knocked these variants into human ovarian KGN cells using CRISPR-Cas9 system (Table S2). Firstly, we sequenced the low-abundance complementary DNA of HSD17B12M2/M2 KGN cells and revealed that the M2 deletion of HSD17B12 exons 5 and 6 (c.392_501del) created a frameshift in canonical HSD17B12 (NP_057226.1: p.V131Dfs*51) (Figure S3). Secondly, quantitative reverse transcription PCR (RT-PCR) demonstrated that HSD17B12+/M2 KGN cells expressed only a half abundance of HSD17B12 when compared with wild-type (WT) controls (Figure 1E), suggesting the possibility of nonsense-mediated mRNA decay.6 Thirdly, we observed a further reduced expression of HSD17B12 in HSD17B12M1/M2 KGN cells (Figure 1E,F). The above experimental evidence suggested a gene dosage effect caused by the combination of paternally and maternally inherited deleterious variants in HSD17B12. Furthermore, we generated gene-edited mouse models to investigate in vivo function of Hsd17b12 (Figure S4). We crossed Hsd17b12+/− and Hsd17b12+/A204P mice of 8 weeks old. However, no Hsd17b12−/A204P mice were found at birth (Table S3), which was due to the embryogenic lethality at E11.5. Coincidentally, we failed to obtain Hsd17b12−/− or Hsd17b12A204P/A204P live mice by intercrossing heterozygous mutants, since they died at E8.5 and P0, respectively (Table S3). These experimental observations indicate that mice are more vulnerable than human subjects to HSD17B12/Hsd17b12 dosage insufficiency. These divergent phenotypes between bi-allelic HSD17B12/Hsd17b12-mutated humans and mice might be attributed by that the 17b-hydroxysteroid dehydrogenases are largely multifunctional enzymes, and their functions depend on the substrates in different species or the isoenzymes.7 Since bi-allelic Hsd17b12-mutated mice were embryonically lethal, heterozygous Hsd17b12-mutated adult mice were recruited to determine the effects of Hsd17b12 variants on female fertility. Although there was no significant difference in ovarian index between heterozygous Hsd17b12-mutated and WT groups (Table S4), significantly decreased mRNA abundances of Hsd17b12 were observed in the ovaries of Hsd17b12+/− and Hsd17b12+/A204P female mice when compared with their WT littermates (Figure 2A). Subsequently, Hsd17b12+/− and Hsd17b12+/A204P female mice of 8 weeks old and their WT control littermates were mated with WT adult males for up to 6 months. Surprisingly, although the offspring fit a conventional pattern of Mendelian inheritance (Table S5), both Hsd17b12+/− and Hsd17b12+/A204P female mice showed significantly longer times between pregnancies and smaller litter sizes than WT controls (Table 2). Additionally, after 2 months daily examination of estrus cycle, Hsd17b12+/− female mice showed inordinate prolongations in the metestrus and diestrus and so that the entire estrous cycle when compared to WT controls (Figure 2B,C). To further visualize the potential female subfertility, the morphologies of follicles at different developmental stages were examined by hematoxylin and eosin staining. Both Hsd17b12+/− and Hsd17b12+/A204P female mice showed significantly reduced numbers of developing-follicles in their ovaries when compared to WT controls (Figure 2D), indicating the correlation between Hsd17b12 dosage insufficiency and female subfertility in mice. As for Hsd17b12 homozygous mutants, Hsd17b12A204P/A204P mice lived till P0; therefore, the P0 female mice were used for three-dimensional structure reconstruction of cleared-ovaries, which showed smaller and thinner ovaries, and consistently, fewer primordial follicles in Hsd17b12A204P/A204P female mice than those in WT controls (Figure S5), further suggesting that Hsd17b12 is essential for ovarian morphology and reserve. HSD17B12 was previously reported in the regulation of female reproduction potentially via the metabolism of arachidonic acid, a precursor of prostaglandins.8-10 Coincidently, the metabolic processes of arachidonic acid and related products are affected in the ovaries of heterozygous Hsd17b12-mutated female mice (Figure S6). In conclusion, our observations based on human POI subjects, gene-edited KGN cell, and mouse models supported the pathogenic roles of HSD17B12 deleterious variants and their associated HSD17B12 dosage insufficiency in POI. HSD17B12 is genetically involved in human early-onset POI and even primary amenorrhea via the autosomal recessive inheritance. The authors would like to thank the family for participating and supporting this study. This work was supported by National Natural Science Foundation of China (grant numbers: 31625015 and 31521003), Shanghai Municipal Science and Technology Major Project (grant number: 2017SHZDZX01), State Key Laboratory of Reproductive Medicine (grant number: SKLRM-K202002), Science and Technology Major Project of Inner Mongolia Autonomous Region of China (grant number: zdzx2018065), and the 111 Project (grant number: B13016). The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Losing of ovarian functions prior to natural menopause age causes female infertility and early menopause. Premature ovarian insufficiency (POI) is defined as the loss of ovarian activity before 40 years of age. Known genetic causes account for 25-30% of POI cases, demonstrating the high genetic heterogeneity of POI and the necessity for further genetic explorations. Here we conducted genetic analyses using whole-exome sequencing in a Chinese non-syndromic POI family with the affected mother and at least four affected daughters. Intriguingly, a rare missense variant of BUB1B c.273A>T (p.G1n91His) was shared by all the cases in this family. Furthermore, our replication study using targeted sequencing revealed a novel stop-gain variant of BUB1B c.1509T>A (p.Cys503*) in one of 200 sporadic POI cases. Both heterozygous BUB1B variants were evaluated to be deleterious by multiple in silico tools. BUB1B encodes BUBR1, a crucial spindle assembly checkpoint component involved in cell division. BUBR1 insufficiency may induce vulnerability to oxidative stress. Therefore, we generated a mouse model with a loss-of-function mutant of Bub1b, and also employed D-galactose-induced aging assays for functional investigations. Notably, Bub1b(+/-) female mice presented late-onset subfertility, and they were more sensitive to oxidative stress than wild-type female controls, mimicking the clinical phenotypes of POI cases affected by deleterious BUB1B variants. Our findings in human cases and mouse models consistently suggest, for the first time, that heterozygous deleterious variants of BUB1B are involved in late-onset POI and related disorders.

Premature ovarian insufficiency (POI) is a major cause of reduced female fertility and affects approximately 1% women under 40 years of age. Recent advances emphasize the genetic heterogeneity of POI. Fanconi anemia (FA) genes, traditionally known for their essential roles in DNA repair and cytogenetic instability, have been demonstrated to be involved in meiosis and germ cell development. Here, we conducted whole-exome sequencing (WES) in 50 Han Chinese female patients with POI. Rare missense variants were identified in FANCA ( Fanconi anemia complementation group A ): c.1772G > A (p.R591Q) and c.3887A > G (p.E1296G). Both variants are heterozygous in the patients and very rare in the human population. In vitro functional studies further demonstrated that these two missense variants of FANCA exhibited reduced protein expression levels compared with the wild type, suggesting the partial loss of function. Moreover, mono-ubiquitination levels of FANCD2 upon mitomycin C stimulation were significantly reduced in cells overexpressing FANCA variants. Furthermore, a loss-of-function mutation of Fanca was generated in C57BL/6 mice for in vivo functional assay. Consistently, heterozygous mutated female mice ( Fanca + / − ) showed reduced fertility and declined numbers of follicles with aging when compared with the wild-type female mice. Collectively, our results suggest that heterozygous pathogenic variants in FANCA are implicated in non-syndromic POI in Han Chinese women, provide new insights into the molecular mechanisms of POI and highlight the contribution of FANCA variants in female subfertility.

OBJECTIVES:X-linked hypophosphataemic rickets (XLHR) is a disease of impaired bone mineralization characterized by hypophosphataemia caused by renal phosphate wasting. The main clinical manifestations of the disorder are O-shaped legs, X-shaped legs, delayed growth, and bone pain. XLHR is the most common inheritable form of rickets, with an incidence of 1/20 000 in humans. It accounts for approximately 80% of familial cases of hypophosphataemia and serves as the prototype of defective tubular phosphate (PO43+) transport, due to extra renal defects resulting in unregulated FGF23 activity. XLHR is caused by loss-of-function mutations in the PHEX gene. The aim of this research was to identify the genetic defect responsible for familial hypophosphataemic rickets in a four-generation Chinese Han pedigree and to analyze the function of this mutation. METHODS:The genome DNA samples of all members in the pedigree were extracted from whole blood. We sequenced all exons of the PHEX and FGF23 genes, as well as the adjacent splice site sequence with Sanger sequencing. Next, we analyzed the de novo mutation c.1692 del A of the PHEX gene with an online digital service and investigated the mutant PHEX with SWISS-MODEL, immunofluorescence, and protein stability detection. RESULTS:Through Sanger sequencing, we found a de novo mutation, c.1692 del A, in exon 16 of the PHEX gene in this pedigree. This mutation can make the PHEX protein become unstable and decay rapidly, which results in familial XLHR. CONCLUSION:We have found a de novo loss-of-function mutation, c.1692 del A, in exon 16 of the PHEX gene that can cause XLHR.Cite this article: J. Huang, X. Bao, W. Xia, L. Zhu, J. Zhang, J. Ma, N. Jiang, J. Yang, Q. Chen, T. Jing, J. Liu, D. Ma, G. Xu. Functional analysis of a de novo mutation c.1692 del A of the PHEX gene in a Chinese family with X-linked hypophosphataemic rickets. Bone Joint Res 2019;8:405-413. DOI: 10.1302/2046-3758.88.BJR-2018-0276.R1.

Premature ovarian insufficiency (POI) is a severe female disorder characterized by primary or secondary amenorrhea before 40 years of age. Genetic factors have been implicated in the pathogenesis of POI, but known POI-associated genes account for only a small fraction of heritability. Here, we performed whole-exome sequencing (WES) to explore pathogenic genes in Han Chinese subjects with POI. Intriguingly, we identified novel or rare heterozygous missense variants of SALL4 (spalt-like transcription factor 4) in 3 (6%) of 50 POI subjects. The SALL4 c.541G>A and c.2279C>T variants were paternally inherited, while c.1790A>G was inherited from an affected mother with early menopause. SALL4 encodes a transcription factor that is highly expressed in oocytes and early embryos. Our in vitro functional assays suggested that all of these SALL4 missense variants had significantly increased SALL4 protein expression with enhanced regulatory activity in regard to its downstream target POU5F1 compared to that of wild-type SALL4. Notably, previous studies demonstrated the genetic involvement of SALL4 loss-of-function variants in Okihiro syndrome and related syndromic developmental disorders. Through our analysis of genotype–phenotype correlations, we suggest that different variation types of SALL4 might have different effects on SALL4 activity, resulting in phenotypic variability. Our findings highlight the genetic contribution of SALL4 missense variants with enhanced regulatory activities to POI and underscore the importance of variant classification and evaluation for molecular diagnosis and genetic counseling.

The DC distribution network can integrate the photovoltaic power generation system with high efficiency, but the random fluctuation of the photovoltaic output power is strong and there is a problem of PV consumption, which has a certain degree of impact on the economic optimization operation of the system. Therefore, the source-network-load-storage optimization interaction concept is introduced in the DC distribution network. Based on the in-depth analysis of the interactive load characteristics, the interaction load and energy storage technology can be fully utilized to the system to consumpt the photovoltaic. Combined with the features of DC distribution network structure, comprehensively consider the impact of the interactive load and energy storage system collaborative optimization on the system operating cost and power flow distribution, so that both the power generation side and the power side can participate in the resource optimization configuration of the grid operation. A multiobjective optimal scheduling model for photovoltaic DC distribution networks with operational cost, network loss and voltage deviation is established. The simulation example shows that: Collaborative optimization using interactive load and energy storage system can greatly reduce system operating cost, network loss and voltage deviation, effectively improve the level of PV consumption, and realize safe and reliable operation of DC distribution network.

Plant homeodomain finger 2 (PHF2) is a JmjC family histone demethylase that demethylates H3K9me2, a repressive gene marker. PHF2 was found to play a role in the differentiation of several tissue types such as osteoblast and adipocyte differentiation. We report here that PHF2 plays a role in the epigenetic regulation of megakaryocytic (MK) and erythroid differentiation. We investigated PHF2 expression during MK and erythroid differentiation in K562 and human CD34+ progenitor (hCD34+ ) cells. Our data demonstrate that PHF2 expression is down-regulated during megakaryopoiesis and erythropoiesis. PHF2 has a negative role in MK and erythroid differentiation of K562 cells; knockdown of PHF2 promotes MK and erythroid differentiation of hCD34+ cells. Similarly, we found that p53 expression is also down-regulated during MK and erythroid differentiation, which parallels PHF2 expression. PHF2 binds to the p53 promoter and regulates the expression of p53 by demethylating H3K9me2 in the promoter region of p53. Taken together, our data show that PHF2 is a negative epigenetic regulator of MK and erythroid differentiation, and that one of the pathways through which PHF2 affects MK and erythroid differentiation is via regulation of p53 expression.

Background: The aim of this study is to investigate the prevalence of the fragile X mental retardation 1 (FMR1) gene premutation in Han Chinese women with primary ovarian insufficiency (POI) using a rapid and cost-effective method. Methods: A total of 153 Han Chinese women with sporadic POI were systematically analyzed for trinucleotide repeats within the FMR1 gene. We employed an improved strategy to screen for cytosine-guanine-guanine repeats in the 5' untranslated region of the FMR1 gene. Before using the previously reported FragilEase polymerase chain reaction (PCR) method for premutation identification, we developed a new cost-effective PCR-based method to exclude most of the normal allele carriers during the initial screening stage. Results: In our initial screening, 62.1% of women with POI were found to carry heterozygous normal alleles of FMR1, which were recognized by our sensitive and cost-effective method. The remaining women were further screened for the presence of the FMR1 premutation. We identified a Han Chinese woman with a premutation allele of FMR1 out of 153 sporadic POI women (0.7%). Conclusions: The frequent FMR1 premutation in Caucasian individuals with POI may not be a common genetic cause of sporadic POI in the Han Chinese population.