Phylogenetic analysis of <Emphasis Type="Italic">Antrodia</Emphasis> species and <Emphasis Type="Italic">Antrodia camphorata</Emphasis> inferred from internal transcribed spacer region

The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.     

A high-throughput apple SNP genotyping platform using the GoldenGate™ assay

EST data generated from 14 apple genotypes were downloaded from NCBI and mapped against a reference EST assembly to identify Single Nucleotide Polymorphisms (SNPs). Mapping of these SNPs was undertaken using 90% of sequence similarity and minimum coverage of four reads at each SNP position. In total, 37,807 SNPs were identified with an average of one SNP every 187 bp from a total of 6888 unique EST contigs. Identified SNPs were checked for flanking sequences of ≥ 60 bp along both sides of SNP alleles for reliable design of a custom high-throughput genotyping assay. A total of 12,299 SNPs, representing 6525 contigs, fit the selected criterion of ≥ 60 bp sequences flanking a SNP position. Of these, 1411 SNPs were validated using four apple genotypes. Based on genotyping assays, it was estimated that 60% of SNPs were valid SNPs, while 26% of SNPs might be derived from paralogous regions.     

Functional characterization of the matrix metalloproteinase-1 cigarette smoke-responsive region and association with the lung health study

Background

Prior studies have demonstrated that the distal 1.5 kb of the MMP-1 promoter is fundamental in directing the induction of the MMP-1 gene by cigarette smoke.

Methods

To characterize the genetic variants in the MMP-1 cigarette smoke-responsive element, deep re-sequencing of this element was performed on DNA samples from participants in the Lung Health Study. Furthermore, evidence of Sp1 binding to the MMP-1 promoter was assessed using chromatin immunoprecipitation assays and the influence of cigarette smoke exposure on this interaction was evaluated in cultured human small airway epithelial cells.

Results

Ten polymorphisms (four novel) were detected in the cigarette smoke-responsive element. Chromatin immunoprecipitation assays to assess the protein-DNA interactions at Sp1 sites in the MMP-1 promoter showed increased binding to the Sp1 sites in the cigarette smoke-responsive element in small airway epithelial cells treated with cigarette smoke extract. In contrast, a Sp1 site outside of the element exhibited the opposite effect. None of the polymorphisms were more prevalent in the fast decliners versus the slow decliners (fast decliners = mean −4.14% decline in FEV1% predicted per year vs. decline in FEV1% predicted per year).

Conclusions

Sequencing analyses identified four novel polymorphisms within the cigarette smoke-responsive element of the MMP-1 promoter. This study identifies functional activity within the cigarette smoke-responsive element that is influenced by cigarette smoke and examines this region of the promoter within a small patient population.     

Polymorphism in environment responsive genes and association with Parkinson disease

Attempts were made in the present case-control study to investigate the association of polymorphism in the genes encoding proteins involved in toxication–detoxication and dopaminergic pathways and susceptibility to Parkinson’s disease (PD). Seventy patients suffering from PD and one hundred healthy controls belonging to the same geographical location and same ethnicity were included in the study. PCR-RFLP and allele-specific PCR-based methodology were used to identify the genotypes. Multivariate logistic regression analysis revealed that heterozygous genotypes of cytochrome P4502D6*4(CYP2D6*4), CYP2E1*5B (RsaI) polymorphism and homozygous mutant genotypes of CYP2E1*6 (Dra1) were found to be overrepresented in PD cases when compared to the controls. Risk was also found to be increased in patients carrying glutathione S-transferase T1 (GSTT1) null or homozygous variant genotypes of GSTP1. Significant association was observed for monoamine oxidase-B(MAO-B) variant allele G and PD, whereas no difference in genotype and allele frequencies was observed for manganese-superoxide dismutase (MnSOD), dopamine receptor-D2(DRD2), and dopamine transporter (DAT) genes between controls and PD cases. Genotype combinations characterized by the presence of two variant genotypes on their corresponding loci revealed that four combinations of GSTT1 null and MnSOD(-9Val) or GST null and MAOB-G or CYP2E1*5B and MAO-B-AG or CYP2E1*5B and DRD2 (Taq1A-het) genotypes in the patients exhibited severalfold higher and significant association with risk to PD. Our data suggest that polymorphism in the genes involved in detoxification and dopamine regulation may modulate the susceptibility to PD and could be important risk factors in the pathogenesis of PD.     

Genetic variants at the resistin locus are associated with the plasma resistin concentration and cardiovascular risk factors

OBJECTIVE: To examine the relation of the single nucleotide polymorphisms (SNPs) of the resistin gene on the plasma resistin concentration and cardiovascular risk factors. METHODS AND RESULTS: Plasma resistin concentrations were measured and SNP -420C>G, +157C>T, and +299G>A genotyped in our Finnish population-based cohort. Association analyses were performed in the control group of the cohort (n=515). In addition, the hypertension group (n=505), representing a high-risk group, was analyzed for these SNP's and the frequencies were compared with the low-risk control group. Resistin concentration differed significantly between the SNP genotypes, the common homozygotes having the lowest concentration in every SNP (p<0.01). After adjustment for age, sex, and BMI SNP -420C-C homozygotes had the lowest IGFBP-1 (p=0.001), the highest GHbA(1c) (p=0.028) in all the subjects and the lowest quick index (p<0.001) and the highest insulin (p<0.001) among female subjects of the control cohort. Plasma triglycerides were the lowest among G-G homozygotes (p=0.006). In addition, the SNP -420C allele was more frequent among hypertensive than control cohort (p=0.018). CONCLUSIONS: The results imply that genetic variation seems to have a role in the determination of plasma resistin level. SNP -420C-C homozygozity status seems to be associated with more deleterious metabolic profile.     

Evaluation of buccal cell collection protocols for genetic susceptibility studies

Buccal cells are increasingly used as a source of quality DNA to improve participation rates in molecular studies. Here, three buccal cell collection protocols were compared to determine factors affecting the yield of cells, total DNA per sample, and DNA yield per cell. In addition, kinetic quantitative polymerase chain reaction (PCR) (TaqMan™) was used to quantify human DNA available for PCR. The method of collection used influenced the overall DNA yield per sample. The collection buffer used influenced the number of cells but not the overall DNA yield per sample. Repeated freezing and thawing did not affect overall DNA yield per sample, DNA yield per cell, or the total number of cells collected. Mouthwashes had the highest DNA yield per sample (20.8 µg) compared with cytobrush samples (1.9 µg from three cytobrushes) and tongue depressors (0.8 µg from three tongue depressors). However, mouthwash samples may contain significant non-human DNA and other contaminants that could interfere with some molecular studies. Spectrometry grossly overestimated the total DNA recovered from mouthwash samples compared with fluorometry or quantitative PCR.     

Genetic variants associated with primary open angle glaucoma in Indian population

Glaucoma is a very common disorder of the eye wherein the disturbance of the structural or functional integrity of the optic nerve causes characteristic atrophic changes in the optic nerve, which may lead to specific visual field defects over time. Primary open angle glaucoma (POAG) is most frequent among the three principle glaucoma subtypes. With well-established role of genes like Myocilin (MYOC), Optineurin (OPTN) and WD repeat Domain 36, (WDR36), at least 29 genetic loci have been found till date to be linked to POAG. Moreover, association studies have found 66 loci with 76 genes associated to POAG till date with conflicting results. This particular study is to summarize the current knowledge regarding the change in glaucoma prevalence worldwide and in India from 1993 onwards and compiles all the studied genes that are involved in POAG pathogenesis in Indian population.     

Genetic polymorphisms of vitamin D receptor (VDR) in Fabry disease

Fabry disease, an X-linked inborn error of metabolism, is characterized by multi-organ involvement including cardiac signs of left ventricular hypertrophy and abnormal intima-medial (IMT) thickening of arteries, progressive renal failure, neurological involvement, and more. The vitamin D receptor (VDR) and an enzyme producing vitamin D3 result in an autocrine loop with direct effects on blood vessels. The purpose of this study is to assess VDR polymorphisms (BsmI, FokI, ApaI, and TaqI) relative to clinically important disease parameters using a disease-specific severity score (MSSI) and haplotype analysis. There were statistically significant differences between females (43% of 74 patients) and males in MSSI total scores, and in general and neurologic sub-scores. There appears to be a protective effect of the TaqI tt genotype so that there were significantly lower scores in clinical categories between those with the tt genotype versus those with the TT genotype. Multivariate models of haplotypes with MSSI scores reveal that T-A-f-B and t-a-F-b haplotypes of the VDR gene polymorphisms are significantly associated with variation in the Fabry phenotype. Despite the limitations of using the MSSI score as a clinical correlate, these results are provocative and further studies in larger cohorts with more males are recommended.     

基于贝叶斯网潜类模型的高维SNPs分析

采用贝叶斯(Bayesian)网的潜类模型对GAW17高维SNPs数据进行分析,为复杂性状疾病遗传以及基因定位等方面的研究提供新的方法支持。本研究从GAW17提供的包含697个个体22条常染色体的上万个SNP中,随机挑选出1号染色体上12个基因的29个SNPs作为研究对象。按照累计信息贡献率达到95%的原则,应用贝叶斯网潜变量模型选出C1S11408,C1S3201,C1S1786等15个与X0互信息量大的SNPs位点来对研究人群进行分类与解释。结果表明697个个体总的被分为2个潜在类别,各类别的概率分别为0.68和0.32。对两类人群的疾病分布状况进行分析,结果表明二者不一致,第二个类别人群患病率(38.64%)明显高于第一个类别人群(25.99%)(χ2=11.46,P=0.001)。由此可见,两类人群疾病患病率的差别正是由选出的15个SNPs造成的,从而有理由认为这些SNPs为可疑致病位点,为进一步的研究提供明确的思路。     

草鱼同工酶基因座位多态性的初步研究

采用垂直淀粉凝胶电泳及特异性组织化学染色技术研究了25尾草鱼的6种同工酶系统(LDH,MDH,ADH,GDH,IDH,EST)约18—23个基因座位的遗传变异型。有7个基因座位(s-Mdh-A,Adh—B,Gdh-1,Gdh-2,Est-1,Est-6,Est-14)具有多态性,在其中4个基因座位(s-Mdh—A,Adh-B,Gdh-1,Est-1)上观察到的基因型频率与Hardy-Weinberg定律相符．实验表明草鱼的同工酶基因座位具有明显的多态性。这一结果为草鱼的人工选种和定向育种的可能性提供了依据。对草鱼GDH,EST同工酶的遗传基础、亚基组成以及酶变异的机理等问题进行了讨论。