[Application of DHPLC screening TGFBR-3 gene in Chinese women with idiopathic premature ovarian failure].

OBJECTIVE: To evaluate clinical value of denaturing high performance liquid chromatography (DHPLC) used in detecting transforming growth factor beta receptor 3 (TGFBR-3) exons 11 and 12 polymorphism in women with idiopathic premature ovarian failure (POF). METHODS: From Feb. 2009 to Dec. 2011, 110 patients with idiopathic POF undergoing treatment at Shenzhen Maternal & Child Health Institute affiliated to Southern Medical University were enrolled as POF group in this study. In the mean time, 110 women under 40 years old with normal hormonal level and menstrual cycles as control group. The exons 11 and 12 of TGFBR-3 gene polymorphism were screened by using DHPLC, and results of DNA sequencing was as golden standard. Some related indexes were calculated, such as sensitivity, specificity, false negative value, false positive value, Youden index, positive predictive value, and negative predictive value. At the same time, 20% of the tested specimens were chosen randomly and detected by DHPLC again. The value of Kappa index were calculated by comparing the results between the first and second DHPLC analysis. RESULTS: The exon 11 of TGFBR-3 were not identified gene polymorphism and two nucleotide polymorphisms were identified in exon 12. For 2022 T/C polymorphism, the frequencies of CC with 0.9% (1/110), TC with 22.7% (25/110), TT with 76.4% (84/110), C with 12.3% (27/220) and T with 87.7% (193/220) in POF group were significantly different from CC with 0, TC with 9.1% (10/110) and TT with 90.9% (100/110), C with 4.5% (10/220) and T with 95.5% (210/220) in control group (all P<0.05). Allelic and genotypic frequencies of 2161-75 C/T were not differed significantly between the two groups (all P>0.05). As DNA sequencing as golden standard, DHPLC showed that the sensitivity was 100%, specificity was 97.9%, Youden index was 97.9%, positive predictive value was 96.3%, negative predictive value was 100%, and Kappa index was 0.888 (P<0.05). CONCLUSION: DHPLC analysis is higher validity, reliability and practicability method in detecting TGFBR-3 polymorphism in idiopathic premature ovarian failure.

Haplotype and mutation analysis of the TGFBR3 gene in Chinese women with idiopathic premature ovarian failure.

This study screened the TGFBR3 mutations in Chinese patients with idiopathic premature ovarian failure (POF) to gain a better understanding the genetic aetiology of POF. One hundred twelve Chinese patients with idiopathic POF and 110 women from normal controls were examined. The coding region and respective flanking intronic regions of the TGFBR3 gene were amplified by the PCR, and the DNA fragments were directly sequenced. Twenty-eight sequence variants, including 12 novel variants, were identified. These novel variants included three missense mutations, two synonymous mutations, and seven mutations in the intronic region. Three novel exonic missense variants were p.E458G, p.P824L, and p.I836V. The c.566-216G>A, c.566-71C>T, c.2022T>C, c.2502A>G, and c.2568G>A variants represented significantly different genotype distribution between POF cases and the controls. The binary logistic regression analysis of c.566-216G>A, c.566-71C>T, and c.2502A>G variants were significantly associated with the POF patients and the ATTAG haplotype was most significantly over-represented as compared with controls (P = 0.00121). The ATTGG and GCTGG haplotypes were significantly higher in controls than in patients (P = 0.00113 and 0.00055, respectively). Other less frequent haplotypes, such as GCCGA, was only present in the patients (P = 0.00066). GTTGG was only present in the controls (P = 0.00001). Significant diversity of genotype distribution and haplotype analysis suggested that TGFBR3 mutations may be responsible for the genetic aetiology of idiopathic POF in Chinese patients.

Pan-Cancer Gene Analysis of m6A Modification and Immune Infiltration in Uterine Corpus Endometrial Carcinoma.

Objective: This investigation was to test the potential role of m6A-related long non-coding RNAs (lncRNAs) and immune infiltration as crucial factors in the diagnosis and treatment of uterine corpus endometrial cancer (UCEC). Method: The UCEC RNA-seq data were downloaded in the Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/). There were 587 samples totally, containing 543 UCEC cases and 35 healthy cases. The clinical information of UCEC cases included survival time, survival status, gender, age, stage, and TMN stage. Twenty-three m6A-related genes were found in published journals. The RNA-seq documents of UCEC were downloaded in the Cancer Genome Atlas (TCGA). The hub gene data of UCEC were downloaded from GEPIA2 database. The different packages of R language were applied to calculate and analyze in this research. Results: Among 587 cases in our study, we discovered 3039 lncRNAs in the TCGA-UCEC database. After the differential analysis, 23 m6A-associated genetics were screened and twenty-one m6A-associated differential genetics were found. In the end, we obtained 20 m6A-related lncRNAs. LNCTAM34A was considered as a predictive gene through univariate and multivariate Cox regression analysis. In addition to the above, patients with high LNCTAM34A expression had better outcomes than those with low LNCTAM34A expression. The high-risk cohort had greater scores of activated dendritic cells (aDCs), B cells, and T cell regulatory (Tregs) than low-risk cohort; in the meanwhile, high-risk cohort had lower scores of DCs and iDCs. Then, the high-risk cohort displayed greater scores in the immune functions of MHC class I, para-inflammation, and type I IFN response than those of low-risk cohort. Among 27 immune-inducible genes, the level of CD244, KIR3DLI, NRP1, PDCD1LG2, and TNFRSF8 was reduced in UCEC samples and the level of CD27, CD28, CD70, CD80, CD86, HAVCR2, ICOS, IDO1, LAIR1, PDCD1, TIGIT, TNFRSF18, -25, -9, -14, and VTCN1 was increased in UCEC samples. Conclusion: The key role of M6A-related lncRNAs in immune microenvironment in high-risk patients of UCEC. The patients with strong expression of LNCTAM34A have a good prognosis, and LNCTAM34A can be used as a prognostic gene for UCEC. m6A-related lncRNAs can be used as a potential treatment for UCEC. Our observations can be used as a hypothetical basis for future in vitro and animal experiments.

FOXE1 polyalanine tract length screening by MLPA in idiopathic premature ovarian failure.

BACKGROUND: FOXE1 is one of the candidate genes for genetic predisposition to premature ovarian failure (POF) and it contains an alanine tract. Our purpose is to assess the influence of length of the alanine tract of FOXE1 on genetic susceptibility to POF. METHODS: The group studied consisted of 110 Chinese patients with idiopathic POF and 110 women from normal controls. The polyalanine tract and flanking sequence of FOXE1 was screened using the Multiple Ligation-dependent Probe Amplification (MLPA) technique and directly sequenced. RESULTS: Three variants of FOXE1-polyalanine length, containing 12, 14, or 16 alanine residues, and 5 different genotypes were identified. There were significantly lower frequencies of the 14/14 genotypes in cases with POF (X2 = 119.73, P = 0.001), as compared with the controls. The incidence of 16/16 genotypes of FOXE1-polyalanine was significantly higher in patients with POF (X2 = 3.403, P = 0.001) in comparison to the controls. The FOXE1 14 alanine allele was significantly less common in the POF patient group (186/220) than the controls (216/220) (X2 = 25.923, P = 0.0001). The FOXE1 16 alanine allele was significantly more common in the POF patient group (28/220) than the controls (4/220) (X2 = 19.412, P = 0.0001). CONCLUSION: This finding provides evidence that polyalanine repeat expansions in FOXE1 may be responsible for the genetic aetiology of POF in Chinese women.

Identification of novel missense mutations of the TGFBR3 gene in Chinese women with premature ovarian failure.

The aim of this study was to assess the association between human transforming growth factor ß receptor, type III (TGFBR3) and idiopathic premature ovarian failure (POF) in a Chinese population. A total of 112 Chinese women with idiopathic POF and 110 normal controls were examined. DNA samples prepared from blood leukocytes were used as templates for polymerase-chain reaction amplification of DNA fragments from TGFBR3. The gene fragments were 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 of TGFBR3. A total of 11 novel variants were identified. Among them, six were found only in the POF patients. Two missense variants, p.E459G and p.P825L, which are conserved in primates, were predicted to have functional and structural impacts on the TGFBR3 protein. The other four variants (c.381+12A>C, c.2431-7A>G, p.S172S and p.C220C) were considered benign. However, further functional studies are necessary to confirm these findings.

[Diagnosis and treatment of endometriosis in adolescents].

OBJECTIVE: To discuss the clinical feature, diagnosis and treatment options of adolescent endometriosis. METHODS: The records of adolescent patients with endometriosis (11-20 years old) who were admitted to First, Second and Third Affiliated Hospital of Zhongshan University and Guangdong Provincial People's Hospital between 1990 and 2003 were retrospectively reviewed. RESULTS: Forty-three patients were diagnosed as endometriosis either by laparotomy or laparoscopy. The chief symptoms leading to the diagnosis were palpable pelvic mass (18/43), dysmenorrhea (15/43), chronic pelvic mass (10/43) and acute pelvic pain (4/43). The majority of patients (53%) presented with the revised AFS-r classification stage III, 8 cases (19%) presented with stage I, 3 cases (7%) with stage II and 9 cases (21%) with stage IV. Nine cases (21%) had complicated genital tract abnormalities. Conservative operations, including salpingo-oophorectomy ins cases, ovarian cystectomy in 31 cases and laparoscopic vaporization in 8 cases, were performed. Surgeries were followed by hormonal suppression using oral contraceptives in a continuous or cyclic manner. CONCLUSIONS: Adolescent endometriosis may occur around 4.6 years after menarche. The chief symptoms are palpable pelvic mass and dysmenorrhea. The treatment of endometriosis in adolescence does not differ principally from that in adult women. In the treatment of endometriosis and for the prevention of recurrence, it is recommended to give 3-6 months of oral contraceptives.

[Clinical features of women with idiopathic premature ovarian failure].

OBJECTIVE: To summarize the clinical features of idiopathic premature ovarian failure (POF) and explore the early diagnosis and intervention. METHODS: A retrospective study was conducted in 39 women with idiopathic POF treated between February, 2009 and January, 2010. The clinical data of the patients including the menstrual feature, POF incidence, vaginal ultrasound and pregnancy outcomes were investigated. RESULTS: One patient had primary amenorrhea and 38 had secondary amenorrhea with an average duration of amenorrhea of 5.82 years. Abrupt cessation occurred after 1-2 menstruations following the menarche in 2 cases (5.1%) and without identifiable preceding signs in 9 cases (23%). The 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. Vaginal ultrasound detected at least one ovary in 89.7% and follicular activity in 79.5% of the POF patients. Evidence of ovulation was found in 12 patients, and spontaneous pregnancy occurred in 2 patients with a pregnancy rate of 5.1%. CONCLUSION: Patients with menstrual disturbance, polymenorrhea and oligomenorrhea are at risk of developing POF, in which case regular detection of the mean uterine volume, ovarian volume and AFC by vaginal ultrasound may help in early POF detection. Close monitoring can be necessary in the course of hormone replacement therapy, and timely intervention with assisted reproductive techniques may increase the chance of pregnancy.