Study On Association Of TGFBR3 Gene Polymorphism With Idiopathic Premature Ovarian Failure

Premature ovarian failure(POF) is a disease related to the ovarian hypo-functioncaused by various factors. Because of the complexity of pathogeny, most cases have no cause to be found, like as idiopathic premature ovarian failure,and that have brought much difficulty to the treatment of the disease. The occurrence of POF has the trend of rejuvenation and more high incidence rate at present.POF is a disease with a long course, affected by many factors and also has individual differences. Patients in different age stages or with different social and cultural background have different clinical manifestations.Discussing the clinical features to provide scientific basis for further prevention and treatment of idiopathic POF.Inhibins are the most important factors directly involved in the down-regulation of FSH via a negative feedback mechanism.Down-regulation of the FSH levels, affect selection of follicles,and ovulation by inhibins is mediated through its receptor TGFBR3 in the gonadotrophs.Taking into account that TGFBR3 is an essential component in mediating the specific antagonist action of inhibins toward activins,which is crucial for normal ovarian function, genetic variants in the TGFBR3 gene could affect the risk of POF.In part 2,we screened the TGFBR3 polymorphism in Chinese patients with idiopathic POF.Of all currently available methods,direct sequencing has the highest sensitivity and the gold standard but also the highest costs, obviously was not suitable for the high throughput screening. DHPLC is a recently developed method for DNA variation detection that uses reversedphase ion pair liquid chromatography to detect DNA heteroduplexes,it represents an attractive consideration as an inexpensive option with a sensitivity that approaches that of direct sequencing.The purpose of Part 3 is to establishing a novel method for the screening of TGFBR3 gene mutations based on DHPLC.To evaluate the sensitivity and potential clinical application value of DHPLC used in detecting TGFBR3 mutation in POF, and provide the theoretical and methodological basis for its subsequent clinical application. Once more studying on association of TGFBR3 gene polymorphism with idiopathic premature ovarian failure based on DHPLC.Part 1 Clinical features of women who have idiopathic premature ovarian failureObjective:Objective:To summarize clinical features in women who have idiopathic premature ovarian failure (POF) to provide scientific basis for further prevention and treatment of idiopathic POF.Methods:A retrospective study of the clinical features was conducted in 110 women with idiopathic POF treated between February,2009 and September,2010. The clinical data of the patients including the menstrual feature, POF incidence, vaginal ultrasound and pregnancy outcomes were investigated.Results: 1. Clinical characteristics of idiopathic POFFive patients had primary amenorrhea and 105 had secondary amenorrhea with an average years of amenorrhea of 28.25±6.97 years. Abrupt cessation occurred after 1-2 menstruations following the menarche in 2 cases (1.8%) and without identifiable preceding signs in 13 cases (11.8%),92 patients with oligomenonrrhea and gradually to cessation of menstruation. There were 110 cases with normal karyotype(46â…©â…©),but 28 cases have polymorphism.there were no significant differences between POF patients and control subjects in age, age at menarche,body mass index, Cultural level, and occupation(all p>0.05).2. The prevalence of idiopathic POF55.2% cases onset before 30 years old,81.9% of the patients within five years course of disease.The starting time of the menstrual cycle change was different, 58.2% of them suffered the menstrual cycle change from no significant incidents,and 37.3% of them suffered the menstrual cycle change in some events, such as pregnancy related events (5.5%),living environment change (9.1%), the pressure of work (8.2%), contacting poisons (9.1%),and postcoital contraceptive (3.6%).3.Pelvic ultrasonographyThe mean uterine and ovarian volume was significantly smaller in POF group than in the control group. Antral follicle count (AFC) was also significantly lower in POF group.The detection of the two ovaries by ultrasound was both visible in 78 patients;only one ovary was identified in five patients;none was noted in 26 patients.The surface area of the ovaries was 1.18±1.36 cm2. Ovaries were considered to have either a normal surface area(≥2cm3), which was the case in 28 patients (25.5%), or a small surface area (<2cm3), this being the case for the 82 other patients (74.5%). Images suggesting the presence of follicles of>2mm were observed in 51 patients. This clearly correlates with the size of the ovaries (P=0.008), since images of follicles were observed in 19 of the 30 patients with ovaries of normal surface area (67.9%), whereas patients with ovarian surface area,<2cm3 (n=82) had images of follicles in only 39% of cases (n=32).Evidence of ovulation was found in 34 patients,and spontaneous pregnancy occurred in 2 patients with a pregnancy rate of 1.8%.ConclusionPatients with menstrual disturbance, polymenorrhea and oligomenonrrhea are at risk of developing POF,in which case regular detection of the mean uterine volume, ovarian volume and AFC by vaginal ultrasound may helpin early POF detection. Close monitoring can be necessary in the course of hormone replacement therapy, and timelyintervention with assisted reproductive techniques may increase the chance of pregnancy.Part 2 Study on association of TGFBR3 gene polymorphism with idiopathic premature ovarian failure Objective:This study screened the TGFBR3 gene polymorphism in Chinese patients with idiopathic premature ovarian failure (POF) to gain a better understanding the genetic aetiology of POF.Methods:One hundred twelve Chinese women with idiopathic POF and 110 from normal controls were examined.DNA samples prepared from blood leukocytes. The coding region and respective flanking intronic regions of the TGFBR3 gene were amplified by the PCR,and the DNA fragments were directly sequenced.Web-based programs, including PolyPhen, Sorting Intolerant from Tolerant (SIFT), Prediction of Pathological Mutations (PMUT), ScanProsite, and ClustalW2, were used to predict the potential functional and structural impacts of the missense variants on TGFBR3.Results:1. Sequence variants detected in the TGFBR3 geneTotally 28 TGFBR3 gene variants were detected in POF and controls group.23 of 28 were base transition(including 13 A/G and 11 C/T)and 3 of 28 were base tansversion(2 A/C and 1 T/A),1 of 28 was double base deletion,and 1of 28 was four base insertion.Twenty-eight sequence variants, including 12 novel variants,were identified. These novel variants included 3 missense mutations,2 synonymous mutations,and 7 mutations in the intronic region.Three novel exonic missense variants were p.E458G,p.P824L,and p.I836V.Two synonymous mutations were c.516C>T (p.S172S) and c.660T>C (p.C220C). Seven mutations in the intronic region were c.382-110A>G,c.566-122A>G,c.734+11C>T,c.59-32T>A,c.381+12A>C,c.2431-7A> C,and c.1408-37₁408-38delGG.2. The pathogenicity assessment of two novel missense mutationsAmong the 110 subjects with idiopathic POF,three novel exonic variants were missense mutations:c.1370A>G (p. Glu459Gly),c.2467C>T (p. Pro825Leu),and c.2502G>A (p. Ile837Val).The variants c.1370A>G (p. Glu459Gly) and c.2467C>T (p. Pro825Leu) were not found in the controls,but c.2502G>A (p. Ile837Val) variant was present. Compared with controls, there were significantly higher frequencies of the p. I837V G/G and A/G genotypes in cases with POF [χ2=6.0517, P=0.014, odds ratio (OR)=2.322,95% confidence interval (CI):1.169-4.613]. Thus, except that the mutation at p.I837V of TGFBR3 gene in the idiopathic POF case was an unreported new single nucleotide polymorphism (SNP), the remaining two were novel missense mutations.(1)Determination of the evolutionary conservation of p. E459G and p.P825L, by multiple amino acid sequence alignment of the human TGFBR3 with the protein sequences derived from Rattus norvegicus,Canis familiaris,Gallus gallus,Bos taurus and Sus scrofa showed that both residues 459 and 825 in TGFBR3 were highly conserved among the primates.(2). We used three web-based programs to evaluate possible biologic effects of the amino acid substitution on the structure and function of the TGFBR3 protein.The impact of variant p. E459G was predicted to be "possibly damaging" by PolyPhen, but as with SIFT, predicted to be "Tolerated". Note that the directionalities of the PolyPhen and SIFT scores are opposite. We therefore applied the PMUT program to provide additional assessment.The impact of variant p. E459G was predicted to be "Tathological" by PMUT, with a maximum reliability index of 1. Based on these assessments, p. E459G is potentially disease causing. For the p.P825L variant, the predictions were "Potentially damaging" and "Intolerant" by PolyPhen and SIFT, and "Pathological" (reliability index of 8) by PMUT. p.P825L should be moderately radical and highly likely to be a functional mutation.(3)PROSITE scanning showed that no structural or functional domain was present at or around the p.P825L residue. However,the variant p. E459G was predicted to affect one zona pellucida (ZP) domain on the TGFBR3 protein.(4)When applying biosoftwares to predict the transmembrane structure of TGFBR3 gene, E459G was in extracellular region,and P825L was in intracellular region.3. Genotype distribution between POF cases and the controls(1). On the basis of Fisher's exact test, genotypic and allelic frequencies for four variants were almost significantly diverse in POF cases as opposed to controls,and are as follows:(1) c.566-71C>T, (2) c.2022T>C, (3) c.2502A>G, and (4) c.2568G>A.The genotypic frequencies for c.566-216G>A variants was also significantly varied (95% confidence level) in patients as compared with controls (P= 0.027).(2).Binary logistic regression analysis was performed for the c.566-71C>T,c.566-216G>A, c.2022T>C,c.2502A>G,and c.2568G>A variants to estimate the relative risk of POF. These variants were chosen for regression analysis,and were considered significant genotypic differences in the patients as compared with the controls at a limit of 95% confidence. The analysis indicated that the c.566-216G>A, c.566-71C>T,and c.2502A>G variants were positively correlated with POF.4. Haplotype analysis of the TGFBR3 gene in Chinese women with idiopathic POFThese five variants—<:.566-71C>T,c.566-216G>A,c.2022T>C,c.2502A>G,and c.2568G>A—were chosen for haplotype analysis.The significance of the haplotype analysis was represented following the Bonferroni's correction guidelines.The GCCGA,GCTAG,and ATTGA haplotype was significantly higher in the patients as compared with the controls.The GTTGG haplotypes were significantly higher in the controls than in patients (P=8.15e-009).5. TGFBR3 SNP in different populationsWe compared c.566-216G>A and c.2022T>C polymorphism in different populations.The distribution of gene variations may be greatly different between various populations. Compared with Indian POF patients, there were significantly higher frequencies of the 566-216G>A GA and 2022T>C TT genotypes in Chinese POF patients(P=0.000 and P=0.004).Conclusions:1.This study provides the first germline case-control status of the TGFBR3 gene in Chinese idiopathic POF patients. We have identified two novel missense mutations (p.E459G and p.P825L).These mutations were predicted to have functional impacts on TGFBR3 and are likely to be pathogenic. However, further functional studies are necessary to confirm our findings. 2. The genotypic distributions were significantly varied for c.566-216G>A, c.566-71C>T,c.2022T>C, c.2502A>G,and c.2568G>A,suggesting their possible importance in the genetic aetiology of POF.3. The haplotype analysis represented a significant association of GCCGA,GCTAG,and ATTGA haplotypes with POF.the GCCGA,GCTAG,and ATTGA haplotypes were associated with a significantly increased risk of POF, in contrast with GTTGG.4. The distribution of c.566-216G>A and c.2022T>C polymorphism may be greatly different between various populations.5. A significant diversity of genotype distribution and haplotype analysis suggested that TGFBR3 polymorphism may be responsible for the genetic aetiology of idiopathic POF in Chinese patients.Part 3 Establishment and Application of Denaturing High Performance Liquid Chromatography Technique Screening TGFBR3 gene in Chinese women with idiopathic premature ovarian failureObjective:The purpose of this study is to establishing a novel method for the screening of TGFBR3 gene mutations based on DHPLC.To evaluate the sensitivity and potential clinical application value of DHPLC used in detecting TGFBR3 mutation in POF, and provide the theoretical and methodological basis for its subsequent clinical application. Once more studying on association of TGFBR3 gene polymorphism with idiopathic premature ovarian failure based on DHPLC.Methods:All the POF specimens were tested with DHPLC.The optima condition of detection were decided by experience eventually,and then some related indexes were calculated, such as sensitivity, specifcity, false negative rate,false positive rate, Youden index, likelihood ratio, positive predictive value, and negative predictive value,taking the results of DNA sequencing as the "gold standard". At the same time, we randomly sampled 20% of the tested specimens, done the DHPLC analysis repeatly,and calculated the value of Kappa index by comparing the results between the first and second DHPLC analysis. Finally,on the basis of all indicators, the sensitivity and potential clinical application value of DHPLC analysis for detecting the idiopathic POF TGFBR3 mutation was evaluated.Results:DHPLC analysis could be optimized in the condition of PCR primer,PCR amplicon length,melting temperatures,and separation gradient.The discrimination of chromatograms to the positive and negative sample were very direct-viewing.The comparison demonstrated that DHPLC detected all alterations found by direct sequencing, no false-negative results were ever obtained.The sensitivity is 100%, specificity is 98.4%,Youden index was 98.4%, false positiverate was 1.6%, false negative rate was 0%, positive likelihood ratio was 62.5,negative likelihood ratio was 0,positive predictive value was 99.0%,negative predictive value was 100%,and Kappa index was 0.888(P<0.05),indicating DHPLC analysis was valid, reliable and practicable.Conclusions:(1)A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the TGFBR3 gene was established.(2)DHPLC analysis had higher validity,reliability and practicability in detecting TGFBR3 mutation in idiopathic premature ovarian failure.(3)TGFBR3 gene polymorphism may be responsible for the genetic aetiology of idiopathic POF in Chinese patients based on DHPLC and directly sequenced.