Karyotype Analysis And Transcription Factors Of SOHLH2and NR5A1in Premature Ovarian Failure

PartⅠCytogenetic Analysis of Chinese Women with Premature Ovarian FailureBackground:Premature ovarian failure (POF) is a complex gynecological endocrinal disorder with significantly genetic and clinical heterogeneity. POF occurs in1%of the general female population prior to age40years; however, the incidence has being increasing in recent years. Given no reliable marker for early diagnosis or effective treatment for improvement of ovarian function, it is of necessity to conduct the etiology study and identify those who are at high risk for POF. Chromosomal abnormalities have long been recognized as a frequent and main cause of POF, with widely varying percentages in reported series. Although numerous different karyotypic anomalies have been found, few large cohorts of POF have been studied. The most common abnormality involved an X chromosome. One half of POF patients with45,X monosomy and anomalies in short arm of X chromosome presented with primary amenorrhea and gonadal dysgenesis. And the majority of patients with45,X mosaicism and anomalies in long arm of X chromosome presented with secondary amenorrhea. Specific abnormal karyotypes with less severe perturbations of ovarian function might be at risk of being transmitted to offspring. Therefore, it is of significance to determine the prevalence and distribution of cytogenetic anomalies in this ethnic group with POF.Objective:The aim of the present study is to analyze the karyotype of large-scale Chinese patients with POF, identify the prevalence and distribution of chromosomal abnormalities, and their association with clinical phenotype, and therefore provide theoretical basis to further etiological research, genetic counseling and family planning.Methods:A total of531women with POF were recruited from2003to2011. Karyotype analysis was performed on GTG-banded metaphase chromosomes using a standard protocol. Analysis of the prevalence and distribution of anomalies, identification of hot region with abnormalities were performed. Distribution differences were compared between women with primary and those with secondary amenorrhea. Association of karyotype with phenotype was determined according to corresponding clinical manifestation.Results:Chromosomal abnormalities were detected in64of531cases (12.1%). Of the64,60(93.7%) involved the X chromosome. The most common abnormality was X-structural aberrations. We detected17terminal deletions [15del(Xq);2del(Xp)],11isochromosomes [6i(Xp);5i(Xq)],1ring X,1inv X,1isodicentric and1complex X arrangement. Nine X-autosome translocations were detected. All but one involved Xq, usually in regions Xq22-Xq24. Aneuploidy without a structurally abnormal X was found in19cases, including745,X monosomy,945,X mosaicisms, followed by347,XXX. Karyotypic abnormalities were more frequent in patients with primary amenorrhea (15/70,21.4%) than those with secondary amenorrhea (49/461,10.6%)(P=0.01).45,X and45,X/46,XX mosaicism were the complements most frequently associated with primary amenorrhea (46.7%). Two of the three cases with46,XY or 45,X/46,XY karyotype presented with’secondary amenorrhea’. One balanced autosomal Robertsonian translocation was also detected.Conclusion:In summary, this report of the largest cohort of Chinese women yet studied found the prevalence of chromosomal abnormalities in POF to be12.1%, most frequent anomalies involving X chromosome (93.7%). This confirms a major role for X chromosome abnormalities in POF, and highlights the importance of routine assessment of chromosomal abnormalities. Chromosomal studies thus provide valuable clinical information for reproductive management and genetic counseling. In addition to providing an etiologic explanation for the individual patient with POF, the cases facilitate identification of genes responsible for POF. PartⅡ Germ Cell-specific Transcription Factor SOHLH2in Premature Ovarian FailureBackground:Establishment of the initial primordial follicle pool and subsequent maintenance is important determinant of female reproductive lifespan and fertility potential. Oogenesis occurs with folliculogenesis through concomitant crosstalk signaling in mammals. This well-orchestrated dynamics must be, in part, dictated by coordinated interactions of genes preferentially expressed in oocytes, which in turn are modulated by germ cell specific transcriptional regulators. Several transcriptional regulators preferentially expressed in oocytes, such as FIGLA, NOBOX, LHX8, SOHLH1, and SOHLH2, established complex specialized regulatory networks to regulate ovary-specific gene (e.g., BMP15, GDF9, ZP1-3) expression pivotal for early folliculogenesis. SOHLH2encodes the spermatogenesis-and oogenesis-specific bHLH transcription factor and plays pivotal roles in early oogenesis and folliculogenesis. Its expression is uniquely confined to germ cell clusters of embryonic ovary and oocytes of primordial to primary follicle stage. Sohlh2-null female mice exhibited defective primordial-to-primary follicle transition, atrophied ovaries devoid of follicles and infertility, mimicking human POF phenotype. Therefore, SOHLH2is an attractive candidate gene of POF.Obejective:The present study was designed to determine whether SOHLH2variants contribute to POF in a large cohort of non-syndromic POF patients of Chinese (n=364) and Serbian (n=197) origin.Methods:A total of561women with POF of Han Chinese (n=364) and Serbian (n=197) origin were recruited. Ethnically matched healthy women served as controls (400Chinese and200Serbian). All were screened for variants in the SOHLH2gene by Sanger sequencing. Novel Variants were sequence-aligned and functional predicted by various online tools.Results:Eleven distinct novel heterozygous variants were identified in cohorts of POF but were absent in matched controls. These included the non-synonymous variants p.Glu79Lys (n=2cases), p.Glu105Gly, and p.Thr321Pro, which were found among four Chinese POF cases, and p.Leu120Phe (n=3cases) and p.Leu204Phe, which were found among four Serbian women. Protein alignments reveal that p.Glu79Lys and p.Glu105Gly involve amino acids highly conserved among mammals, both of which are predicted to be deleterious. The c.-210G>T found in the Chinese POF cohort lies in the core promoter region, which is enriched with transcription factor binding sites and CpG islands. In the Serbian cohort, the variant most likely to have a deleterious effect is c.530+6T>G, which is predicted to affect RNA splicing and result in nonsense mediated decay of transcripts. Disturbing the expression, transactivation or homo-/hetero-dimerization of the SOHLH2protein could result in ovarian failure.Conclusion:Our identification of novel variants in the SOHLH2gene, in women with POF of both Chinese and Serbian origin, suggests a contribution to the etiology of POF. This indicates the pivotal role of the transcription factor SOHLH2in early oogenesis and folliculogenesis. PartⅡSomatic Transcription Factor NR5A1Mutation in Premature Ovarian FailureBackground:The etiology of premature ovarian failure (POF) is genetically heterogenous, and no predominant explanation has been documented. Nuclear receptor subfamily5, group A, member1(NR5A1) encodes steroidogenic factor1(SF1), a nuclear receptor involved in adrenal and gonadal development, steroidogenesis, and reproduction. SF1serves as a master transcriptional regulator of multiple genes, including STAR, CYP11A1, CYP19A1, AMH, INHA, and LHB. Human SF1protein is mainly expressed in ovarian somatic cells of growing follicles and corpus lutea. Granulosa cell-specific Nr5al knockout mice exhibit infertility, hypoplastic ovaries with decreased follicles and absence of corpora lutea. These findings indicate a critical role of NR5A1in ovarian development and function and its disruption might involve in the pathogenesis of human POF.Objective:The present study was designed to determine whether NR5A1variants account for women with nonsyndromic POF in Han Chinese.Methods:Mutation screening in the NR5A1gene in a large cohort of Chinese women with non-syndromic POF (400cases) were performed with direct sequencing. Subsequently, functional prediction using Align GVGD, Polyphen-2and SIFT, transactivation activity assay using Dual-Luciferase Reporter Assay System, and protein expression and nuclear localization with immunofluorescence were conducted to determine the causative role of the novel variants identified in vitro.Results:A novel heterozygous missense variant-c.13T>G (p.Tyr5Asp) in exon2was identified. The mutated tyrosine, located seven residues upstream of the DNA-binding domain (DBD), was highly conserved among SF1orthologs. This non-synonymous variant impaired transcriptional activation on Amh, Inhibin-a, Cypl1al and Cypl9al gene, as shown by transactivation assays. However, no dominant negative effect was observed, nor was there impact on protein expression and nuclear localization.Conclusion:A novel heterozygous variant p.Tyr5Asp, in a novel non-domain region of NR5A1, was identified in the large cohort of Chinese women with POF. This missense variant is presumed to result in haploinsufficiency and impairment of transactivation activity on different SF1target genes. Irrespectively, perturbation in NR5A1is not a common explanation for POF in Chinese.