[Prenatal diagnosis and clinical counseling for fetal chromosomal reciprocal translocations].

OBJECTIVE: To analyze the clinical effect of fetal chromosomal reciprocal translocation in order to optimize procedures for prenatal diagnosis and clinical counseling. METHODS: Conventional G-banding karyotype analysis was performed on 7901 amniotic fluid samples. For fetuses found to have carried a reciprocal translocation, karyotypes of their parents were checked. Fetuses with de novo translocations also underwent microarray analysis to exclude small deletions, and were subjected to prenatal ultrasound monitoring till birth and one year follow-up. Those with de novo translocations were followed till 3 years old. RESULTS: A total of 24 fetal reciprocal translocations have been identified, which gave a detection rate of 0.30%. Analysis of parental karyotypes has found reciprocal translocations in 17 cases, including 9 maternal and 8 paternal cases. The remaining 4 were of de novo mutations, for which parental examination was refused in three cases. For fetuses with inherited translocations, prenatal ultrasound monitoring and follow-up results were all normal. For those with de novo translocations, although gene chip analysis has failed to detect copy number variations (CNVs), prenatal ultrasound and follow-up results had found three with abnormal outcome. These included 1 case with reciprocal translocation involving the X chromosome and an autosome. CONCLUSION: For prenatally detected reciprocal chromosome translocations, parental origin should be traced. Gene chip analysis can help to exclude small deletions and duplications. However, ultrasound monitoring and follow-up after birth are equally important. Based on comprehensive analysis of the results of combined testing, accurate counseling can be provided.

[SNP-chip technology for identification of origins for prenatally detected marker chromosomes].

OBJECTIVE: To determine the origin of 1 prenatally detected small supernumerary marker chromosome (sSMC) using SNP-chip technology, and to deduce the underlying mechanism. METHODS: The fetal sample was subjected to karyotype analysis. The identified sSMC was subjected to genom wide scan using a SNP microarray chip. The results were validated with fluorescence in situ hybridization (FISH). RESULTS: The karyotype of the fetus was determined as 46, X, +mar, which was verified by SNP microarray chip analysis as Yp11.2-11.3 duplication, along with loss of Yq11.2 region, FISH analysis has confirmed that the sSMC has derived from the Y chromosome. CONCLUSION: The karyotype of the fetus was determined as 46, X, idic(Y) (pter→ p11.2::11.2→ pter). Regional deletion of Yq11.2 has been associated with male azoospermia. SNP chip analysis can exclude minor deletions and duplications with a size of more than 1 Mb, which may be applied for verifying difficult cases as well as microdeletion and duplication syndromes upon prenatal diagnosis.

[Rapid and high-throughput multiplex ligation-dependent probe amplification for diagnosis of chromosome aneuploidy].

OBJECTIVE: To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice. METHODS: The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. RESULTS: The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples, the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of >1.3. CONCLUSION: Thedata suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.

Next-generation sequencing identifies rare pathogenic and novel candidate variants in a cohort of Chinese patients with syndromic or nonsyndromic hearing loss.

BACKGROUND: Hearing loss (HL) is a common sensory disorder in humans characterized by extreme clinical and genetic heterogeneity. In recent years, next-generation sequencing (NGS) technologies have proven to be highly effective and powerful tools for population genetic studies of HL. Here, we analyzed clinical and molecular data from 21 Chinese deaf families who did not have hotspot mutations in the common deafness genes GJB2, SLC26A4, GJB3, and MT-RNR1. METHOD: Targeted next-generation sequencing (TGS) of 127 known deafness genes was performed in probands of 12 families, while whole-exome sequencing (WES) or trio-WES was used for the remaining nine families. RESULTS: Potential pathogenic mutations in a total of 12 deafness genes were identified in 13 probands; the mutations were observed in GJB2, CDH23, EDNRB, MYO15A, OTOA, OTOF, TBC1D24, SALL1, TMC1, TWNK, USH1C, and USH1G, with eight of the identified mutations being novel. Further, a copy number variant (CNV) was detected in one proband with heterozygous deletion of chromosome 4p16.3-4p15.32. Thus, the total diagnostic rate using NGS in our deafness patients reached 66.67% (14/21). CONCLUSIONS: These results expand the mutation spectrum of deafness-causing genes and provide support for the use of NGS detection technologies for routine molecular diagnosis in Chinese deaf populations.

Mutation analysis of common deafness-causing genes among 506 patients with nonsyndromic hearing loss from Wenzhou city, China.

OBJECTIVES: The frequency and spectrum of mutations in deafness-causing genes differs significantly according to the ethnic population and region under investigation. The molecular etiology of nonsyndromic hearing loss (NSHL) in Wenzhou, China, has not yet been systematically elucidated. To provide accurate genetic testing and counseling in this area, we investigated the molecular etiology of NSHL in a deaf population from Wenzhou. METHODS: A total 506 unrelated patients with NSHL were enrolled in this study. Nine hotspot mutations in four major deafness genes were investigated by sequencing (Group I: 187 patients enrolled between 2011 and 2015) or allele-specific PCR-based universal array (Group II: 319 patients enrolled between 2016 and 2017). The investigated genes included GJB2 (c.35delG, c.176_191del16, c.235delC, c.299-300delAT), SLC26A4 (c.2168A > G, c.919-2A > G), mtDNA 12SrRNA (m.1555A > G, m.1494C > T), and GJB3 (c.538C > T). Furthermore, whole coding region sequencing or improved multiplex ligation detection reaction (IMLDR) were performed for patients who carried mono-allelic variants of GJB2 and SLC26A4, in order to detect other mutations among these patients. RESULTS: GJB2 mutations were detected in 22.92% (116/506) of the entire cohort and SLC26A4 mutations were found in 6.52% (33/506) of the cohort. GJB3 mutations were detected in 0.79% (4/506) of the cohort. The mutation rate of mitochondrial DNA 12SrRNA in our patients was 17.40% (88/506), including 17.00% (86/506) with the m.1555A > G mutation and 0.40% (2/506) with the m.1494C > T mutation. The allelic frequency of the c.235delC mutation was 14.62% (148/1012), which is significantly higher than that of c.109G > A (33/1012, 3.26%), c.299_300delAT (13/1012, 1.28%), and c.176_191del16 (6/1012, 0.59%). The most common pathogenic mutation of SLC26A4 was the c.919-2A > G mutation (37/1012, 3.66%), followed by c.2168A > G (6/1012, 0.59%), and c.1229C > T (4/1012, 0.40%). Moreover, five rare pathogenic variants of GJB2 and eight rare pathogenic variants of SLC26A4 were identified. CONCLUSION: GJB2 is the primary deafness-causing gene in deaf patients from Wenzhou, China; this is consistent with what is observed in most Chinese populations. However, the surprisingly high rate of the m.1555A > G mutation (17.00%) in patients from Wenzhou was significantly higher than in other populations in China. These findings highlight the specificity of the common deafness-causing gene mutation spectrum in the Wenzhou area. This information may be of benefit for genetic counseling and risk assessment for deaf patients from this area.

[Effects of trabeculectomy combined with amniotic membrane and extracellular matrix of conjunctiva on filtering bleb in rabbits eyes].

OBJECTIVE: To investigate the effect of trabeculectomy combined with amniotic membrane and extracellular matrix (ECM) of conjunctiva on filtering bleb in rabbits. METHODS: Glaucoma was induced by injecting alpha-chymotrypsin into the posterior chamber of rabbit eyes. 27 rabbits were randomly divided into the following three groups: group (1) trabeculectomy alone as control; group (2) trabeculectomy combined with amniotic membrane transplantation; group (3) trabeculectomy combined with extracellular matrix (ECM) transplantation. IOPs were measured by a Schiötz tonometer and filtering bleb observed under a microscopy and electric microscopy at day 1, 7, 14, 21, 28, 35, 42, 56 after surgery. RESULTS: Compared with pre-operation, the IOP in the control group (group 1) was reduced from (34.59 +/- 4.44) mm Hg (1 mm Hg = 0.133 kPa) to (11.31 +/- 2.76) mm Hg, (19.20 +/- 5.27) mm Hg, (21.17 +/- 4.36) mm Hg, (22.22 +/- 1.39) mm Hg, (23.90 +/- 1.97) mm Hg, (23.67 +/- 1.73) mm Hg, respectively, after surgery. In the group 2, the IOP was decreased from (34.38 +/- 4.21) mm Hg to (10.48 +/- 2.45) mm Hg, (12.80 +/- 3.41) mm Hg, (13.50 +/- 2.25) mm Hg, (16.17 +/- 1.73) mm Hg, (17.22 +/- 1.32) mm Hg, (16.71 +/- 1.52) mm Hg, respectively. In the group 3, the IOP was decreased from (34.66 +/- 4.49) mm Hg to (10.94 +/- 2.75) mm Hg, (11.29 +/- 2.40) mm Hg, (13.93 +/- 3.55) mm Hg, (15.63 +/- 3.54) mm Hg, (15.70 +/- 2.44) mm Hg, (15.12 +/- 3.65) mm Hg, respectively, at day 1, 7, 14, 28, 42, 56, post-operatively. IOPs in the group 2 and 3 were significantly (P < 0.01) lower than that in the control group (group 1) at post-operative day 7, 14, 28, 42, 56. However, there was no IOP difference between the group 2 and 3 post-operatively at any time. Morphologically, the filtering blebs were flatded at day 28 in the group 1, while were normal in the group 2 and 3 at day 56. Less scar formation of filtering blebs was observed in group 2 and 3 compared with group 1. CONCLUSIONS: Trabeculectomy combined with the application of amniotic membrane or conjunctiva ECM can improve filtering bleb function and reduce scar formation that lead to a better IOP control after surgery.

Identification and characterization of Bimgamma, a novel proapoptotic BH3-only splice variant of Bim.

BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In this study, a novel human BH3-only protein, Bcl-2-interacting mediator (Bim)gamma, was identified during our study of regulation of prostate cancer cell death by Bcl-2 family proteins. Bimgamma shares the highest amino acid sequence homology to BimEL and BimL, two proapoptotic BH3-only Bcl-2 proteins derived from alternative mRNA splicing. Genomic studies indicate that Bimgamma is a novel splice variant of Bim and is generated as a result of the retention of a 126-bp intron of the bim gene. Bimgamma mRNA displays a tissue-specific expression pattern distinct from those of the other Bim isoforms. Subcellular fractionation studies indicate that Bimgamma is localized both in intracellular membranes and cytosol. Interestingly, Bimgamma mRNA, similar to the BimEL protein, is up-regulated in the majority of the prostate cancer cell lines studied, whereas several other proapoptotic Bcl-2 proteins, including Bax, Bak, and Bad, are down-regulated in prostate cancer cells. Functional studies indicate that Bimgamma inhibits clonal growth in prostate cancer cells and promotes apoptosis, which is inhibited by overexpressing Bcl-2. Because both Bimgamma and BimEL are proapoptotic BH3-only proteins and both are up-regulated in prostate cancer cells, they may play a unique role in prostate cancer development.

[Clinical Therapeutic Effect of Oblique Needling with Tuina in Relieving Sacroiliac Joint Injury].

OBJECTIVE: To observe the therapeutic effect of oblique needling in combination with Tuina at the sacroiliac joint for patients experiencing sacroiliac joint injury. METHODS: One hundred and twenty patients with sacroiliac joint injury were randomized into routine Tuina group and oblique needling combined with Tuina (Acu+ Tuina) group (n = 60 in each group). For patients of the Tuina group, routine Tuina as rotating, pressing-rubbing, digital pressing, articular moving, etc. was manipulated at Shangliao (BL 31), Ciliao (BL 32), Zhongliao (BL 31), Xialiao (BL 30), Huantiao (GB 30), Zhibian (BL 54), Weizhong (BL 40) and sacroiliac joint area. For patients of the Acu+Tuina group, the anatomical points between the bilateral iliac crests and the sacral joints were punctured obliquely with disposable acupuncture needles. The treatment was conducted for 30 min every time, once daily for 3 weeks except weekends. The Visual Analogue Scale (VAS) and Oswestry Disability Index (ODI, concerning intensity of pain, lifting, ability to care for oneself, ability to walk, ability to sit, sexual function, ability to stand, social life, sleep quality, and ability to travel) were employed to evaluate the patients' reactions and functional activity changes before and after the treatment. RESULTS: Following the treatment, of the two 60 patients in the Tuina and Acu + Tuina groups, 12 and 26 cases were cured, 20 and 20 experienced marked improvement, 16 and 11 were effective, 12 and 3 invalid, with the effective rates being 80% and 95%, respectively. The effective rate of the Acu+ Tuina group was significantly superior to that of the Tuina group (P<0.05). The VAS scores and OD were considerably decreased in both groups after the treatment and were significantly lower in the Acu+Tuina group than in the Tuina group (P<0.01). CONCLUSION: Oblique needling the anatomical points in the sacroiliac joint region combined with Tuina manipulation is evidently better than simple Tuina in reducing pain and in improving functional activity and life quality in sacroiliac joint injury patients.

Preoperative serum visfatin levels and prognosis of breast cancer among Chinese women.

Visfatin is identified a pro-inflammatory cytokine and its serum level is increased in various cancers. This study aimed to evaluate the prognostic value of preoperative serum visfatin level in breast cancers. Preoperative serum visfatin levels of 248 patients with breast cancer and serum visfatin levels of 100 healthy individuals and 100 benign women controls were determined using enzyme-linked immunosorbent assay. Unfavorable outcome was defined as first local recurrence, distant metastasis, second primary cancer of another organ, or death from any cause. Disease-free survival was defined as the time between surgery and the date of unfavorable outcome whichever appeared first. Overall survival was defined from surgery to death for any cause. The association of serum visfatin level with outcomes including mortality, unfavorable outcome, disease-free survival and overall survival was investigated by univariate and multivariate analyses. Preoperative serum visfatin level was substantially higher in patients than in healthy subjects and benign controls respectively. Elevated preoperative serum level of visfatin was identified an independent predictor of mortality, unfavorable outcome, disease-free survival and overall survival. Receiver operating characteristic curve analysis showed that serum level visfatin had high predictive value for mortality and unfavorable outcome. Thus, our results suggest that high preoperative serum visfatin level is associated with poor patient outcomes as well as may play a role as prognostic biomarker in breast cancer survival.

First trimester, second trimester, and integrated screening for Down's syndrome in China.

OBJECTIVE: To compare the effectiveness of first trimester, second trimester, and integrated screening for Down's syndrome. SETTING: Two prenatal diagnosis centres in China. METHODS: A total of 11,966 pregnant women (≥18 years) were screened over 21 months. First trimester screening (11-13 weeks) comprised measurement of serum free beta-human chorionic gonadotrophin (ß-hCG) and pregnancy-associated protein-A concentrations, and fetal nuchal translucency thickness. Second trimester screening (15-20 weeks) comprised measurement of ß-hCG and alpha fetoprotein concentrations. Computer software was used to calculate the risk of carrying a Down's syndrome fetus. RESULTS: The overall incidence of Down's syndrome was 0.2% (23/11,966). When the false-positive rate was fixed at 5%, detection rates for first trimester, second trimester, and integrated screening were 73.9%, 69.6%, and 82.6%, respectively. When the false-positive rate was fixed at 3%, detection/sensitivity rates for first trimester, second trimester, and integrated screening were 65.2%, 56.5%, and 73.9%, respectively. CONCLUSIONS: These findings suggest that integrated screening was the most effective means of screening for Down's syndrome in a Chinese population.