Analysis of copy number variations of WNT4 gene in a Chinese population with Müllerian anomalies

Background To investigate the genetic contribution of copy number variations (CNVs) in Wingless-type MMTV integration site family, member 4 (WNT4), in a Chinese population with Müllerian anomalies (MA), copy number analysis of WNT4 by Multiplex ligation‐dependent probe amplification (MLPA) was performed on 248 female patients. Some studies have shown that heterozygous missense mutation of WNT4 can lead to MA. However, few studies on the relationship between WNT4 CNVs and MA have been performed. Results Among the 248 Chinese women affected by MA in this study, heterozygous deletion of WNT4 was detected in a single patient. Conclusions MLPA identified one heterozygous deletion in WNT4 in a single female patient among 248 Chinese women affected by MA. This study firstly reports CNVs of WNT4 in a large sample of MA patients from the Chinese population, which suggests that CNVs of WNT4 cannot be excluded in the occurrence of MA. This provides a genetic basis for precise treatment in the future.

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Background
Müllerian anomalies (MA) are among the most common diseases in gynecology and obstetrics. They include various malformations of fallopian tubes, vagina, cervix, and corpus uteri [1]. The estimated incidence of MA is 5.5% in the general population, but 24.5% in women with infertility and miscarriage [2,3]. This disease seriously affects women's reproductive function and causes a huge social burden.
According to guidance of the European Society of Human Reproduction and Embryology/European Society for Gynaecological Endoscopy (ESHRE/ESGE) in 2013 [4], Müllerian duct abnormalities can be classified into 36 different combinations as the main class for the uterine body (U) and subclasses for the cervix (C) and vagina (V). Compared to the classification method put forward by American Fertility Society, ESHRE/ESGE classified uterine morphology into 7 classes: U0, normal; U1, dysmorphic; U2, septate; U3,bicorporeal; U4, hemiuterus; U5, aplastic; U6, unclassified cases.
Different types of Müllerian duct anomalies have different effects on reproductive function. People with mild symptoms can have normal fertility, but most patients with uterine malformations exhibit serious effects such as primary amenorrhea, recurrent abortion, and infertility. Septate uterus is one of the milder symptoms in patients with uterine malformation, and the rate of live births from such patients is about 62.5%. After operation, this rate can be significantly increased [5].
Many studies have also shown that the heterozygous missense mutation of WNT4 can lead to MA and hyperandrogenism in humans [15][16][17][18]. However, few studies have been performed on the relationship between WNT4 copy number variations (CNVs) and MA. Therefore, here we detected the copy number of WNT4 in 248 female patients with MA.

Materials and methods
Participants 248 unrelated MA patients were recruited from the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University. All patients were Chinese, ranging in age from 22 to 48 years (29.32 ± 4.87). All patients showed a normal karyotype of 46, XX and had been diagnosed with abnormal Müllerian ducts using physical examination, ultrasonographic investigations, hysteroscopy and laparoscopy. The study protocol was approved by the Ethics Committee of the National Research Institute for Family Planning, and written informed consent was obtained from all participants.

DNA extraction
Genomic DNA was extracted from the peripheral blood of 248 patients using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany).

MLPA assay
Three probes designed in-house targeted the region of the WNT4 gene. The first probe covered exon 1 and exon 2, second probe covered exon 3 and exon 4, third probe covered exon 5. Fluorescence data on the PCR products analyzed by capillary electrophoresis were collected using an Applied Biosystems 3730xl DNA Analyzer, and the peak area data were output. Relative peak area of WNT4 was compared with that of the reference gene. Dosage quotient (DQ) was used as the signal result of CNVs. The classification was as follows: normal (0.8-1.2), homozygous deletion (0), heterozygous deletion (0.4-0.65), and heterozygous duplication (1.3-1.65).

Analysis of CNV pathogenicity
The pathogenicity of CNVs was evaluated in accordance with the CNV interpretation scoring metric from the American College of Medical Genetics and Genomics (ACMG) [19]. Online databases including the Database of Genomic Variants (DGV, http:// dgv. tcag. ca/ dgv/ app/ home/) and the Genome Aggregation Database (Gno-mAD, http:// gnomad. broad insti tute. org/) were used to retrieve the frequency of CNVs. Public databases, including PubMed (https:// pubmed. ncbi. nlm. nih. gov/) and Online Mendelian Inheritance in Man (OMIM, https:// www. omim. org/), were used to evaluate the significance of CNVs of WNT4.

MLPA results of MA patients
One deletion was identified in WNT4 in a single female patient among the 248 Chinese women affected by MA in this study (Fig. 1). The DQ values of three probe recognition sequences were all between 0.4 and 0.65, reflecting a heterozygous deletion of WNT4 (Table 1).

Clinical characteristics of the patient with CNVs
The patient with a single copy of WNT4 was 28 years old, had regular menstruation, and exhibited normal levels of estradiol (E2) and testosterone (T). She suffered from secondary infertility, having been diagnosed with partial septate uterus but no renal abnormalities. She had undergone transcervical incision of septa (TCIS) in 2016 (see Table 1 for details).

Online database analysis
No identical overlapping variant was found in the DGV database and GnomAD database. Therefore, this heterozygous whole deletion of WNT4 was considered to be rare in the general population. In the OMIM database, the mutation of WNT4 was reported to be associated with abnormal Müllerian ducts in women. The heterozygous deletion of WNT4 was considered variant of uncertain significance (VUS) according to the ACMG guidelines (see Table 2 for details).

Discussion
WNT4 has five exons and is located at 1p36.12 [genome coordinates (GRCh38): 1:2217307-22143980] (NCBI, https:// www. ncbi. nlm. nih. gov/). It is a member of the WNT family of signaling molecules. The signaling protein encoded by this gene plays an important role in early embryonic sex determination, nephrogenesis, and female reproductive tract development [6,13]. WNT4 −/− female mice have been reported to show the phenotypes of sex reversal, Müllerian duct aplasia, hyperandrogenism, and follicle depletion [6,14]. WNT4 monomeric cherry/monomericcherry mice were shown to form Müllerian ducts in only 45% of cases [20]. The abnormal Müllerian ducts caused by WNT4 mutation are rare, but are pathological. Four missense mutations of WNT4 (p.L12P, p.E226G, p.R83C, p.A233) were found in four unrelated women with MA between 2004 and 2011 [15][16][17][18]. However, Chang et al. did not find any significant WNT4 mutation in 189 Chinese women with MA in 2012, although they did not analyze the CNVs [21]. Meanwhile, we identified only one heterozygous deletion in WNT4 in a single female patient among 248 Chinese women affected by MA. This shows the rarity of CNVs in MA patients. To our best knowledge, it is the first report of CNVin WNT4 in MA patients overall. And the deletion was assessed as VUS. Only missense variants have so far been associated with the phenotype, so that loss-of-function is not a known mechanism of disease. A VUS is not a benign or likely benign variant, the deletion we observed may possibly contributed to MA. So further evidence might accumulate future recognition of LOH as a pathogenic mechanism.
We also found that the abnormality of the Müllerian duct caused by missense mutation of WNT4 differed from that caused by CNVs of WNT4. All of the women carrying the missense mutation showed Müllerian duct abnormalities (two patients showed absent vagina and uterus, while one 15-year-old patient showed uterine agenesis), hyperandrogenemia, and primary amenorrhea [15][16][17][18]. However, our patient with a septate uterus showed normal menstruation and testosterone (T). WNT4 inhibits steroidogenic enzymes by upregulating DAX1. Steroidogenic enzymes [including 3b-hydroxysteroid dehydrogenase 2 (HSD3B2) and 17a-hydroxylase (CYP17A1)] are necessary for testosterone production. The four missense mutations found in previous studies have been proven to increase the levels of HSD3B2 and CYP17A1 in cells transfected with the mutant alone or in combination with the wildtype protein. It was concluded that mutation of WNT4 showed a dominant negative effect [15][16][17][18]. The loss of heterozygosity (LOH) in the patient that we identified may only led to haploinsufficiency. Therefore, we believe that the phenotype caused by LOH of WNT4 might be less severe than that caused by a dominant negative effect.
In addition, some rearrangements of other genes or regions found in MA patients should not be overlooked. Some of these regions have been shown to be clinically significant, such as 1q21.1, 7p14.3, 16p11.2, 17q12, and 22q11. In addition, some candidate genes have been proposed, includingLHX1, BBS9, HNF1B, and TBX6 [22][23][24][25] [26]. Thus, some other genes or regions should be noted in CNV analysis of MA patients.

Conclusions
One heterozygous deletion in WNT4 was identified by MLPA in a single female patient among 248 Chinese women affected by MA. This study reports CNVs of WNT4 in a large sample of MA patients from the Chinese population for the first time. This suggests that CNVs of WNT4 cannot be excluded in the occurrence of MA. This provides a genetic basis for precise treatment in the future.