Deregulation of microRNAs in blood and skeletal muscles of myotonic dystrophy type 1 patients.

INTRODUCTION: MicroRNAs (miRNAs) are short RNA molecules of approximately 22 nucleotides that function as post-transcriptional regulators of gene expression. They are expressed in a tissue-specific manner and show different expression patterns in development and disease; hence, they can potentially act as disease-specific biomarkers. Several miRNAs have been shown to be deregulated in plasma and skeletal muscles of myotonic dystrophy type 1 (DM1) patients. METHODS: We evaluated the expression patterns of 11 candidate miRNAs using quantitative real-time PCR in whole blood (n = 10) and muscle biopsy samples (n = 9) of DM1 patients, and compared them to those of normal control samples (whole blood, n = 10; muscle, n = 9). RESULTS: In DM1 whole blood, miRNA-133a, -29b, and -33a were significantly upregulated, whereas miRNA-1, -133a, and -29c were significantly downregulated in the skeletal muscles compared to controls. CONCLUSIONS: Our findings align to those reported in other studies and point towards pathways that potentially contribute toward pathogenesis in DM1. However, the currently available data is not sufficient for these miRNAs to be made DM1-specific biomarkers because they seem to be common to many muscle pathologies. Hence, they lack specificity, but reinforce the need for further exploration of DM1 biomarkers.

Lack of correlation between X-ray repair cross-complementing group 1 gene polymorphisms and the susceptibility to colorectal cancer in a Malaysian cohort.

X-ray repair cross-complementing group 1 (XRCC1) is one of the key components in the base excision repair pathway that repairs erroneous DNA lesions and removes nonbulky base adducts for the maintenance of genome integrity. Studies have revealed that differences in individual DNA repair capacity can impact the interindividual variation in cancer susceptibility, tumour aggressiveness and treatment response. The relationship between XRCC1 and sporadic colorectal cancer (CRC) susceptibility, which is hitherto inconclusive, has been explored in many association studies of different populations. In view of the conflicting findings generated, we aimed to investigate the association between XRCC1 and genetic predisposition to CRC among Malaysians. The present case-control association study was conducted on 130 CRC patients and 212 age-matched healthy controls. The genotyping of XRCC1 Arg194Trp, Arg280His and Arg399Gln single nucleotide polymorphisms was performed with allele-specific real-time PCR approach. This was followed by basic statistical analysis on the single nucleotide polymorphisms and haplotype data obtained. No significant difference in the allele and genotype frequencies was observed between CRC patients and healthy controls (P>0.05). There was also no association observed between XRCC1 haplotypes and CRC (P>0.05). In conclusion, a positive association between XRCC1 gene polymorphisms and CRC risk was not established in our Malaysian population.

Association between genetic polymorphisms in interferon regulatory factor 5 (IRF5) gene and Malaysian patients with Crohn's disease.

OBJECTIVE: The study aimed to investigate the association between the interferon regulatory factor 5 (IRF5) gene polymorphisms and the onset of Crohn's disease (CD) in a Malaysian cohort. METHODS: Genomic DNA was extracted from blood samples collected from 91 CD patients and 100 healthy individuals via a conventional phenol-chloroform extraction method. Screening of the four target single nucleotide polymorphisms (SNPs), including rs3807306, rs4728142, rs10954213 and rs11770589 was carried out in a real-time polymerase chain reaction (PCR) thermal cycler using TaqMan genotyping assay. The genetic data obtained was subsequently subjected to statistical analysis to relate the SNPs to the onset of CD in the Malaysian population. The genotyping assay and data were further validated selectively by conventional PCR amplification of the SNP sites and DNA sequencing. RESULTS: The rs3807306 G allele was a risk factor for CD (OR 2.3630, P = 0.00004), whereas the homozygous T genotype was protective against the disease (OR 0.2038, P = 0.00004). The heterozygous A/G genotype of rs10954213 was significantly associated with CD (OR 4.319, P = 0.0377). On the other hand, the homozygous A and heterozygous A/G genotypes of the rs11770589 were significant in the controls (OR 0.4242, P = 0.0166) and patients (OR 2.000, P = 0.0179), respectively. In the ethnic-stratification analysis, the rs11770589 homozygous A genotype was protective in Indians (OR 0.1551, P = 0.0112). CONCLUSION: IRF5 gene polymorphisms may play a role in the development of CD in the Malaysian population.

Genetic polymorphisms in the one-carbon metabolism pathway genes and susceptibility to non-Hodgkin lymphoma.

Corroborating evidence related to the role of aberrations on one-carbon metabolism (OCM) genes has been inconsistent. We evaluated the association between polymorphisms in 12 single nucleotide polymorphisms (SNPs) in 8 OCM genes (CBS, FPGS, FTHFD, MTRR, SHMT1, SLC19A1, TCN1, and TYMS), and non-Hodgkin lymphoma (NHL) risk in a multi-ethnic population which includes Malay, Chinese and Indian ethnic subgroups. Cases (N = 372) and controls (N = 722) were genotyped using the Sequenom MassARRAY platform. Our results of the pooled subjects showed a significantly enhanced NHL risk for CBS Ex9 + 33C > T (T versus C: OR 1.55, 95% CI 1.22-1.96, P = 0.0003), CBS Ex18-319G > A (A versus G: OR 1.15, 95% CI 1.14-1.83; P = 0.002), SHMT1 Ex12 + 236 T > C (T versus C: OR 1.44, 95% CI 1.15-1.81, P = 0.002), and TYMS Ex8 + 157C > T (T versus C: OR 1.29, 95% CI 1.06-1.57, P = 0.01). Haplotype analysis for CBS SNPs showed a significantly decreased risk of NHL in subjects with haplotype CG (OR 0.69, 95% CI 0.56-0.86, P = <0.001). The GG haplotype for the FTHFD SNPs showed a significant increased risk of NHL (OR 1.40, 95% CI 1.12-1.76, P = 0.002). For the TYMS gene, haplotype CAT at TYMS (OR 0.67, 95% CI 0.49-0.90, P = 0.007) was associated with decreased risk of NHL, while haplotype TAC (OR 1.29, 95% CI 1.05-1.58, P = 0.01) was found to confer increased risk of NHL. Our study suggests that variation in several OCM genes (CBS, FTHFD, SHMT1, TCN1, and TYMS) may influence susceptibility to NHL.

Effect of polymorphisms within methotrexate pathway genes on methotrexate toxicity and plasma levels in adults with hematological malignancies.

AIM: Pharmacogenetics of methotrexate (MTX) contributes to interindividual differences in toxicity. We aimed to evaluate the impact of SNPs within the MTX pathway genes on MTX-induced toxicity and MTX plasma levels at 48 h following treatment in Asian adults with acute lymphoblastic leukemia or non-Hodgkin lymphoma. PATIENTS & METHODS: Patients (n = 71) were genotyped for MTHFR C677T, MTHFR A1298C, SLC19A1 G80A, ABCG2 C421A and ABCB1 C3435T using the Sequenom MassARRAY platform. Plasma MTX concentrations at 48 h were measured by fluorescence polarization immunoassay. RESULTS: Forty-eight patients had hematopoietic toxicity, 51 had hepatic toxicity and 36 had mucositis. Patients homozygous for MTHFR 677TT were associated with increased risk of both hematopoietic (odds ratio [OR]: 9.03; 95% CI: 2.28-36.16; p = 0.002) and hepatic (OR: 3.92; 95% CI: 1.01-15.11; p = 0.036) toxicities. Hepatic toxicity was associated with SLC19A1 G80A (OR: 5.27, 95% CI: 1.21-22.72; p = 0.032) and ABCB1 C3435T (OR: 8.62; 95% CI: 1.96-37.57; p = 0.004). However, polymorphisms in MTHFR A1298C and ABCG2 C421A were not associated with any of the toxicities, and mucositis was not associated with any polymorphisms of the MTX pathway genes. Patients with MTHFR C677T and ABCB1 C3435T polymorphisms appear to have significantly higher MTX plasma concentrations (p < 0.05). CONCLUSION: Our results in Asian adults provides evidence for the contribution pharmacogenetics to the toxicity of high-dose MTX and plasma MTX concentrations at 48 h following treatment in patients with acute lymphoblastic leukemia or non-Hodgkin lymphoma. These results will contribute towards the effort of MTX therapy individualization.

Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas.

OBJECTIVES: To characterise the mRNA expression patterns of early and advanced stage colorectal adenocarcinomas of Malaysian patients. DESIGN: Comparative expression analysis. SETTING AND PARTICIPANTS: We performed a combination of annealing control primer (ACP)-based PCR and reverse transcription-quantitative real-time PCR for the identification of differentially expressed genes (DEGs) associated with early and advanced stage primary colorectal tumours. We recruited four paired samples from patients with colorectal cancer (CRC) of Dukes' A and B for the preliminary differential expression study, and a total of 27 paired samples, ranging from CRC stages I to IV, for subsequent confirmatory test. The tumouric samples were obtained from the patients with CRC undergoing curative surgical resection without preoperative chemoradiotherapy. The recruited patients with CRC were newly diagnosed with CRC, and were not associated with any hereditary syndromes, previously diagnosed cancer or positive family history of CRC. The paired non-cancerous tissue specimens were excised from macroscopically normal colonic mucosa distally located from the colorectal tumours. PRIMARY AND SECONDARY OUTCOME MEASURES: The differential mRNA expression patterns of early and advanced stage colorectal adenocarcinomas compared with macroscopically normal colonic mucosa were characterised by ACP-based PCR and reverse transcription-quantitative real-time PCR. RESULTS: The RPL35, RPS23 and TIMP1 genes were found to be overexpressed in both early and advanced stage colorectal adenocarcinomas (p<0.05). However, the ARPC2 gene was significantly underexpressed in early colorectal adenocarcinomas, while the advanced stage primary colorectal tumours exhibited an additional overexpression of the C6orf173 gene (p<0.05). CONCLUSIONS: We characterised two distinctive gene expression patterns to aid in the stratification of primary colorectal neoplasms among Malaysian patients with CRC. Further work can be done to assess and compare the mRNA expression levels of these identified DEGs between each CRC stage group, stages I-IV.

Polymorphisms in methylenetetrahydrofolate reductase gene and risk of non-Hodgkin lymphoma in a multi-ethnic population.

An imbalance in folate metabolism can adversely affect DNA synthesis and methylation systems which can lead to susceptibility to non-Hodgkin lymphoma (NHL). Whether single nucleotide polymorphisms (SNPs) and their haplotypes in the methylenetetrahydrofolate reductase (MTHFR) are associated with NHL, remain inconclusive. We investigated the association between MTHFR C677T and A1298C SNPs and NHL risk in a population which is made up of Malay, Chinese and Indian ethnic subgroups. A total of 372 NHL patients and 722 controls were genotyped using the Sequenom MassARRAY platform. Our results of the pooled subjects failed to demonstrate significant association between the MTHFR C677T and A1298C SNPs with NHL and its subtypes. The results were in agreement with the previous meta-analyses. In the Indian ethnic subgroup however, single locus analysis of MTHFR A1298C appears to confer risk to NHL (Odds ratio (OR) 1.91, 95% confidence interval (95% CI) 1.22-3.00, P=0.006). The risk is almost doubled in homozygous carrier of MTHFR 1298CC (OR 4.03, 95% CI 1.56-10.43, P=0.004). Haplotype analysis revealed higher frequency of CC in the Indian NHL patients compared with controls (OR 1.86, 95% CI 1.18-2.93, P=0.007). There is lack of evidence to suggest an association between MTHFR C677T and A1298C with the risk of NHL in the Malays and Chinese. In the Indians however, the MTHFR A1298C confers risk to NHL. This study suggests ethnicity modifies the relationship between polymorphisms in the folate-metabolizing gene and NHL.

Association between inflammatory bowel disease gene 5 (IBD5) and interleukin-23 receptor (IL23R) genetic polymorphisms in Malaysian patients with Crohn's disease.

OBJECTIVE: This study was aimed to investigate the possible association of Crohn's disease (CD) with inflammatory bowel disease gene 5 (IBD5) IGR2198a_1 (rs11739135), IGR2096a_1 (rs12521868) and interleukin-23 receptor (IL23R) genetic variant (rs1004819) in the Malaysian population. METHODS: Blood samples from 80 CD patients and 100 healthy controls were recruited. Genomic DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The results revealed that there was an increased frequency of IGR2198a_1 C allele (8.8% in CD, 1.5% in controls, P < 0.05, OR 6.30, 95% CI 1.77-22.31) and IGR2096a_1 T allele (6.9% in CD, 1.5% in controls, P < 0.05, OR 4.85, 95% CI 1.33-17.69) in the CD patients as compared to the controls, suggesting the two variants were potential risk factors of CD. Both risk alleles (C and T) were highest in Indians. In contrast, no significant difference was observed for the IL23R gene variant (rs1004819) between these two groups (P = 0.941). All genotypes and alleles of this gene variant were present in equal ratios in the CD and control groups (OR 1.02, 95% CI 0.66-1.57 for T allele and OR 0.98, 95% CI 0.64-1.52 for C allele). CONCLUSIONS: There is a strong association between both IBD5 locus variants but not the IL23R gene variant with CD in the Malaysian population. The IBD5 locus variants were highest in Indians, which may explain the increased susceptibility of this particular ethnic group to the disease.

Identification of DLG5 and SLC22A5 gene polymorphisms in Malaysian patients with Crohn's disease.

OBJECTIVE: The aim of this study was to investigate the association of DLG5 and SLC22A5 gene polymorphisms with the onset of Crohn's disease (CD) in a Malaysian cohort. METHODS: Genomic DNA of 80 CD patients and 100 healthy unrelated control individuals was extracted and analyzed via polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on DLG5 (4136 C/A), DLG5_e26 and SLC22A5 (-207 G/C) genetic polymorphisms. Data obtained from the study were then subjected to statistical analysis to test for risk association. RESULTS: Significant associations of both DLG5 polymorphisms with the development of CD in the Malaysian patients were observed in this study. The homozygous C genotype of the DLG5 polymorphism was significantly related to CD patients (P = 0.0023, OR = 2.5320), while the homozygous A was significant in control individuals (P = 0.0224, OR = 0.4480). In DLG5_e26 polymorphisms, we found a significant distribution of the homozygous insA genotype in CD patients (P = 0.0006, OR = 2.8916), whereas the heterozygous insA/delA genotype was significant in controls (P = 0.0007, OR = 0.3487). We hypothesized that there might be a complex interaction of both alleles, which confered a protective effect against the onset of CD. However, we did not observe any significant correlation of SLC22A5 polymorphisms with this disease. CONCLUSIONS: In our study, both polymorphisms in the DLG5 gene were found to be associated with CD patients in Malaysia. Therefore, these loci can be potentially used as susceptibility markers in the Malaysian population.