NOD2 gene mutations associate weakly with ulcerative colitis but not with Crohn's disease in Indian patients with inflammatory bowel disease

Background

Three mutations (two missense and one frameshift) in the NOD2 gene are associated with Crohn's disease (CD) in a proportion of patients with Crohn's disease in North America, Europe and Australia. These three mutations are not found in Indian patients with CD. We undertook new studies to identify polymorphisms in the NOD2 gene in the Indian population and to detect whether any of these were associated with inflammatory bowel disease (IBD) in this population.

Methods

Individual exons of the NOD2 gene were amplified by PCR and subjected to denaturing high performance liquid chromatography (DHPLC) to detect heteroduplex formation. All 12 exons of the NOD2 gene were amplified and Sanger-sequenced to detect polymorphisms in the NOD2 gene. 310 patients with CD, 318 patients with ulcerative colitis (UC) and 442 healthy controls (HC) were recruited for association studies. DNA from these participants was evaluated for the identified eight polymorphisms by Sequenom analysis.

Results

Heteroduplex formation was noted by DHPLC in exons 2 and 4 of the NOD2 gene. Sequencing of the entire NOD2 gene data revealed eight polymorphisms – rs2067085, rs2066842, rs2066843, rs1861759, rs2111235, rs5743266, rs2076753, and rs5743291 – of which the latter four were described for the first time in Indians. None of these polymorphisms was associated with CD. The SNPs rs2066842 and rs2066843 were in significant linkage disequilibrium. Both SNPs showed a significant association with UC (P = 0.03 and 0.04 respectively; odds ratio 1.44 and 1.41 respectively).

Conclusion

Four NOD2 polymorphisms were identified for the first time in the Indian population. Of 8 NOD2 polymorphisms, none were associated with CD but two were weakly associated with UC. NOD2 polymorphisms do not play a major role in CD genesis in India.     

Snipping polymorphisms from large EST collections in barley (<Emphasis Type="Italic">Hordeum vulgare </Emphasis>L.)

The public EST (expressed sequence tag) databases represent an enormous but heterogeneous repository of sequences, including many from a broad selection of plant species and a wide range of distinct varieties. The significant redundancy within large EST collections makes them an attractive resource for rapid pre-selection of candidate sequence polymorphisms. Here we present a strategy that allows rapid identification of candidate SNPs in barley (Hordeum vulgare L.) using publicly available EST databases. Analysis of 271,630 EST sequences from different cDNA libraries, representing 23 different barley varieties, resulted in the generation of 56,302 tentative consensus sequences. In all, 8171 of these unigene sequences are members of clusters with six or more ESTs. By applying a novel SNP detection algorithm (SNiPpER) to these sequences, we identified 3069 candidate inter-varietal SNPs. In order to verify these candidate SNPs, we selected a small subset of 63 present in 36 ESTs. Of the 63 SNPs selected, we were able to validate 54 (86%) using a direct sequencing approach. For further verification, 28 ESTs were mapped to distinct loci within the barley genome. The polymorphism information content (PIC) and nucleotide diversity () values of the SNPs identified by the SNiPpER algorithm are significantly higher than those that were obtained by random sequencing. This demonstrates the efficiency of our strategy for SNP identification and the cost-efficient development of EST-based SNP-markers.The first two authors contributed equally to this work     

不明原因男性不育人群中睾丸特异性乳酸脱氢酶基因突变的发现及其意义

为了研究睾丸特异性乳酸脱氢酶,即乳酸脱氢酶C4(LDH-C4)基因突变在男性不育发病中的作用,利用LDH-C4特异性底物对100名不明原因男性不育症患者的精子LDH-C4进行活性显色,用变性高效液相色谱(DHPLC)技术对LDH-C4活性低下的患者进行LDHC基因PCR产物的突变筛查,对DHPLC峰形异常的PCR产物进行序列测定.筛选到一组精子LDH-C4活性明显下降的患者,其中1名患者的LDHC基因PCR产物在DHPLC中呈异常洗脱峰.对这一PCR产物进行序列测定,发现患者LDHC基因第5外显子的115位碱基发生了T→A的杂合改变(GenBank登录号GU479375),该突变使LDHC基因的178位密码子由原来的TTG(编码亮氨酸)变为TAG(终止密码子),形成截短的C亚基.T克隆-测序进一步证实了该无义突变的杂合状态.这是在人类LDHC基因上发现的第一个突变,提示LDHC基因突变可能是男性不育发病的原因之一.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.     

Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.     

Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome

BACKGROUND: The retinoic acid (RA)-catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1-null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS: Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by chi(2) analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS: Mutational screening allowed us to identify 6 SNPs, 4 of which (447 C>G, 1134 G>A, IVS 1+10 G>C, and IVS 4+8 AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS: Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects.     

单核细胞增生李斯特氏菌PCR-DHPLC检测新技术的建立

应用PCR结合变性高效液相色谱(DHPLC)技术建立食品中单核细胞增生李斯特氏菌fimY的快速检测方法。根据单核细胞增生李斯特氏菌prfA和hlyA基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以单核细胞增生李斯特氏菌等61株参考菌株做特异性试验;单核细胞增生李斯特氏菌菌株稀释成不同梯度,进行灵敏度试验。试验结果表明该方法具有很好的特异性,灵敏度较高,检测低限可达到为181CFU/ml。可以快速、准确检测单核细胞增生李斯特氏菌,是食品中致病菌快速检测的新技术。     

利用DHPLC研究我国几个地方绵羊品种黑素细胞刺激素受体基因单核苷酸多态性

利用测序及变性高效液相色谱(DHPLC)研究了蒙古羊、哈萨克羊、滩羊和藏绵羊黑素细胞刺激素受体（MSHR）基因单核苷酸多态性（SNP）。结果表明,在扩增片断长度为415bp范围内存在一个T317C突变,DHPLC可检测到该突变并被证明是一种高通量且简便的筛选方法。通过两次DHPLC可确定两个杂合子和一个纯合子,第一次DHPLC可迅速检测出由于形成异源双链而呈肩峰的杂合子（TC）,但不能区分两个均呈单峰的纯合子（TT或CC）。第二次DHPLC将未知纯合子与已知序列的纯合子混合后进行,通过判定单峰或肩峰即可推断未知纯合子的基因型。所研究的4个绵羊群体在该突变位点均处于Hardy-Weinberg平衡状态。蒙古羊与哈萨克羊较近的遗传亲缘关系以及滩羊与藏绵羊较近的遗传亲缘关系与线粒体DNA的研究结果一致。