Novel insertion mutation p.Asp610GlyfsX23 in APC gene causes familial adenomatous polyposis in Chinese families

Objective

The aim of the study was to identify the causative gene defects associated with familial adenomatous polyposis (FAP) in two Chinese pedigrees.

Methods

The diagnosis of FAP patients was confirmed by clinical manifestations, family histories, colonoscopy and pathology examinations. Blood samples were collected and genomic DNA was extracted. The mutation analysis of the adenomatous polyposis coli (APC) and human mutY homolog (MUTYH) genes was conducted by direct polymerase chain reaction (PCR) sequencing and multiplex ligation-dependent probe amplification (MLPA).

Results

In pedigree A, the results of direct PCR sequencing revealed a heterozygous insertion mutation at codon 610 in exon 15 of APC gene (c.1828_1829insG), which resulted in frameshift change (p.Asp610GlyfsX23) in all 4 patients, but was absent in the unaffected familial members and controls. In pedigree B, we didn't identify that causative mutations cosegregated with the clinical phenotype in the APC and MUTYH genes.

Conclusions

We identified a novel insertion mutation as the pathogenic gene of FAP in Chinese population, which could enrich the germline mutation spectrum of APC gene, and the prophylactic proctocolectomy for the mutation carrier in family should be considered.     

Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran

Background

Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.

Methods

Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.

Results

A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).

Conclusion

With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen.     

Role of the RAD51 G172T polymorphism in the clinical outcome of cervical cancer patients under concomitant chemoradiotherapy

Introduction

Cervical cancer is one of the most common cancers diagnosed in women worldwide. Mammalian cells are constantly exposed to a wide variety of genotoxic agents from both endogenous and exogenous sources. The RAD51 protein is required for meiotic and mitotic recombination and plays a central role in homology-dependent recombinational repair of double-strand breaks (DSBs). Given the functional relevance of the DNA repair system on carcinogenesis, potential associations between genetic polymorphisms of DNA repair genes, cancer risk and response to therapy have been intensively evaluated. This is the first study evaluating the role of the RAD51 G172T genetic variants in cancer prognosis and clinical outcome of cervical cancer patients.

Material and methods

We analyzed RAD51 G172T polymorphism genotypes in cervical cancer patients who underwent a platinum-based chemotherapy in combination with radiotherapy. Genotyping was performed by Taqman™ Allelic Discrimination methodology.

Results and discussion

Concerning the overall survival rates found using Kaplan–Meier method and Log Rank Test, we observed that the mean survival rates were statistically different according to the patients RAD51 genotypes. The group of patients carrying the T allele present a higher mean survival rate than the other patients (102.3 months vs. 86.4 months, P = 0.020). Using the Cox regression analysis, we found an increased overall survival time for T-carrier patients, when compared with GG genotype, with tumor stage, age and presence of lymph nodes as covariates [hazard ratio (HR), 0.373; 95% CI, 0.181–0.770; P = 0.008]. Among patients (n = 193), RAD51 genotype frequency distributions were not under the influence of clinicopathologic characteristics, namely, treatment response (P = 0.508), recurrence (P = 0.150) and tumor stage (P = 0.250).

Conclusions

This is the first study evaluating the role of the RAD51 G172T genetic variants in cancer prognosis and clinical outcome of cervical cancer patients. Our results indicate an influence of the RAD51 genetic variants in overall survival of cervical cancer. Thereby, RAD51 G172T genotypes may provide additional prognostic information in cervical cancer patients who underwent cisplatin-based chemotherapy in combination with radiotherapy.     

A functional germline variant in the P53 polyadenylation signal and risk of esophageal squamous cell carcinoma

Aim

P53 plays a critical role in the maintenance of genomic stability as well as the control of cell growth and apoptosis. Recently, an uncommon P53 genetic variant (rs78378222) was reported to be significantly associated with multiple cancers in Caucasians in a genome-wide association study. rs78378222 locates in the 3′-untranslated region of the P53 gene, and this A-to-C polymorphism results in changes of the AATAAA polyadenylation signal to AATACA, which leads to impaired 3′-end processing of P53 mRNA and decreased P53 expression.

Methods

We evaluated the association between this polymorphism and esophageal squamous cell carcinoma (ESCC) risk in a case–control cohort consisting of 405 ESCC patients and 810 healthy controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression.

Results

We did observe this polymorphism with low minor allele frequency in Chinese Han population. Additionally, significantly increased ESCC risk was associated with P53 rs78378222 A > C polymorphism. Compared with rs78378222AA carriers, the OR of developing ESCC for AC carriers was 3.22 (95% CI = 1.71 − 6.33, P = 1.34 × 10^− 4).

Conclusion

These results suggest that this functional uncommon P53 rs78378222 variant is associated with ESCC risk in the current Han Chinese population.     

Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer

The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05). However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05). There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively), but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively). Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR): 0.51, 95% confidence interval (95% CI): 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan.     

家族性腺瘤息肉病的遗传病因学研究进展

家族性腺瘤息肉病(FAP)是第二常见的遗传性结直肠癌综合征,多在青春期发病,发病率约1/10000,主要临床表现为大肠中多发的腺瘤性息肉,是一种结直肠癌的癌前病变,如果不予治疗,几乎100%的患者会发展成为结直肠癌。一直以来,FAP被认为是一种常染色体显性遗传疾病,发病由APC基因胚系突变引起。根据临床特点的不同,FAP患者可以分为经典型FAP(CFAP)和轻表型FAP(AFAP)。然而近年来,在一些无APC基因胚系突变的FAP患者中发现了Mut YH基因的双等位基因突变。这种由于Mut YH基因双等位基因突变而无APC生殖突变所引起的临床综合征定义为Mut YH基因相关性息肉病[2](MAP)。MAP为常染色体隐性遗传,是一种特殊类型的FAP。另外,很多研究表明,APC基因的突变位点与结肠腺瘤病的严重程度、癌变的风险程度和某些肠外表现相关。MAP的发现和对FAP基因型-表型相关性的研究,完善了对FAP遗传病因学的认识,对于FAP患者及高危亲属的合理防治和预后具有重要的意义。     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Combined genetic mutations have remarkable effect on deep venous thrombosis and/or pulmonary embolism occurence

Purpose

Although deep vein thrombosis and thromboembolic diseases differ among various races, they are still important in our day. The difficulties in treatment and following-up of these diseases are caused by secret genetic mutations rather than predisposing factors.

Methods

Between January 2011 and May 2013, patients who were traced for deep vein thrombosis and/or pulmonary embolism were evaluated retrospectively. 84 patients (53.6% males and 46.4% females) were included in the study. Their family histories, predisposing factors and treatments were researched. Factor V Leiden (G 1691A), Factor II G20210A, Plasminogen Activator Inhibitor-Type 1 (4G/5G), and Methylene Tetrahydrofolate Reductase (C677T, A1298C) mutations were investigated from peripheral venous blood.

Results

Among the genetic mutations we searched, the incidence of single mutation rate was observed at 11.9%, double mutation collocation at 44%, triple mutation collocation at 29.8%, quadruple mutation collocation at 13.1%, and finally, quintuplet mutation collocation at 1.2%. Our approximate mutation number was found as 2.47 ± 0.91.

Conclusion

We observed that multiple mutations were high in number compared to single genetic mutations. The patients who have multiple mutations should be more in the front line considering their diagnosis, treatment and following up, and also in terms of decreasing mortality, morbidity and recurrence.     

Polymorphism of the methylenetetrahydrofolate reductase gene C677T and its influence on the antihypertensive and vascular protective effects of short-term lercanidipine treatment

Objective

To determine whether the antihypertensive and vascular protective effects of short-term treatment with lercanidipine, a calcium channel blocker, are modulated by the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism.

Methods

In a self-controlled study, a total of 143 essential hypertensive patients, all permanent residents of Shanghai, were included. All of them were treated orally with lercanidipine at a single daily fixed dosage of 10 mg for 28 consecutive days and the genotypes of the MTHFR C677T polymorphism were determined. Blood pressures, ankle-brachial index values (ABI), and pulse wave velocity (PWV) were measured at baseline and on the 29th day.

Results

The 110 subjects for whom complete genotype and phenotype information were available were used for final data analysis. Patients with the TT genotype showed higher baseline diastolic blood pressure (DBP) than those with the CC and CT genotypes (P = 0.018). Within each genotype group, SBP, DBP and PWV showed significant difference between baseline and after treatment (P < 0.05). However, ABI showed significant difference between baseline and after treatment only within the CT and TT groups (P < 0.05) but not in the CC group (P > 0.05). Patients with the TT genotype presented a greater reduction in normalized PWV than those with the CC and CT genotypes (P = 0.02). Patients in all genotype groups had statistically similar changes in normalized SBP, DBP and ABI (P > 0.05).

Conclusion

The MTHFR gene polymorphism C677T might be associated with the vascular protective effects of short-term lercanidipine treatment. However, the MTHFR C677T polymorphism might not affect the antihypertensive effects of the lercanidipine treatment.     

Association study of AGER gene polymorphism and hypertension in Han Chinese population

Background

Advanced glycation end products (AGEs) are produced by non-enzymatic glycation or glycoxidation of proteins, lipids and nucleic acids. The bond of AGEs and the receptor of AGE (AGER) in a pro-oxidant environment could induce immune and inflammation reaction involved in progress of microvascular disease. Accumulated evidence warrant further study on AGE–AGER pathway and genetic susceptibility to hypertension (HT).

Methods

We designed a two-stage association study to evaluate the association of AGER polymorphism and HT. In stage 1, seven tagSNPs were tested in 524 cases and 531 controls and the significant SNPs (P < 0.05) would enter into stage 2 including 807 cases and 869 controls. Furthermore, joint analysis was performed for all 2731 subjects including 1331 cases and 1400 controls, and meta-analysis was applied to evaluate combined estimations from the subgroups of stage 1 and stage 2.

Results

In stage 1, rs204994 had significant association with HT (P < 0.05) and enter stage 2. Neither joint analysis nor meta-analysis found statistical association of rs204994 with HT after adjusted for the covariates in the whole population. However, further stratification analysis found that rs204994 was significantly associated with HT in < 50 years and ≥ 50 years groups, ORs (95%CI) of dominant model were 1.623 (1.054–2.500) and 0.721 (0.546–0.952) respectively. No significant correlation was found between blood pressure and the polymorphisms of rs204994.

Conclusions

Our data suggests that age might modulate the genetic effects of variation of rs204994 in AGER on HT and further replications in other populations and functional studies should be warranted.     

Polymorphisms of folate metabolism-related genes and survival of patients with colorectal cancer in the Korean population

Background

5-Fluorouracil (5-FU) is a cornerstone of chemotherapy for colorectal cancer (CRC), and the major targets of 5-FU are thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), and reduced folate carrier 1 (RFC1). We hypothesized that polymorphisms in the genes encoding these proteins would be associated with CRC patient survival.

Patients and methods

We genotyped the following polymorphisms in 372 CRC patients: TS enhancer region (TSER), TS 1494del6, MTHFR 677C > T and 1298A > C, and RFC1 − 43T > C, 80G > A, and 696C > T. Using Kaplan–Meier curves, log-rank tests, and Cox proportional hazard models, we evaluated associations between these polymorphisms and overall survival (OS).

Results

The combined TS 1494 0bp6bp + 6bp6bp genotype was associated with reduced OS compared to the TS 1494 0bp0bp genotype. Among rectal cancer patients, the RFC1 − 43CC and 80AA genotypes were associated with favorable OS.

Conclusions

Our data suggest that TS and RFC1 polymorphisms are associated with CRC prognosis in Korean patients. Further studies are needed to verify these findings.     

NQO1 609C > T polymorphism interaction with tobacco smoking and alcohol drinking increases colorectal cancer risk in a Chinese population

Background

NAD (P)H:quinone oxidoreductase (NQO1) catalyzes the activation of some environmental procarcinogens present in tobacco smoke or the diet. We conducted a hospital-based case–control study to evaluate the potential association between NQO1 609C > T polymorphisms and colorectal cancer risk in a Chinese population.

Methods

The study population comprised 672 histologically confirmed colorectal cancer patients and 672 frequency-matched control subjects without cancer or systemic illness. We used PCR restriction fragment length polymorphism-based methods for genotyping analyses and unconditional logistic regression model for statistical evaluations.

Results

The risk of colorectal cancer increased with the level of smoking and decreased with the consumption of tea, fresh fruits, and vegetables. In addition, we found that the NQO1 609 CT and TT genotypes were associated with an increased risk of colorectal cancer (CT: adjusted OR = 2.02, 95% CI = 1.55–2.57; TT: adjusted OR = 2.51, 95% CI = 1.82–3.47), compared with the CC genotype. Moreover, NQO1 609C > T appeared to have a multiplicative joint effect with both tobacco smoking and alcoholic drinking (P for multiplicative interactions were 0.0001 and 0.013, respectively) on colorectal cancer risk.

Conclusion

Our findings suggest that the NQO1 609C > T polymorphism plays an important role in the development of colorectal cancer in the Chinese population, which is strengthened by alcohol drinking or tobacco smoking.     

Significance of MEFV gene R202Q polymorphism in Turkish familial Mediterranean fever patients

Objective

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent attacks of fever and inflammation in the peritoneum, synovium, or pleura, accompanied by pain. The disease is associated with mutations in the Mediterranean fever (MEFV) gene, which encodes for the pyrin protein. The aim of this study was to explore the frequency and clinical significance of the R202Q (c.605G>A) polymorphism in exon 2 of the MEFV gene in a cohort of Turkish patients with FMF.

Methods

The study included 191 patients with FMF and 150 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay for the MEFV gene R202Qpolymorphism.

Results

The genotype and allele frequencies of R202Q polymorphism showed a statistically significant difference between FMF patients and controls (p < 0.0001 and p = 0.0004, respectively) and especially the homozygous AA genotype was significantly higher in FMF patients than healthy controls (p = 0.0002; odds ratio = 6.27; 95% CI = 2.1–18.3). However no significant association was observed between clinical and demographic features of FMF patients and R202Qpolymorphism.

Conclusion

The results of this study showed that there was a high association between MEFV gene R202Q polymorphism and FMF. R202Q polymorphism should be included in routine molecular diagnosis of FMF patients.     

Clinical and genetic characteristics of patients with multiple polyposis of the large bowel negative for APC and MYH gene mutations using conventional methods

A genealogical analysis and molecular and clinical investigations were carried out in 19 probands with multiple colorectal adenomas (numbering near 100 or more). Twelve of these patients (63.1%) were negative for the APC and MYH gene mutations using conventional methods. A hereditary form of the disorder was confirmed in three (25%) probands. The median age of the disease onset in APC-negative patients was somewhere between the mean age of the disease onset in APC- and MYH-positive polyposis. Extraintestinal signs of the disorder were less frequent in APC-negative than in APC-positive probands. Half of APC- and MYH-negative probands with multiple polyposis developed colon cancer. APC- and MYH-negative patients formed a genetically heterogeneous group.     

Mutational analysis of OGG1, MYH, MTH1 in FAP, HNPCC and sporadic colorectal cancer patients: R154H OGG1 polymorphism is associated with sporadic colorectal cancer patients

MYH, OGG1 and MTH1 are members of base excision repair (BER) families, and MYH germline mutations were recently identified in patients with multiple adenomas or familial adenomatous polyposis (FAP). A total of 20 APC-negative Korean FAP patients were analyzed for OGG1, MYH and MTH1 germline mutations. A total of 19 hereditary nonpolyposis colorectal cancer (HNPCC), 86 suspected HNPCC, and 246 sporadic colorectal cancer cases were investigated for OGG1 and MYH mutations. A total of 14 R154H OGG1 polymorphisms were identified in hereditary, sporadic colorectal cancers, and normal controls. For the case-control analysis of OGG1 R154H, a total of 625 hereditary or sporadic colorectal cancer patients and 527 normal controls were screened. R154H was a rare polymorphism associated with sporadic colorectal cancer patents (OR: 3.586, P= 0.053). R154H does not segregate with cancer phenotypes. Upon examining the possibility of recessive inheritance of R154H, we could not identify any complementary mutations in OGG1, MYH or MTH1. Samples with R154H were further screened for mutations of K-ras, -catenin, APC, p53, BRAF and the microsatellite instability (MSI) status. Eight somatic mutations were identified in these genes and G:C to T:A transversion mutations were not dominant in samples harboring R154H. This result raises the possibility that OGG1 R154H may function as a low/moderate-penetrance modifier for colorectal cancer development.I.-J. Kim and J.-L. Ku contributed equally to this work     

A rational,non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Context

Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward.

Objective

To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis.

Patients and methods

We studied 99 patients from 90 families with salt-wasting (SW; n = 32), simple-virilizing (SV; n = 29), and non-classical (NC; n = 29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated.

Results

ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3′-end of CYP21A1P, C4B, and the 5′-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation.

Conclusion

Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.     

Is Surveillance Colonoscopy Necessary for Patients with Sporadic Gastric Hyperplastic Polyps?

Background

Gastric polyps, such as adenomas and hyperplastic polyps, can be found in various colonic polyposis syndromes. Unlike in sporadic gastric adenomas, in which the increased risk of colorectal neoplasia has been well characterized, information in sporadic gastric hyperplastic polyps was limited.

Aim

To evaluate the association of sporadic gastric hyperplastic polyps with synchronous colorectal neoplasia in a large cohort.

Methods

Patients with sporadic gastric hyperplastic polyps who underwent colonoscopy simultaneously or within six months were consecutively enrolled. Each patient was compared with two randomly selected age and sex matched controls without gastric polyps who also underwent colonoscopy in the same period. Data of patients’ demographics and characteristics of the gastrointestinal polyps were documented.

Results

A total of 261 cases in 118,576 patients who underwent esophagogastroduodenoscopy were diagnosed as sporadic gastric hyperplastic polyps, and 192 of 261 (73.6%) patients underwent colonoscopy. Colorectal neoplasias were identified in 46 (24.0%) of 192 cases and in 40 (10.4%) of 384 controls (P＜0.001). The mean size and distribution of colorectal neoplasias were not significantly different between the two groups. There was a significantly higher rate of colorectal adenoma (odds ratio [OR] 3.2, 95% confidence interval [CI] 1.9–5.3) in the gastric hyperplastic polyps group than in the control group, while the prevalence of colorectal cancer was similar in the two groups. Logistic regression analysis also suggested that the presence of gastric hyperplastic polyps (OR 2.5, 95% CI 1.5–4.0) was an independent risk factor for colorectal neoplasias.

Conclusion

The risk of colorectal adenoma increases in patients with sporadic gastric hyperplastic polyps, and surveillance colonoscopy for these patients should be considered.     

Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23-25 years

Objective

To evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs).

Methods

Samples of human BMCs that were cryopreserved for 23–25 years (n = 20) were thawed to obtain an initial culture and a primary culture (P₀) that was propagated through five passages (P₁–P₅) to obtain MSCs. Freshly collected human bone marrow samples (n = 20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P₃ cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing.

Results

In the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p < 0.05) while the relative rate of recovery of MSCs was only 48.5 ± 8.6% in P₀. At the end of P₃, fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p > 0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P₃ from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells.

Conclusion

Using the methods described here, long-term (23–25 years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.     

Association study of microRNA polymorphisms with hepatocellular carcinoma in Korean population

Background

Recent studies have suggested that common genetic polymorphisms alter the processing of microRNA (miRNA) and may be associated with the development and progression of cancer.

Patients and methods

The association of miRNA polymorphisms with HCC survival was analyzed in 159 HCC patients and 201 controls by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Results

The risk of HCC was significantly lower for the miR-499A>G, AG + GG in HCC patients (AOR = 0.603, 95% CI = 0.370–0.984) and hepatitis B virus (HBV)-related HCC patients (AOR = 0.561, 95% CI 0.331–0.950). In addition, the risk of HCC was significantly lower for the miR-149C>T, CT and CT + CC in HCC patients (CT; AOR = 0.542, 95% CI = 0.332–0.886, CT + CC; AOR = 0.536, 95% CI = 0.335–0.858) and HBV-related HCC patients (CT: AOR = 0.510, 95% CI 0.305–0.854, CT + CC: AOR = 0.496, 95% CI 0.302–0.813). The miR-149C>T polymorphism was also associated with survival rate of HCC patients in OKUDA II stage.

Conclusions

miR-149C>T and miR-499A>G were associated with HBV-related HCC. Further studies on larger populations will need to be conducted to confirm these results.     

rs4215 SNP in zinc-α2-glycoprotein gene is associated with obesity in Chinese north Han population

Objective

Zinc-α2-glycoprotein (ZAG) has been identified recently as a novel adipokine due to its close link with lipid and glucose metabolism, as well as regulation of body weight. The aim of our present study is to investigate the ZAG genetic polymorphism association with obesity in Chinese north Han population.

Design and methods

Five SNPs of ZAG gene including rs2247607 (A>T), rs4727442 (G>T), rs4215 (A>G), rs2527923 (C>T) and rs2527882 (C>T) were genotyped in 648 overweight/obese patients and 313 healthy controls by TaqMan-PCR methods. Crosstabs statistical analysis method with subjects stratifying by age (≦ 30 y, 31–45 y, ≧ 46 y) and gender was used.

Results

The results showed the constitution of three genotype frequencies in rs4215 (A>G) site significantly differs in male subgroup (aged 31–45 y) between overweight/obese and healthy control group (χ² = 6.401, P = 0.041). GG genotype frequency in overweight/obese group is 19.3% which is much higher than 6.1% in healthy control group. Further statistical analysis under a recessive inheritance model demonstrated odd ratio (OR) for GG vs. AA+AG in overweight/obese group was 3.674 (95% CI 1.049–12.866; P = 0.035). Among three genotypes of rs4215, the subjects with GG genotype have much more higher body weight, BMI, waist circumference and SBP.

Conclusion

Our data, for the first time, suggest the genotypes of rs4215 in ZAG gene are significantly associated with obesity in Chinese north Han population. GG genotype subjects in rs4215 site have an increased susceptibility to obesity when compared with the AA+AG genotype subjects.