Beili Chen

A novel mutation of HOXA10 in a Chinese woman with a Müllerian duct anomaly

BACKGROUND Müllerian duct anomalies consist of a set of congenital structural malformations that occur when the Müllerian ducts do not develop properly during embryonic life. Their molecular genetic basis is poorly understood. METHODS In this study, we conducted mutation analysis of the HOXA10 gene in a cohort of 109 Chinese women with Müllerian duct anomalies. RESULTS We identified a novel mutation (Y57C) in one patient with a didelphic uterus. The mutation affected the transcriptional regulation capacity of HOXA10. CONCLUSIONS Our study showed that mutation of HOXA10 gene may contribute to the development of Müllerian duct anomalies and confirmed that HOXA10 is an important transcription factor in reproductive tract development.

Differential Expression of Long Noncoding RNAs in Human Cumulus Cells Related to Embryo Developmental Potential A Microarray Analysis

Long noncoding RNAs (lncRNAs), which are prevalently transcribed in the genome, are involved in a variety of biological functions, yet little is known about their abundance in human cumulus cells (CCs) during oocyte development. Here, we describe the expression profile of lncRNAs in 3 pairs of cumulus cells from mature oocytes that result in high-quality embryo (H-CCs) and from oocytes that result in poor-quality embryo (P-CCs) using microarray analysis. In this study, a total of 20 563 lncRNAs were expressed in human CCs. One hundred and twenty four lncRNAs were consistently upregulated, and 509 lncRNAs were consistently downregulated in all samples analyzed (fold change ≥ 2.0, P < .05). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate 5 upregulated and 7 downregulated lncRNAs. The qRT-PCR results in the study were confirmed to be consistent with the microarray results. Network analysis was used for further research. The results displayed the differentially expressed lncRNAs in P-CCs between H-CCs, which suggested that lncRNAs may contribute to the processes of oocyte and early embryo development.

New candidate gene POU5F1 associated with premature ovarian failure in Chinese patients.

Premature ovarian failure (POF) is defined as a cessation of ovarian function in women before the age of 40 years. POU5F1 has a critical role in regulating pluripotency in embryonic development and POU5F1 transcription factor is down-regulated more than 30-fold in the POF candidate gene Nobox knockout ovaries. In order to identify the potential correlation between POU5F1 and the development of POF, the exon regions of POU5F1 were amplified and sequenced in 115 POF patients and 149 healthy controls. One non-synonymous variant of POU5F1 (c. C37A, p. Pro13Thr) was identified and confirmed in one of the POF patients. The substitution replacing a hydrophobic amino acid, proline, with a hydrophilic amino acid, threonine. As far as is known, the present study is the first to identify a potential association between POU5F1 and the development of POF.

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 INTEREST(S) 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.

Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure.

BackgroundThe WNT4 gene plays an important role in female sex determination and differentiation. It also contributes to maintaining of the ovaries and the survival of follicles.MethodsWe sequenced the coding region and splice sites of WNT4 in 145 Han Chinese women with premature ovarian failure (POF) and 200 healthy controls.ResultsOnly one novel variation, in Exon 2 (195C > T), was detected among the women with POF. However, this synonymous variation did not result in a change in amino acid sequence (65 Asp > Asp). No further variants were found in any of the samples.ConclusionAlthough we cannot provide any evidence that it is a possible disease-causing gene, this study is the first attempt to investigate the possible role of WNT4 in Han Chinese women with POF.

Increased New lncRNA–mRNA Gene Pair Levels in Human Cumulus Cells Correlate With Oocyte Maturation and Embryo Development

The close relationship between cumulus cells and oocyte indicates that the analysis of cumulus gene expression is a potential noninvasive method to aid embryo selection and in vitro fertilization outcome. Long noncoding RNAs (LncRNAs) could regulate essential pathways that contribute to human oocyte maturation, fertilization, and embryo development, which indicates that lncRNA would be valuable biomarkers. In our previous study, AK124742 is a newly detected lncRNA that was identified as being natural antisense to PSMD6, but its role in oocyte and embryo development is still not elucidated and needs to be investigated. Here, the expression of AK124742 and PSMD6 was measured in 40 pairs of cumulus cells from oocytes that result in high-quality embryos (HCCs) and from oocytes that result in poor-quality embryos (PCCs) by real-time quantitative reverse transcriptase polymerase chain reaction. The predictive value of AK124742 and PSMD6 was evaluated using a receiver–operating characteristic (ROC) curve. Notably, elevated expression levels of AK124742 and PSMD6 were observed in HCCs compared to PCCs (72.5% and 62.5%, respectively; P < .01). Expression of AK124742 was potentially positively associated with the PSMD6 levels. The relative expression levels of AK124742 and PSMD6 in the pregnancy group were significantly higher than those in the nonpregnancy group (P < .01).The area under the ROC curve of AK124742 was 0.78 (95% confidence interval: 0.64-0.93). In conclusion, AK124742 and PSMD6 as a new lncRNA–messenger RNA gene pair in human cumulus cells may be considered as potential biomarkers to aid embryo selection.

Haplotype analysis of chemokine CXCL12 polymorphisms and susceptibility to premature ovarian failure in Chinese women

BACKGROUND Chemokine (C-X-C motif) ligand 12 (CXCL12/stromal cell-derived factor 1) has been suggested to play an essential role in primordial germ cell migration, colonization and survival, and in the primordial to primary follicle transition. This study was performed to investigate an association of polymorphisms in CXCL12 with the risk of premature ovarian failure (POF) in Chinese patients. METHODS Tagging single nucleotide polymorphisms (SNPs) were selected using the Chinese HapMap database. Five SNPs (rs4948878, rs1801157, rs266087, rs266093 and rs1029153) were genotyped by direct sequencing in 111 patients with POF and 183 healthy controls recruited from the First Affiliated Hospital, Anhui Medical University, China. RESULTS Compared with controls, there were significantly higher frequencies of the rs1801157 A allele and haplotype C-T-A-T-T in cases with POF [P = 6.38E-07, odds ratio (OR) = 3.10, 95% confidence interval (CI) 1.955-4.890 by allele; P = 7.0E-04, OR = 2.39, 95% CI 1.43-3.97 by haplotype]. No differences were observed for the other four SNPs between POF cases and controls. CONCLUSIONS A strong association between a CXCL12 polymorphism and POF was established in Chinese patients, suggesting that CXCL12 might be a new candidate gene involved in POF. The A allele of CXCL12 polymorphism rs1801157 is a possible risk factor for developing POF. However, further independent studies are necessary to confirm our findings.

Sequence variants of KHDRBS1 as high penetrance susceptibility risks for primary ovarian insufficiency by mis-regulating mRNA alternative splicing

STUDY QUESTION Does a novel heterozygous KHDRBS1 variant, identified using whole-exome sequencing (WES) in two patients with primary ovarian insufficiency (POI) in a pedigree, cause defects in mRNA alternative splicing? SUMMARY ANSWER The heterozygous variant of KHDRBS1 was confirmed to cause defects in alternative splicing of many genes involved in DNA replication and repair. WHAT IS KNOWN ALREADY Studies in mice revealed that Khdrbs1 deficient females are subfertile, which manifests as delayed sexual maturity and significantly reduced numbers of secondary and pre-antral follicles. No mutation of KHDRBS1, however, has been reported in patients with POI. STUDY DESIGN SIZE, DURATION This genetic and functional study used WES to find putative mutations in a POI pedigree. Altogether, 215 idiopathic POI patients and 400 healthy controls were screened for KHDRBS1 mutations. PARTICIPANTS/MATERIALS, SETTING, METHODS Two POI patients were subjected to WES to identify sequence variants. Mutational analysis of the KHDRBS1 gene in 215 idiopathic POI patients and 400 healthy controls were performed. RNA-sequencing was carried out to find the mis-regulation of gene expression due to KHDRBS1 mutation. Bioinformatics was used to analyze the change in alternative splicing events. MAIN RESULTS AND THE ROLE OF CHANCE We identified a heterozygous mutation (c.460A > G, p.M154V) in KHDRBS1 in two patients. Further mutational analysis of 215 idiopathic POI patients with the KHDRBS1 gene found one heterozygous mutation (c.263C > T, p.P88L). We failed to find these two mutations in 400 healthy control women. Using RNA-sequencing, we found that the KGN cells expressing the M154V KHDRBS1 mutant had different expression of 66 genes compared with wild-type (WT) cells. Furthermore, 145 genes were alternatively spliced in M154V cells, and these genes were enriched for DNA replication and repair function, revealing a potential underlying mechanism of the pathology that leads to POI. LIMITATIONS REASONS FOR CAUTION Although the in vitro assays demonstrated the effect of the KHDRBS1 variant on alternative splicing, further studies are needed to validate the in vivo effects on germ cell and follicle development. WIDER IMPLICATIONS OF THE FINDINGS This finding provides researchers and clinicians a better understanding of the etiology and molecular mechanism of POI. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Ministry of Science and Technology of China (2012CB944704; 2012CB966702), National Research Institute for Family Planning (2017GJZ05), the National Natural Science Foundation of China (31171429) and Beijing Advanced Innovation Center for Structural Biology. The authors declare no conflict of interest.

The miR-449b polymorphism, rs10061133 A>G, is associated with premature ovarian insufficiency.

Objective:To determine if the miR-449b polymorphism, rs10061133 A>G, is associated with premature ovarian insufficiency (POI) pathogenesis. Methods:From January 2011 to December 2014, a total of 148 individuals with POI and 225 age-matched controls were collected from the Center for Reproductive Medicine, 1st Affiliated Hospital of Anhui Medical University (Hefei, China). Genotyping of miR-449b rs1006113 was performed using matrix-assisted laser desorption ionization time-of-flight-based mass spectrometry. Results:Rs10061133 A>G is a highly conserved SNP locus in the mature area of miR-449b. Association analysis shows that the rs10061133 AA genotype is a risk factor for POI. Conclusions:Our study provides the first evidence that the miR-449b rs10061133 AA genotype is associated with POI risk.

Successful preimplantation genetic diagnosis by targeted next-generation sequencing on an ion torrent personal genome machine platform

Hearing loss may place a heavy burden on the patient and patients family. Given the high incidence of hearing loss among newborns and the huge cost of treatment and care (including cochlear implantation), prenatal diagnosis is strongly recommended. Termination of the fetus may be considered as an extreme outcome to the discovery of a potential deaf fetus, and therefore preimplantation genetic diagnosis has become an important option for avoiding the birth of affected children without facing the risk of abortion following prenatal diagnosis. In one case, a couple had a 7-year-old daughter affected by non-syndromic sensorineural hearing loss. The affected fetus carried a causative compound heterozygous mutation c.919-2 A>G (IVS7-2 A>G) and c.1707+5 G>A (IVS15+5 G>A) of the solute carrier family 26 member 4 gene inherited from maternal and paternal sides, respectively. The present study applied multiple displacement amplification for whole genome amplification of biopsied trophectoderm cells and next-generation sequencing (NGS)-based single nucleotide polymorphism haplotyping on an Ion Torrent Personal Genome Machine. One unaffected embryo was transferred in a frozen-thawed embryo transfer cycle and the patient was impregnated. To conclude, to the best of our knowledge, this may be the first report of NGS-based preimplantation genetic diagnosis (PGD) for non-syndromic hearing loss caused by a compound heterozygous mutation using an Ion Torrent Personal Genome Machine. NGS provides unprecedented high-throughput, highly parallel and base-pair resolution data for genetic analysis. The method meets the requirements of medium-sized diagnostics laboratories. With decreased costs compared with previous techniques (such as Sanger sequencing), this technique may have potential widespread clinical application in PGD of other types of monogenic disease.