Variants Of The WNT7A、WNT9B Gene In Chinese Patients With Mullerian Duct Abnormalities

BACKGROUND:Mullerian duct abnormalities (MDAs) consists of a miscellaneous group of congenital absence or severe hyperplasia of the female reproductive tract, such as Mayer-Rokitansky-Kuster-Hauser syndrome (congenital absence of the uterus and vagina), unicornuate uterus, uterus didelphys, bicornuate uterus and septate uterus, et al. The clinical presentation of MDAs varies including primary amenorrhea, infertility, recurrent pregnancy loss, malpresentation, fetal intrauterine growth restriction and retained placenta. Previous researches reported that1-3%infertile patients had evidence of uterine abnormalities, and the prevalence of MDAs is up to15%within patients being evaluated with hysterosalpingogram because of recurrent spontaneous abortion. Several hypotheses for the mechanisms of MDAs have been proposed, but apart from environmental and iatrogenic causes, majority of cases are considered to be idiopathic. Despite that the MDAs are supposed to be sporadic and multifactorial, genetic factors are still an importance facet to elucidate this uncharted disease.As one member of WNT family, WNT7A gene (wingless-type MMTV integration site family, member7A) was reported to be critical for the sexually dimorphic development of the Mullerian ducts, and for maintaining the appropriate boundaries in a molecular and morphological basis of cellular populations along the anterior-posterior axes of female reproductive tract. Mutations in the WNT7A gene not only affect the limb bud development in chick and mouse models but also are involved in murine Mullerian duct abnormalities. Female mammals with a deficiency of the WNT7A gene were infertile because of small uteru, incompletely differentiated Mullerian duct and thin muscular development in the uterus.The WNT9B gene (wingless-type MMTV integration site family, member9B) is a common organizing signal regulating diverse components of the mammalian urogenital system and plays a primary role in the development of the female reproductive tract, particularly. The WNT9B-/-mice showed that this gene was crucial in the development of mesonephric and metanephric tubules and caudal extension of the mullerian duct. Histological examination revealed that males lacked the epididymis and vas deferens and females lacked parts of the oviduct, uterus, and upper vagina. However, peripheral mesenchyme and rudimentary ureteric epithelia still existed and the gonads were normal. These observations suggested that the WNT9B gene might be required for the induction of the mammalian kidney and reproductive system and was essential in posterior extension of the Mullerian duct.OBJECTIVE:The aim of the present work is to examine whether mutations of the WNT7A, WNT9B gene exist in Chinese women with Mullerian duct abnormalities (MDAs).METHODS:In the first stage, we recruited191Chinese MDAs patients, including uterine agenesis (n=15), unicornuate uterus (n=55), uterus didelphys (n=38), bicornuate uterus (n=43) and septate uterus (n=40). One hundred and ninety-two women without Mullerian duct abnormalities were recruited as controls, who asked for infertility treatment because of tubal obstruction or male factors. Genomic DNA was extracted from peripheral blood samples. Subsequently the four exons of the WNT7A, WNT9B gene were amplified using polymerase chain reaction (PCR) with specific primers respectively. The PCR product was sequenced on an automated sequencer. To identify the results in the first stage, the number of patients and ethnic-matched controls were enlarged to542and563respectively in the second stage.RESULTS:Four variants were identified in the WNT7A gene:3known SNPs and1novel variant. Three known SNPs included rs3749319, rs12639607and rs3762719. Comparison of genotype and allelic frequencies between the controls and MDAs cases showed no significant differences in the frequency of rs12639607and rs3762719(p>0.05). One novel synonymous variation (c.342C→T), which was detected in one patient with bicornuate uterus, was located in exon3and not present in any of the192controls.As regards to the WNT9B gene, one known single-nucleotide polymorphisms (SNP)(rs34072914) and four novel variations were identified in the first stage. Among these novel ones, two were synonymous:c.12G→T and c.588C→T; the other two rare non-synonymous novels were:c.566G→A (p.Arg189G1n) and c.773G→A (p.Arg258His). None of the four novel variations was found in controls. In the second stage, sequence analysis revealed that both of the two novel non-synonymous variants were detected in MDAs cases as well as in control groups, and comparison of genotype and allelic frequencies between these two groups showed no significant differences in the frequency of c.566G→A and c.773G→A (p>0.05).CONCLUSION:The results indicate that mutations in the coding sequence of the WNT7A, WNT9B gene are not responsible for Mullerian duct abnormalities in Chinese population.