[Application of single-sperm sequencing technology in preimplantation genetic testing for men with autosomal dominant polycystic kidney disease].

OBJECTIVE: To investigate the value of single-sperm sequencing technology in preimplantation genetic testing. METHODS: Haplotypes were constructed by single-sperm isolation combined with single-sperm sequencing for a patient with autosomal dominant polycystic kidney disease (ADPKD) caused by de novo mutation of the PKD1 gene c.3815T>G. 50. Single-sperm samples were isolated by mechanical braking, whole-genome amplification was performed, and mutation loci and their 187 upstream and downstream single nucleotide polymorphisms (SNP) were designed. The amplified products were verified for determination of the chromosome haplotypes carrying or not carrying pathogenic mutations. The embryos carrying pathogenic mutations were identified in 7 embryonic trophectoderm cell biopsy samples by high-throughput sequencing after whole-genome amplification. Available blastocysts were selected for embryo transfer, and amniotic fluid samples were collected at 18 weeks of gestation to determine whether the fetuses carried pathogenic mutations. RESULTS: A total of 30 SNPs were identified by single-sperm sequencing, and haplotypes were successfully constructed. Preimplantation haplotype analysis indicated that 5 embryos carried pathogenic mutations and 2 did not. mid-gestation amniotic fluid genetic testing revealed no PKD1 gene c.3815T>G mutation in the fetuses. CONCLUSION: SNPs can be identified by single-sperm sequencing in males carrying de novo pathogenic mutation, and haplotypes can be constructed by linkage analysis for preimplantation genetic testing of embryos.

Characterization of bridging integrator 1 (BIN1) as a potential tumor suppressor and prognostic marker in hepatocellular carcinoma.

It has been shown that bridging integrator 1 (BIN1) can interact with c-myelocytomatosis (c-Myc) oncoprotein in cancer. However, the role of BIN1 in hepatocellular carcinoma (HCC) is not clear. In the present study, we investigated the expression and prognostic role of BIN1 in primary HCC and evaluated the function of BIN1 in hepatocarcinogenesis. Using real-time polymerase chain reaction and Western blot analysis, we found significantly decreased expression of BIN1 in primary HCC tumor tissues (n = 42) compared with adjacent normal tissues and in HCC cell lines. Immunohistochemistry analysis also found decreased BIN1 expression in HCC tumor tissues (n = 117). In clinicopathological analysis, loss of BIN1 expression correlated significantly (P < 0.05) with differentiation scores and tumor size. Importantly, decreased expression of BIN1 in tumors was found to be closely associated with a poor prognosis, and we conclude that BIN1 was an independent prognostic factor in a multivariate analysis. In mechanistic studies, restoring BIN1 expression in BIN1-null HCC cells significantly inhibited cell proliferation and colony formation and induced apoptosis of HCC cells. Furthermore, we found that BIN1 overexpression could significantly suppress the motility and invasion of HCC cells in vitro. Our results indicate that BIN1 may function as a potential tumor suppressor and serve as a novel prognostic marker in HCC patients. The BIN1 molecule might play an important role in tumor growth, cell motility and invasion. Modulation of BIN1 expression may lead to clinical applications of this critical molecule in the control of hepatocellular carcinoma as well as in early and effective diagnosis of this aggressive tumor.

[Analysis of fetal chromosomal karyotype and etiology in 252 cases of early spontaneous abortion].

OBJECTIVE: To investigate the relationship between fetal chromosomal karyotype and early spontaneous abortion, and the effect of the environmental factors on spontaneous abortion. METHODS: Choronic villi from 252 cases of missed abortion were sampled as patient group and 50 normal pregnancies as control group. Chorionic villi were cultured and karyotype analysis was performed by G-banding. Clinical information was collected. RESULTS: The rate of chorion chromosome abnormality in the patient group was 58.09%, significantly higher than that in the control group (4.17%) (P<0.01). Among the 140 cases of karyotype abnormalities, 81 were trisomy, 29 were monosomy X and 17 were polyploidy, accounting for 57.86%, 20.71% and 12.14% of total abnormalities, respectively. Long time and low dose radiation exposure of the pregnant female seemed to be related with spontaneous abortion(P<0.01). CONCLUSION: Chorion chromosome abnormality is a major reason of early spontaneous abortion and karyotype analysis of chorionic villus is of clinical importance. For pregnant women, long-term exposure to computers and television seems a risk factor for missed abortion.

Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice.

BACKGROUND: Aging and aging-related disorders impair the survival and differentiation potential of bone marrow mesenchymal stem cells (MSCs) and limit their therapeutic efficacy. Induced pluripotent stem cells (iPSCs) may provide an alternative source of functional MSCs for tissue repair. This study aimed to generate and characterize human iPSC-derived MSCs and to investigate their biological function for the treatment of limb ischemia. METHODS AND RESULTS: Human iPSCs were induced to MSC differentiation with a clinically compliant protocol. Three monoclonal, karyotypically stable, and functional MSC-like cultures were successfully isolated using a combination of CD24(-) and CD105(+) sorting. They did not express pluripotent-associated markers but displayed MSC surface antigens and differentiated into adipocytes, osteocytes, and chondrocytes. Transplanting iPSC-MSCs into mice significantly attenuated severe hind-limb ischemia and promoted vascular and muscle regeneration. The benefits of iPSC-MSCs on limb ischemia were superior to those of adult bone marrow MSCs. The greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo differentiation and paracrine mechanisms. CONCLUSIONS: Functional MSCs can be clonally generated, beginning at a single-cell level, from human iPSCs. Patient-specific iPSC-MSCs can be prepared as an "off-the-shelf" format for the treatment of tissue ischemia.

[Karyotype analysis of chorionic villi from pregnant women with missed abortion using multiplex ligation-dependent probe amplification].

OBJECTIVE: To evaluate the clinical value of multiplex ligation-dependent probe amplification (MLPA) technique used in karyotype analysis of chorionic villi from missed abortion. METHODS: Feb 2008 to Oct 2008, 91 patients with missed abortion diagnosed by hormonal measurement, type B ultrasound and physical exam matched with 20 normal pregnant women undergoing artificial abortion were enrolled in this study. Chorionic villi was obtained by suction dilation and curettage in aseptic condition, then those villi was cultured and analyzed by traditional cytogenetic karyotyping method, in the mean time, the DNA extracted from villi was detected by MLPA. The results of chromosomal G-banding of chorionic villi were compared between two methods. RESULTS: The diagnostic concordance of MLPA and traditional karyotyping was observed in 92% (84/91) cases, there were 84 cases in the case group with diagnostic concordance by traditional karyotyping and MLPA except 7 cases of euploidy could not be detected by MLPA. The 84 cases included 40 normal karyotype, 29 trisomy of euchromosome, 1 double trisomy of euchromosome, 10 monosomy X, 1 monosomy X combined with trisomy of euchromosome, 2 chimaera of X chromosome, 1 structural abnormity of euchromosome. Among 7 cases with discordance diagnosis, 2 cases with trisomy and 5 cases with tetrasomy of euchromosome were identified in traditional karyotyping, however, they were all diagnosed with normal disomy by MLPA. Of 20 villi from normal pregnancy, two methods got the consistent results. CONCLUSION: The MLPA was rapid and efficacy method used for analyzing aneuploids in chorionic villi.

The pivotal role of p38 and NF-kappaB signal pathways in the maturation of human monocyte-derived dendritic cells stimulated by streptococcal agent OK-432.

OK-432, a streptococcal preparation, has been shown as an effective activator to induce human monocyte-derived dendritic cells maturation. During this process, the activation of Toll-like receptor 4 plays an important role. However, the signaling pathway involved in has not been fully understood. In the present study, we investigated the underlying mechanisms, by which OK-432 induced maturation of human monocyte-derived DCs (MoDCs). We observed that exposure of immature MoDCs to OK-432 activated the p38 MAPK and NF-kappaB pathway, accompanied up-regulated the surface expression of maturation markers CD80, CD83, CD86 and HLA-DR, increased secretion of TNF-alpha, IL-12 and chemokine, IP-10. In addition, T cells stimulatory capacity was also enhanced. The maturation of MoDCs stimulated by OK-432 was inhibited by treatment with p38 pathway inhibitor, SB203580, or NF-kappaB pathway inhibitor, BAY-117082. Whereas, blocking of JNK pathway with SP600125 or ERK pathway with PD98059 did not influence OK-432-induced DCs maturation. Taken together, our data indicated that OK-432-induced DCs maturation was due, at least partly to the activation of p38 and NF-kappaB pathway.

[Expression and clinical significance of TESTIN in primary gastric cancer].

BACKGROUND & OBJECTIVE: Previously, we identified frequent loss of heterozygosity (LOH) at D7S486 of chromosome 7q31 in gastric carcinoma. The TESTIN (TES) gene is a candidate tumor suppressor gene at 7q31.2. This study aimed to investigate the expression of TES in gastric carcinomas, and explore its correlations to clinicopathologic features and prognosis of gastric cancer. METHODS: Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to investigate the expression of TES in 140 specimens of primary gastric carcinomas and corresponding adjacent normal tissues; Western blot was performed to evaluate the expression of TES protein in 50 specimens of gastric carcinomas and adjacent normal tissues. The correlations of TES to clinicopathologic features and prognosis of gastric carcinoma were analyzed. RESULTS: The mRNA level of TES was down-regulated in 96 (68.6%) of the 140 carcinomas as compared with that in matched normal tissues. The positive rate of TES protein was significantly lower in gastric cancer than in normal tissues (22.9% vs. 85.7%, P<0.001). The protein level of TES was down-regulated in 36 (72.0%) of 50 carcinomas as compared with corresponding normal tissues. TES expression was positively correlated with the differentiation of gastric carcinoma (r=0.178, P=0.035). The median survival was significantly shorter in TES-negative patients than in TES-positive patients (P=0.035). CONCLUSIONS: The expression level of TES is significantly down-regulated in primary gastric cancer. TES may be a valuable marker for assessing the prognosis of gastric carcinoma.

[Cytogenetic and molecular genetic alterations on chromosome 4q in human hepatocellular carcinoma].

The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factor, multi-step, multi-gene and complicated process. Specific chromosome losses and corresponding inactivation of tumor suppressor genes (TSGs) are frequently detected during the development of HCC. A high frequency of loss on chromosome 4q in HCC has been reported, suggesting that the dysfunction of specific TSGs on this chromosome arm is involved in the development and progression of HCC. In this article, we reviewed the studies on chromosomal loss of 4q in HCC patients using cytogenetic and molecular genetic technologies, such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) analysis; we also summarized the regions of chromosome 4q with high frequency of loss in HCC patients from different countries, and discussed their relationships with clinical parameters including hepatitis B virus (HBV) infection, tumor differentiation and tumor size, and listed potential TSGs on chromosome 4q in HCC patients.

Expression and prognosis role of indoleamine 2,3-dioxygenase in hepatocellular carcinoma.

PURPOSE: Immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) plays an important role in immune tolerance. In some malignancies, the evidences were given to show that overexpression of IDO was related to poor prognosis of cancer patients. METHODS: In this study, we investigated IDO expression in hepatocellular carcinoma (HCC) cell lines and 138 primary HCC clinical surgical specimens, and correlation of IDO expression with clinical outcomes and prognosis of HCC patients. RESULTS: Reverse transcription-PCR analysis showed that in vitro IDO expression in HCC cell lines and non-cancerous liver cell line were dependent on IFN-gamma. Immunohistochemical detection revealed that IDO was overexpressed in 49 of 138 (35.5%) tumor resection samples, whereas 89 of 138 (64.5%) cases showed weak immunostaining. IDO overexpression was significantly correlated with high metastasis rates (P = 0.049). Kaplan-Meier survival curves showed that overexpression of IDO resulted in significantly poor prognosis (P = 0.017, log-rank test). Multivariate Cox's analysis showed that IDO expression was an independent prognostic factor for overall survival of HCC patients (P = 0.010). CONCLUSION: Our data indicated that IDO may be a novel favorable prognostic indicator and candidate adjuvant therapeutic target for HCC.

Decreased expression of ING2 gene and its clinicopathological significance in hepatocellular carcinoma.

The inhibitor of growth (ING) family member 2 (ING2) is a newly discovered member of ING family that can regulate a wide range of cellular processes including cell growth arrest, apoptosis, and DNA repair. Researches have shown that ING2 can activate p53 and p53-mediated apoptotic pathway involved in the hepatocarcinogenesis. To investigate the role of ING2 in hepatocellular carcinoma (HCC) pathogenesis, we analyzed the correlations between the ING2 expression level and clinicopathologic factors and studied its prognostic role in primary HCC. Using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, ING2 transcription and post-transcription level was found to be downregulated in the majority of tumors compared with matched non-tumors liver tissues (p=0.004 and p=0.014, respectively). The immunohistochemistry data indicated significant reduction of ING2 expression level in 44 of 84 (52.4%) HCC cases. In addition, the expression level of ING2 correlated with tumor size, histopathologic classification, serum AFP (p<0.05). Kaplan-Meier curves demonstrated that patients with reduced ING2 expression were at significantly increased risk for shortened survival time (p=0.009). Using multivariate analysis, ING2 expression was found to be an independent prognostic factor. Our data suggest that ING2 is involved in the progression of HCC, therefore it is considered to be a candidate tumor suppressor gene and its significantly decreased expression in HCC may lead to an unfavorable prognosis.

Increased polycomb-group oncogene Bmi-1 expression correlates with poor prognosis in hepatocellular carcinoma.

PURPOSE: Recent studies have identified polycomb-group gene Bmi-1 as oncogene in the generation of mouse pre-cell lymphomas, and overexpression of Bmi-1 has been found in several human tumor with the disease progress and poor prognosis of the cancer patients. METHODS: In present study, we investigated Bmi-1 expression and its prognostic significance in hepatocellular carcinoma (HCC) by performing immunohistochemical analysis, using a total of 137 HCC clinical tissue samples. RESULTS: High Bmi-1 expression (Bmi-1 2+ or 3+) was shown in 29.9% cases. The positive immuno-staining of Bmi-1 was not only in well/moderately-differentiated tumor cells, but also in surrounding noncancerous or cirrhotic liver tissue. Bmi-1 expression level did not correlate with any clinicopathological parameters. However, survival analysis showed that the high-Bmi-1 group had a significantly shorter overall survival time than the low-Bmi-1 group (P=0.047). Multivariate analysis after 24 months revealed that Bmi-1 expression was a significant and independent prognostic parameter (P=0.002) for HCC patients. CONCLUSIONS: Our study indicated that Bmi-1 could be a candidate biomarker for long-term survival in HCC.