| Objective:Müllerian duct anomalies(MDA) are disorders characterized by congenital absence or malformations of the female reproductive tract. The müllerian ducts will develop into the uterus, cervix, fallopian tubes and the upper vagina, so if the unilateral or bilateral müllerian suffer from arrest of development, dysplasias, improper fusion or abnormal resorption due to various factors during the embryonic period, it will result in a series of congenital absence or malformations of the female reproductive tract, such as the congenital absence of the uterus and vagina(Mayer-Rokitansky-Küster-Hauser syndrome), uterus didelphys, uterus unicornis, uterus septus, longitudinal septum of vagina, vaginal atresia and so on. The patients of müllerian ducts anomalies suffer from the amenorrhea, difficulties in sexual intercourse and adverse pregnancy outcomes which impact the physical and psychological health of the patients. The etiology of the müllerian ducts anomalies is quite unclear until now, and the polygenic/multifactorial inheritance is supported. To today, several candidate genes have been found to be related to the müllerian ducts anomalis. TBX6, belonging to the T-box family, is one of the candidate genes. This gene is essential to regulate the embryonic development and organ formation. Our study aims to screen the mutation of the TBX6 gene in the Han patients with müllerian ducts anomalies in Hebei province of China and discuss the correlations between them.Methods:1 Clinical data collection: The case group: 97 patients of müllerian ducts anomalies who visited or had a follow up in the Second Hospital of Hebei Medical University between March 2013 and November 2014, who all had gynecological examinaions and at least two kinds of the auxiliary examinations from the B ultrasound, hysterosalpinography, hysteroscopy, laparoscopy, then diagnosed by two gynecological chief physicians. The control group: 110 women visiting the Second Hospital of Hebei Medical University in the same period because of salpingocyesis, with at least one normal pregnancy, without any other underlying diseases and congenital deformities, and the family history of the genital abnormalities was denied. All participants in the case group and the control group came from Hebei province with a similar distribution and belonged to the same race, the Han. Moreover, they all had normal karyotype(46, XX) and normal secondary sexual characteristics. Gonadal abnormalities and the congenital adrenal cortical hyperplasia were excluded. The patients in the case group included MRKH syndrome(congenital absence of the uterus and vagina, n=32), primordial uterus and absence of vagina(n=3), uterus didelphys with double cervix and complete longitudinal septum of vagina(n=1), uterus didelphys with double or one cervix without longitudinal septum of vagina(n=4), uterus septus(complete or partial) complicated with longitudinal septum of vagina(n=12), uterus septus(complete or partial) without longitudinal septum of vagina(n=11), uterus bicornis(n=4), uterus unicornis complicated with rudimentary uterus or not(n=16), arcuate uterus(n=2), oblique vaginal septum syndrome(OVSS, characterized by uterus didelphys, double cervix, Oblique vaginal septum resulting complete or imperfect vagina atresia and the unilateral renal agenesis at the same side)(n=6), atresia of vagina complicated with cervix aplasia or not(n=3), ransverse septum of vagina(n=2). All the participants of the study were phlebotomized peripheral venous blood after obtaining the informed consent. The blood samples were numbered and cryopreserved.2 Extrating genome DNA, gene amplification and sequencing: the genome DNA from the patients and controls was extracted from the peripheral venous blood. Nine exons and their exon-intron boundaries were amplified by polymerase chain reaction(PCR) repectively. The objective fragments were indentified by the comparison with the DNA marker through agarose gel electrophoresis, purified and detected by Sanger sequencing.3 Statistical analysis: Sequences analysis in comparison with the normal sequence obtained from the NCBI(National Center for Biotechnology Information) was performed using the Mutation Surveyor software. Statistical analysis was performed using SPSS 13.0 software. The computer software programs SIFT(Sorting Intolerant from Tolerant) and Poly Phen2(Polymorphism Phenotyping v2) were used to predict the impact of the MISsence mutation detected in the study. Measurement data was expressed by median and the frequency data was compared by using Chi-square test. The P-value <0.05 was considered to have statistical significance.Results: 1 The distribution of the native place of the participants in the case group and the control group had no significant difference(P=0.755, P>0.05). 2 The clinical data was analyzed and there were 24(24/97, 24.74%) patients of müllerian ducts anomalies complicated with the deformities of the other systems in total in the case group, 6(6/97, 6.19%) of whom had malformations of at least two other systems. The associated malformations found in the case group included congenital scoliosis(n=5, 5/97, 5.15%), congenital vertebral body dysplasia(n=1, 1/97,1.03%), spina bifida(n=1, 1/97, 1.03%), congenital rib deformities(n=4, 4/97, 4.12%), congenital limbs deformities/ polydactyly/ oligodactyly(n=4, 4/97, 4.12%), unilateral renal agenesis(n=12, 12/97, 12.37%), ectopia of kidney(n=2, 2/97, 2.06%), congenital urinary bladder neck stenosis(n=1, 1/97, 1.03%), congenital heart disease(n=2, 2/97, 2.06%), congenital imperforate anus(n=1, 1/97, 1.03%), congenital Meckel’s diverticulum(n=1, 1/97, 1.03%), congenital inguinal hernia(n=2, 2/97, 2.06%). In all the associated malformation types, the skeletal anomalies accounted for 40.00%(14/35), the urinary system anomalies for 42.86%(15/35), the heart anomalies for 5.71%(2/35), digestive system anomalies for 5.71%(2/35), the other system deformities for 5.71%(2/35). 3 The mutation(c.484G>A) was found in the exon4, which was recorded in the database of single nucleotide polymorphisms(db SNP) as the rs56098093. 3.1 The frequency of the genotype(GA+AA) of the patients(9/97, 9.3%) was higher than that of the controls(3/110, 2.7%) and it had statistical significance(P=0.044, P<0.05). The minor allele frequency(MAF) of this SNP in patients was 5.2%(10/194), higher than that in controls(3/220, 1.4%), and it had statistical significance(P=0.027, P<0.05). 3.2 c.484G>A was a MISsense mutation, resulting in an alteration of the amino acid sequence(p.Gly162Ser). The score of this mutation by the SIFT analysis was 0, and 1 by the Poly Phen analysis. 3.3 There were 8 patients who had the heterozygous mutation(genotype GA), including MRKH syndrome(n=4), primordial uterus and absence of vagina(n=1), uterus unicornis with rudimentary uterus(n=1), uterus septus with longitudinal septum of vagina(n=1) and oblique vaginal septum syndrome(n=1). In addition, a patient with MRKH syndrome in the case group was found to have the homozygotic mutation(genotype AA), but there was no genotype AA in the control group. 3.4 The rate of this mutation in the patients with MRKH syndrome(5/32, 15.6%) and that in the patients with other kinds of MDA(4/65, 6.2%) had no statistical significance(P=0.151, P>0.05). 3.5 There were 4 patients with other associated malformations who had this mutation in the case group. The rate of this mutation in the MDA patients with associated anomalies(4/24, 6.2%) and that in the MDA patients without any other associated anomalies(5/73, 6.8%) had no statistical significance(P=0.538, P>0.05). c.1227G>A was synonymous and had no impact on the amino acid sequence(p.Pro409=). 5 The genotype frequencies of the SNP rs56090093 and rs2289292 in controls were conducted to have the Hardy-Weinberg test, and the P-value were 0.99 and 0.89 respectively, meaning that the controls obeyed the Hardy-Weinberg(P=0.219, P>0.05). 4 The mutation(c.1227G>A) was found in the exon9, or rs2289292 which was known in the database of single nucleotide polymorphisms. The frequency of the genotype(GA+AA) of the patients(61/97, 66.5%) and it had no statistical difference compared with that in control group(71/110, 64.5%)(P=0.804, P>0.05). In addition, the MAF of this SNP in patients(71/194, 36.6%) and that in controls(87/220, 39.5%) had no statistical difference law(P>0.05).Conclusions:1 The distribution of the native place of the participants in the case group and control group was similar, and all the participants in the control group came from a cohort with genetic stability, so the comparison in the study was meaningful.2 The phenotype of müllerian ducts anomalies of the Han women in Hebei province was complex and diverse, often associating with other malformations, and the skeletal deformities and urinary system malformations were more common associated anomalies.3 The SNP(rs56098093) situated in the exon4 of TBX6 gene were most likely to impact the gene expression and the protein function and it was detrimental. This SNP might have a correlation with the müllerian ducts anomalies of Han women in the area of Hebei province. It was not responsible for the phenotype of MDA whether it was MRKH syndrome or other kind of MDA. Moreover, it was not related to whether the MDA was complicated with malformations of other systems.4 The SNP(rs2289292) situated in the exon9 of TBX6 gene might have no correlations with the müllerian ducts anomalies of Han women in the area of Hebei province. |
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