Genotyping of Novel SNPs in BMPR1B,BMP15, and GDF9 Genes for Association with Prolificacy in Seven Indian Goat Breeds

Goats form the backbone of rural livelihood and financial security systems in India but their population is showing decreasing trend. Improvement of reproductive traits such as prolificacy offers a solution to stabilize the decreasing goat population and to meet the nutritional needs of growing human population. In the present study, six novel SNPs in three candidate genes for prolificacy (BMPR1B, BMP15, and GDF9) were genotyped in seven breeds of Indian goats to evaluate their association with litter size. Tetra primer ARMS-PCR and PCR-RFLP based protocols were developed for genotyping six novel SNPs, namely, T(-242)C in BMPR1B; G735A and C808G in BMP15; and C818T, A959C, and G1189A in GDF9 gene. The effect of breed was highly significant (p ≤ 0.01) on litter size but the effect of genotype was nonsignificant. The effect of parity on litter size was also significant in the prolific Black Bengal breed. The litter size differences observed between breeds are attributed to breed differences. Novel mutations observed at different loci in GDF9, BMP15, and BMPR1B genes do not contribute to the reproductive capability of the investigated breeds. Further studies with more number of breeds and animals exploring association of these novel SNPs with reproductive traits may be fruitful.     

Functional polymorphisms in the interleukin-12 gene contribute to cancer risk: Evidence from a meta-analysis of 18 case–control studies

Background

Emerging evidence from preclinical and clinical studies has shown that interleukin-12 (IL-12) has some effectiveness against endogenously arising carcinogenesis. Several potentially functional polymorphisms of IL-12 gene have been implicated in cancer risk, but individually published studies showed inconclusive results. The aim of this study was to investigate the association between IL-12 polymorphisms and cancer risk.

Methods

The MEDLINE, EMBASE, Web of science and CBM databases were searched for all articles published up to June 10, 2012 that addressed IL-12 polymorphisms and cancer risk. Statistical analyses were performed using RevMan 5.1.6 and STATA 12.0 softwares.

Results

Eighteen studies were included with a total of 6463 cancer cases and 7412 healthy controls. We found that the 3'UTR A > C (rs3212227) polymorphism of IL-12B gene was associated with significantly increased overall risk of cancers using random effects model (C vs A: odds ratio [OR] = 1.14, 95% confidence interval [CI]: 1.02-1.27; AC + CC vs AA: OR = 1.20, 95%CI: 1.01-1.43). However, the 3'UTR G > A (rs568408), IVS2 T > A (rs582054) and 5'UTR T > G (rs2243115) polymorphisms of IL-12A gene did not appear to have an influence on cancer susceptibility. Further subgroup analyses showed that the 3'UTR A > C (rs3212227) polymorphism was associated with increased cancer risks in the subgroups of Asians, cervical and nasopharyngeal cancers.

Conclusions

Results from the current meta-analysis indicates that the 3'UTR A > C (rs3212227) polymorphism of IL-12B gene might be a potential biomarker for cancer risk among Asians, especially for cervical and nasopharyngeal cancers.     

The effect of tumor necrosis factor alpha (-308G/a) and interferon gamma (+874T/a) polymorphisms on susceptibility to coronary heart disease

Abstract

Background: Coronary heart disease (CHD) is a chronic inflammatory disease, which is still regarded as a major cause of morbidity and mortality worldwide. Several studies have suggested that polymorphisms in cytokine genes are associated with the pathogenesis of CHD. The genotype distribution of Tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) genes polymorphisms have been shown to be different in various ethnic populations. This study was aimed to investigate the association of TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms with risk of CHD in an Iranian population.

Methods: A total of 187 unrelated subjects comprised 96 CHD patients and 91 healthy controls were enrolled in this cross-sectional study. The TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms were genotyped using amplification refractory mutation system-PCR (ARMS-PCR). The chi-square and logistic regression tests were used to calculate the odds ratios (ORs) as a measure of differences in genotype frequencies.

Results: A significant differences in the allelic and genotypic distribution of TNF-α-308?G/A and IFN-γ?+?874T/A polymorphisms was found between CHD patients and healthy controls (P?=?0.017, P?=?0.011, P?=?0.006 and P?=?0.002, respectively). Logistic regression analyses were also revealed statistically significant risk for CHD with respect to TNF-α-308?A and IFN-γ?+?874?T carriers either in crude or after adjustment for potential confounders (P?=?0.003 and P?=?0.006, respectively).

Conclusion: This study provides strong evidence supporting the association of TNF-α-308G/A and IFN-γ?+?874T/A polymorphisms with the increased risk of CHD. Therefore, these two cytokine polymorphisms may play a role in predisposition to coronary heart disease.     

Polymorphisms of transforming growth factor beta 1 (RS#1800468 and RS#1800471) and esophageal squamous cell carcinoma among Zhuangese population,China

Epidemiological evidence has shown two polymorphisms (namely RS#1800468G > A and RS#1800471G > C) of transforming growth factor-beta 1 (TGF-β1) gene may be involved in the cancer development. However, their role in the carcinogenic process of esophageal squamous cell carcinoma (ESCC) has been less well elaborated. We conducted a hospital-based case-control study including 391 ESCC cases and 508 controls without any evidence of tumors to evaluate the association between these two polymorphisms and ESCC risk and prognosis for Zhuangese population by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS)-PCR techniques. We found that individuals with the genotypes with RS#1800471 C allele (namely RS#1800471-GC or -CC) had an increased risk of ESCC than those without above genotypes (namely RS#1800471-GG, adjusted odds ratio 3.26 and 5.65, respectively). Further stratification analysis showed that this polymorphism was correlated with tumor histological grades and TNM (tumor, node, and metastasis) stage, and modified the serum levels of TGF-β1. Additionally, RS#1800471 polymorphism affected ESCC prognosis (hazard ratio, 3.40), especially under high serum levels of TGF-β1 conditions. However, RS#1800468 polymorphism was not significantly related to ESCC risk. These findings indicated that TGF-β1 RS#1800471G > C polymorphism may be a genetic modifier for developing ESCC in Zhuangese population.     

A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice

The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.     

Association of interleukin (IL)-4 intron-3 and IL-6 −174 G/C gene polymorphism with susceptibility to end-stage renal disease

Earlier studies suggest that end-stage renal disease (ESRD) is associated with inflammatory state and have become a major cause of morbidity and mortality worldwide. This study speculated the role of interleukins (IL)-2, -4, and -6 cytokines gene polymorphism with risk of susceptibility to ESRD. Polymorphism in IL-2 (−330 T/G, polymerase chain reaction [PCR]-restriction fragment length polymorphism), IL-4 (intron-3, variable number of tandem repeat, variable number tandem repeats analysis), and IL-6 (-174 G/C, amplification refractory mutation system, i.e. ARMS-PCR) were genotyped in 193 ESRD patients and 180 controls. Significant difference was observed in genotype frequencies of IL-4 and IL-6 between ESRD patients and control group (p < 0.001 and p = 0.032, respectively). Patients had higher frequency of homozygous B2B2 genotype (IL-4) than controls (62.7% vs 46.7) and GG genotype of IL-6 (73.1% vs 60.6%). The genotypic frequencies of IL-2 were comparable in patients and controls (p = 0.102). Significant association of IL-4 was also observed in patients with glomerulonephritis (p = 0.001). Combination of low IL-4 and high IL-6 genotypes were significantly associated with ESRD showing the highest risk, i.e. >threefolds risk (odds ratio=3.48, 95%CI=1.88–6.42; p < 0.001) among the four possible combinations taking high IL-4 and low IL-6 as reference. Our study suggests that polymorphism in IL-4 and IL-6 may be associated with susceptibility to ESRD. Further, combined analysis implicated a higher risk in ESRD patients with low IL-4 and high IL-6 producing genotypes. This study provided the basis for defined anti-inflammatory approaches to limit renal disease progression.     

Polymorphisms of IL-10 gene in patients infected with HCV under antiviral treatment in southern Brazil

Interleukin-10 (IL-10) is a cytokine that plays an important role in the regulation of the immune system. Gene polymorphisms of IL-10 have been associated with the different expression levels of this cytokine. In hepatitis C virus infection, IL-10 appears to interfere with the progression of disease, viral persistence and the response to therapy. This study investigated genetic variability in the IL-10 gene promoter between patients infected with hepatitis C virus (HCV) and healthy individuals, associating the frequency of polymorphisms with different aspects of viral infection. This is a case-control study with 260 patients who were infected with HCV and 260 healthy individuals. Genotyping of the polymorphisms was performed using the technique of amplification refractory mutation system PCR (ARMS-PCR) for regions of the IL-10 gene promoter (-1082 G/A, -819 C/T, -592 C/A). The frequencies of alleles and genotypes related to polymorphisms in the IL-10 gene promoter showed a higher frequency of the G allele and genotype GG in the -1082 region between the infected group and the control group (p = 0.005 and p = 0.001, respectively), whereas the AA genotype was significantly more frequent in the control group. The frequencies of the haplotypes GTA and GCC were higher in the group of infected individuals, whereas the haplotype ATA was more frequent in the healthy group (p < 0.006). It was also observed that the genotypes GG and AG in the region -1082 were significantly more frequent among patients infected with HCV who were in advanced stages of fibrosis and cirrhosis (p = 0.042). No association was observed between polymorphisms of IL-10 and sustained virologic response (SVR).     

A simple and rapid technique for the authentication of the ginseng cultivar, Yunpoong, using an SNP marker in a large sample of ginseng leaves

Yunpoong is an important Korean ginseng (Panax ginseng C. A. Meyer) cultivar, but no molecular marker has been available to identify Yunpoong from other cultivars. In this study, we developed a single nucleotide polymorphism (SNP) marker for Yunpoong based on analysis of expressed sequence tags (ESTs) in an exon region of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. This SNP marker had high specificity to authenticate Yunpoong in twelve different main ginseng cultivars. For application of the molecular marker, a rapid identification method was established based on the NaOH-Tris method and real-time polymerase chain reaction (PCR) in order to ensure more efficiency in the cultivar selection. The biggest feature of the NaOH-Tris method was that it made the extraction of DNA very simple and rapid in young leaf tissues. We only spent 1 min to extract DNA and directly used it to do PCR. In this report, the conventional DNA extraction method was used to develop molecular marker process, and the NaOH-Tris method was applied in screening large numbers of cultivars. Moreover, the greatest advantage of the real-time PCR compared with traditional PCR, is time saving and high efficiency. Thus, this strategy provides a rapid and reliable method for the specific identification of Yunpoong in a large number of samples.     

Evaluation of IFN-γ polymorphism +874 T/A in patients with recurrent tonsillitis by PCR Real Time Mismatch Amplification Mutation Assay (MAMA Real Time PCR)

Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP) + 874 1 T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor’s blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p < 0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p = 0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.