Novel WT1 Missense Mutations in Han Chinese Women with Premature Ovarian Failure

Premature ovarian failure (POF) is a heterogeneous disease. Though dozens of candidate genes have been identified for the genetic etiology of POF, it is largely unexplained in majority of patients. Recently, Wt1+/R394W mice was found to present POF-like phenotype, which indicates that WT1 might be a plausible candidate gene for non-syndromic POF. The coding region of WT1 gene was screened in 384 patients with POF and 6 novel variations were identified, including two missense mutations (p. Pro126Ser in exon1 and p. Arg370His in exon7) and four intronic variants (c.647-27C > T, c.647-13G > C, c.647-13G > A in intron1 and c.950 + 14T > C in intron 4). In vitro experiments showed that both mutant p. Pro126Ser and p. Arg370His repressed the expression of Amh and Cdh1, and induced the expression of Fshr and Cyp19 in mRNA level (P < 0.05). The expression changes of AMH, FSHR, CYP19 and CDH1 were confirmed by western blot. These genes (AMH, FSHR, CYP19 and CDH1) are required for granular cells (GCs) proliferation, differentiation and oocyte-GCs interaction. The novel mutant p. P126S and p. R370H in the WT1 gene potentially impaired GCs differentiation and oocyte-GCs interaction, which might result in loss of follicles prematurely. Therefore, WT1 is a plausible causal gene for POF.

WT1 is expressed in Sertoli cells of the tests and granulosa cells (GCs) of ovarian follicles 13 . In rat, the expression of WT1 in GCs decreased along with the follicular development. WT1 was speculated as a repressor of genes responsible for GCs proliferation and differentiation 14 . More recently, the Wt1 +/R394W mice (a common WT1 missense mutation in Denys-Drash syndrome) has been shown to be subfertile due to defect in follicle development. Compared to controls, the Wt1 +/R394W mice have smaller ovaries and significantly fewer follicles, which is similar to the characteristics of patients with POF 15 . These findings indicate that the WT1 gene play a critical role in follicle development and might be a plausible candidate for POF. Therefore, we screened the coding region of WT1 gene in 384 Chinese women with POF to determine its contribution involved in the etiology of POF.

Results
Clinical characteristics of patients and two novel missense mutations identified within gene WT1. Clinical characteristics of the 384 subjects were showed in Table 1. Two novel heterozygous missense mutations c.376C > T (p. Pro126Ser, p. P126S) in exon 1 and c.1109G > A (p. Arg370His, p. R370H) in exon 7 were identified in two patients, respectively (Fig. 1A). The two variants were absent in either dbSNP database or 384 control females. The amino acids involved were highly conserved among WT1 orthologs (Fig. 1B).
The patient carrying p. P126S mutation was 28 years old. She suffered from primary amenorrhea with elevated serum FSH (77.23 IU/L). The other patient with p. R370H developed spontaneous menarche at the age of 16, and experienced irregular menses and subsequent amenorrhea at the age of 31. Trans-vaginal ultrasonography showed normal uterus and small ovaries with severe fibrosis. No other family member of the two carriers had POF or a form of 46, XY disorders of sex development (DSD).
Mutant p. P126S and p. R370H impaired the regulations of AMH, FSHR, CYP19 and CDH1 expression. To explore the effect of mutant p. P126S and p. R370H on transcription, adenovirus vectors containing Wt1 gene were generated and transfected to differentiated GCs from three-weeks-mouse. Transcription level of down-stream genes including Amh, Fshr, Lhr, Cyp19, Cdh1 and Par6b were detected by qRT-PCR. As shown in Fig. 2A,B, in the wild type (WT) Wt1-overexpressing differentiated GCs, the expression of Amh, Cdh1, and Par6b was up-regulated while Fshr, Lhr, and Cyp19 down-regulated by Wt1, which was consistent with previous studies (15). In both p. P126S and p. R370H transfected GCs, the expression of Amh and Cdh1 was decreased and expression of Fshr and Cyp19 was increased (P < 0.05). Given the heterozygous status of two patients carrying p. P126S and p. R370H, dominant negative effect of the mutant over WT protein was further investigated. Co-transfection of WT with mutant p. P127S and p. R387H did not interfere with the transcription of WT protein ( Fig. 2A,B), which did not support the dominant negative effect.
Western blot was performed to compare the expression of AMH, FSHR, CYP19 and CDH1 between WT Wt1 and mutant Wt1-overexpressed GCs. As shown in Fig. 2C, the expression of FSHR and CYP19 were repressed in WT Wt1-transfected GCs; whereas higher expression was observed in GCs transfected either with p. P126S or with p. R370H. The level of AMH and CDH1 was significantly lower in GCs overexpressed either with p. P126S or with p. R370H compared with WT Wt1. These findings confirmed the impaired regulations of mutant p. P126S and p. R370H on AMH, FSHR, CYP19 and CDH1.

Discussion
The p. P126S mutant located in the proline/glutamine rich DNA-binding domain and p. R370H was adjacent to the four zinc-finger domains. The zinc-finger domains are crucial for WT1 to acquire the ability to interact with transcription and splice factors 16 . In mice, Wt1 plays a key role in follicular development regulating these genes associated with GCs proliferation and differentiation, e.g. Amh, Fshr, Lhr and Cyp19, and GCs polarity, e.g. Cdh1 and Par6b 15 . Dysregulation of these genes could result in immature differentiation of GCs and impaired establishment of polarity, and then aberrant follicular development.
FSHR is detected in GCs from primary follicle and remains present throughout folliculogenesis 17 . CYP19, encoding aromatase cytochrome P450 (P450arom), is expressed in GCs of large follicles and corpus luteum 18 . FSHR and CYP19 trigger the proliferation and differentiation of GCs, which is important for normal follicular growth [19][20][21] . As a transcriptional repressor of FSHR and CYP19, WT1 inhibits the premature proliferation and differentiation of GCs. In this study, mutant p. P126S and p. R370H induced FSHR and CYP19 expression, resulting in possible impaired estrogen synthesis, immature proliferation and differentiation of GCs.
AMH is expressed in primary follicles and preantral/small antral follicles [22][23] . AMH inhibits the recruitment of primordial follicles and decreases the responsiveness of growing follicles to FSH 24 . Our results revealed that mutant p. P126S and p. R370H repressed the expression of AMH, which would result in increased sensitivity to FSH. Therefore, it was speculated that p. P126S and p. R370H increased the FSH-responsiveness of follicles and promote the activation of primordial follicles.
In the early growing follicle, adherent junctions (AJs) are key adhesion complex 25 . Cadherins and nectins are two important molecules associated with AJs. The most well-characterized cadherins are E-cadherin (official symbol CDH1) and N-cadherin (official symbol CDH2) 26 . CDH1 is a calcium-dependent cell adhesion protein localized at the adherent junctions and serves as a marker for epithelial differentiation 27 . It is essential for maintaining the cuboidal shape of GCs, providing a means of rotating the orientation of mitotic axes and enabling multi-layering of GCs 28 . In this study, mutant p. P126S and p. R370H repressed the expression of CDH1 protein. It is speculated that declined CDH1 levels could not maintain the oocyte-GC interaction and the loss of granulosa cell adhesion would result in follicular atresia 25,29 .
To our knowledge, this is the first study to explore the role of the WT1 gene in human POF. Two novel heterozygous mutations p. P126S and p. R370H were identified, and resulted in impaired transcription on downstream genes, including AMH, FSHR, CYP19 and CDH1. They could interfere with the proliferation and differentiation of GCs and disrupt the oocyte-GC interaction. Further studies focusing on the signaling pathways associated with WT1 in ovary are warranted to reveal the mechanism involved in POF.

Patients.
A total of 384 unrelated Han Chinese women with POF were recruited. Inclusion criteria consisted of primary amenorrhea (PA) or secondary amenorrhea (SA) for at least 6 months before 40 years of age and at least two serum FSH measurements exceeding 40 IU/L. Women with known karyotype abnormalities, previous chemotherapy or radiotherapy, ovarian surgery, or autoimmune diseases were excluded. Patients accompanying with somatic anomalies were excluded, particularly any reported as associated with pleiotropic Mendelian disorders (e.g., blepharophimosis-ptosis-epicanthus inversus syndrome, neurosensory deafness and cerebellar ataxia). Three hundred and eighty-four Han Chinese women with regular menses and normal hormone level were included as controls. Written informed consent was obtained from each subject.
Mutation Screening of WT1 Gene. Genomic DNA was extracted from peripheral blood samples with QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. All 10 exons and exon-intron boundaries of the WT1 gene (NM_000378.4) were amplified using polymerase chain reaction (PCR). All WT1 primers and PCR conditions are presented in Supplementary Table S1. The PCR product was first analyzed by agarose gel electrophoresis and then sequenced on an automated sequencer (ABI 3730 × 1 DNA Analyzer; Applied Biosystems, Foster City, CA). The novel sequence variants were confirmed by three independent PCR runs, followed by sequencing in both forward and reverse directions. Nomenclature of variants identified was established according to Human Genome Variant Society (HGVS, www.hgvs.org/mutnomen). LaJolla, CA) with WT mouse Wt1 expression vector as a template. A recombinant adenovirus plasmid was generated by GV adenovirus vector system, pHelper 1.0 vector and pHelper 2.0 vector (Genechem, Shanghai, CH) and then packaged by AdMax system (Microbix, CA). A high titer (1 × 1011 PFU/ml) of adenovirus was obtained by two rounds of amplification and purified.

Granulosa cells isolation and quantitative RT-PCR analysis.
Female mouse at the age of three weeks were injected with 5 IU pregnant mare serum gonadotropin (PMSG), and the ovaries were harvested at 46-48 h after PMSG injection. GCs from pre-ovulatory follicles were collected and cultured overnight for adhesion. Twenty-four hours after adenovirus vectors containing Wt1 gene infected, GCs were harvested and RNA was extracted. After reverse transcription (Takara, Japan), quantitative real-time PCR (qRT-PCR) was performed on the Roche Lightcycle 480 using specific primers (listed in Supplementary Table S2) with PrimeScript RT Master Mix (Takara). The mRNA levels were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed using the comparative cycle threshold method.
Western blot. The nuclear extracts from infected GCs of empty (Mock), wild-type (WT), mutant p.

Statistical analysis. Results of qRT-PCR were expressed by bar graphs plotted in MS Excel and
represented the Mean ± S.D. of 3 independent experiments, each performed in triplicate. Experiments were repeated at least tree times. The data were performed by chi-squared test and one-way ANOVA. Statistical differences were considered significant when P < 0.05.