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Data storage and data coordination are important aspects of project design and execution. Pyrosequencing technology allows thousands of data-points to be collected per day. Consequently, a consistent and reliable method of data input and storage is vital. This chapter discusses the strengths and weaknesses of data storage systems.
Experimental and epidemiological data have implicated a potential chemoprotective role of green tea polyphenols and a potential enhancing role of angiotensin II in the development of breast cancer in humans. Angiotensin II is converted from its precursor by angiotensin-converting enzyme (ACE). Women with low-activity genotype of the ACE gene had a reduced risk of breast cancer compared with those possessing high-activity ACE genotype. Experimental data showed that green tea polyphenols could inhibit angiotensin II-induced reactive oxygen species production. We reasoned that if this is one of the mechanisms by which green tea polyphenols protect against human breast cancer, then their effect should be more prominent among women possessing high-activity ACE genotype than women with low-activity ACE genotype. In other words, we predict a stronger inverse green tea-breast cancer association among the former versus the latter subgroup of women. To test this hypothesis, we conducted a nested case-control study involving 297 incident breast cancer cases and 665 control subjects within the Singapore Chinese Health Study. There was no association between intake frequencies of green tea and risk of breast cancer among all women or those with low-activity ACE genotype. Among women with high-activity ACE genotype, however, intake frequency of green tea was associated with a statistically significant decrease in risk of breast cancer (P for trend=0.039); the odds ratio (95% confidence interval) was 0.33 (0.13-0.82) for women drinking green tea at least monthly and 0.29 (0.10-0.79) for those drinking green tea at least weekly compared with non-drinkers. There was a statistically significant interaction effect between green tea intake and ACE genotype on risk of breast cancer (P for interaction=0.01). Black tea intake was unrelated to breast cancer risk irrespective of the ACE genotype. The findings of the present study highlight the importance of genetically determined factors in evaluating the role of green tea intake in the development of breast cancer.
We investigated the association of paraoxonase (PON) gene polymorphism with both the occurrence of coronary heart disease (CHD) and the severity of coronary artery stenosis in Japanese subjects. PON is a protein associated with plasma HDL. It has been hypothesized an A/B (Gln 192-->Arg) polymorphism of PON may be involved in the pathogenesis of CHD, especially among subjects with non-insulin-dependent diabetes mellitus (NIDDM). The polymorphism was determined in 134 patients with myocardial infarction (MI) or angina pectoris, and in 252 healthy subjects as controls. The frequencies of the AA, AB, and BB genotypes in the patients were 15, 50 and 35%, respectively, and these frequencies did not differ from those in control subjects (14, 49, and 37%). The relative risk of CHD was not found to be associated with these genotypes. These data also were similar among selected subgroups (patients with MIs, those with a low-risk lipoprotein profile for CHD, and those with NIDDM). Neither the number of affected vessels nor Gensini's scores differed among the genotype groups. Our case-control study in Japanese subjects did not show that the PON A/B polymorphism is associated with a risk of CHD.
Oxidative stress may be one mechanism by which tobacco smoke causes lung cancer. A common oxidative damage to DNA is the highly mutagenic 7,8-dihydro-8-oxoguanine adduct, which can be repaired by 8-oxoguanine glycosylase I (OGG1). A Ser326Cys substitution polymorphism in the hOGG1 gene has been suggested, based on in vitro data, to reduce the activity of the enzyme. We tested the association of this polymorphism with lung cancer in a population-based, case control study of 298 cases and 405 controls of Caucasian, Japanese, or Native Hawaiian ancestry in Hawaii. Subjects were genotyped with a PCR-RFLP assay, and odds ratios were estimated by logistic regression after adjustment for other observed risk factors, including smoking and vegetable intake. We found marked differences in the frequencies of the hOGG1 Cys variant allele among ethnic groups (45% in Hawaiians, 42% in Japanese, and 22% in Caucasians). The homozygous Cys/Cys genotype was also found to be more common in cases than controls (P = 0.008), with an odds ratio of 2.1 (95% confidence interval: 1.2-3.7) for this genotype compared with the Ser/Ser genotype. Heterozygous individuals were not at increased risk. This association with the Cys/Cys genotype was observed for each sex, ethnic group, and lung cancer cell type. There was also the suggestion that vegetable intake may not be protective against lung cancer among subjects with the Cys/Cys genotype. These data suggest that the presence of two hOGG1 326Cys alleles confers a 2-fold increased risk of lung cancer. Additional studies need to be conducted to confirm this association.
Currently, more than 1,000 mutations have been identified in the cystic fibrosis transmembrane regulator (CFTR) gene. While some mutations are common worldwide, the majority are restricted in certain ethnic groups. We have found that in Southern Italy, the 852del22 mutation is well represented with a frequency of 3.5%. We have screened, by reverse dot blot, denaturing gradient gel electrophoresis (DGGE), and gene sequencing, the entire coding regions of CFTR gene in 371 consecutive cystic fibrosis (CF) patients from Southern Italy and have identified 17 patients carrying rare genotypes, among which 13 [6 M; median age 21.7 years (range: 4.5-47.7 years)] carry the 852del22 mutation. To assess the phenotypic expression of CF in patients with the 852del22 mutations we have compared these patients with a group of age and gender matched patients homozygous for the DeltaF508 mutation [n = 34; 19 M; median age 19.9 years (range: 3.8-34.6 years)]. Overall, we found no difference in terms of complications, patient survival (17.6% vs. 30.7%; P = NS), estimated time needed to develop a severe lung disease (22.1 vs. 24.5 years; P = NS), nutritional status, and rate of infection or colonization by most common pathogens between patients in the two groups. Finally, we have found that a late diagnosis was associated with a poor outcome (severe lung disease) regardless of genotype. Our data show that 852del22 mutation results in a phenotypic expression of disease as severe as that determined by the more typical DeltaF508 and, as in the latter case, there is no strict genotype/phenotype correlation.
Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C-->T transition at -1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 -1306 C-->T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055-3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT+TT genotypes. Our data suggest that MMP-2 -1306 C-->T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population.
OBJECTIVE: To understand the relationship of apolipoprotein E (APOE) polymorphism to dementia in Parkinson disease (PD) because the APOE epsilon4 allele is linked to Alzheimer disease. DATA SOURCE: We reviewed MEDLINE, BIOSIS Previews, and ISI Web of Science from January 1, 1966, to May 7, 2004, supplemented by citation analysis from retrieved articles. STUDY SELECTION: Case-control studies using clinical or pathologic criteria for PD and dementia, and with complete APOE genotype frequencies data. DATA EXTRACTION: We compared estimated prevalence odds ratios for dementia in PD in relation to each allele. We also looked for evidence of heterogeneity and publication bias and performed a stratified analysis on several study characteristics. DATA SYNTHESIS: Data analyses suggest publication bias and heterogeneity of source data for the epsilon4 allele (homogeneity P = .2; Begg and Mazumdar, P = .06; and Egger et al, P = .1). The estimated odds ratios for development of dementia in PD are 1.6 for epsilon4 (95% confidence interval, 1.0-2.5); 1.3 for epsilon2 (95% confidence interval, 0.73-2.4); and 0.54 for epsilon3 (95% confidence interval, 0.18-1.6). The odds ratio estimates for epsilon4 were higher for studies published in 1996 or later (2.3 vs 1.0) and for studies conducted outside North American sites (2.4 vs 1.2). CONCLUSIONS: The APOE epsilon4 allele appears to be associated with a higher prevalence of dementia in PD. Publication bias and heterogeneous source data may, however, confound this conclusion. Confirmatory studies that use standardized and validated diagnostic criteria for dementia in PD are needed.
Ten mice were individually exposed to 30 male and 30 female cercariae of Schistosoma intercalatum from Cameroon and to 30 male and 30 female cercariae of S. intercalatum from Zaire. Ten weeks later, the nine surviving mice were killed, and a total of 263 adult schistosomes (228 pairs, 7 unpaired males, 28 unpaired females) were perfused. All worms were identified utilising the random amplified polymorphic DNA technique using primer OPG06 which enables the differentiation of the Cameroon and Zaire genotypes. The data demonstrate that there were no significant differences between the number of homospecific pairs observed compared with the expected number under the null hypothesis. Matings occurred in a random manner indicating that mate choice did not exist for the two genotypes of S. intercalatum. Hence there is no prezygotic isolation mechanism which would prevent the interaction of the two genotypes if they were sympatric. However, as they are allopatric, the possibility of the production of unfit hybrids does not arise. Consequently, the reinforcement of a prezygotic isolating mechanism does not play a significant role in the speciation process.
Impaired platelet-derived nitric oxide (NO) contributes to acute coronary syndromes by enhancing platelet recruitment and thrombus formation. Polymorphic variants of the endothelial NO synthase (eNOS) gene have been associated with cardiovascular diseases. To examine whether eNOS variants affect platelet-derived NO and platelet function, and to assess the effects of estradiol on platelet function, we studied platelets from 47 healthy caucasians who were genotyped for eNOS polymorphisms in the promoter region (T-786 C), in intron 4, and in exon 7 (Glu298Asp). Platelet aggregation, platelet-derived NO and superoxide production were measured in control samples and samples pretreated with 17-alpha-estradiol (10 nmol/l). The occurrence of variants in the promoter region (P = 0.002) or in exon 7 (P = 0.007), but not in intron 4 (P > 0.05), were associated with lower levels of platelet-derived NO. An increased (P = 0.047) release of superoxide was observed with platelets from subjects with the variant in the promoter region, but not with other eNOS genetic variants. The eNOS gene polymorphisms did not affect ADP-induced platelet aggregation (P > 0.05). However, estradiol significantly increased platelet aggregation (P = 0.004), and platelet-derived superoxide (P = 0.047) in individuals homozygous for the variant in exon 7, but not in subject with other genotypes. These data demonstrate that the eNOS variants in the promoter region and in exon 7 decrease platelet-derived NO and that estradiol significantly increases platelet aggregation in homozygous for the variant in exon 7 but not in subjects with other genotypes, suggesting that eNOS variants may influence the thrombotic response.
STUDY OBJECTIVES: The association between genotype and phenotype in cystic fibrosis (CF) has been clearly established for pancreatic status, but not for lung disease. DESIGN: Retrospective study. SETTING: A respiratory unit of a teaching hospital. PATIENTS: We studied 51 adult CF patients for whom current data and genotype were available. Thirty-seven patients carried two severe mutations associated with pancreatic insufficiency phenotype (group S). Fourteen patients carried at least one mild (and dominant) mutation associated with pancreatic sufficiency phenotype (group M). MEASUREMENTS: We compared the course of the disease between the two groups, looking for a genotype/phenotype association for lung disease. RESULTS: The mean age of the population was 30 years. Patients with two severe mutations presented more severe disease with earlier onset (1.7 years vs 7.9 years, p = 0.0001). They presented with a more severe respiratory impairment, with a lower mean FEV(1) (29% of predictive value vs 58% of predictive value, p < 0.001); a higher Pseudomonas colonization rate (97% vs 57%, p < 0.01); a more frequent end-stage respiratory insufficiency, defined by a FEV(1) < 30% (73% vs 29%, p < 0.05); and a more marked yearly decline of FEV(1) (3% vs 1.4%, p < 0.001). By multivariate logistic regression analysis, carrying two severe mutations was the only independent predictor of a terminal respiratory insufficiency (relative risk, 6.75; 95% confidence interval, 1.79 to 26.50; p = 0.003). CONCLUSION: This study suggests that pulmonary disease appears to be associated with the severity of CF transmembrane regulator mutations.
Cryptosporidium meleagridis is a common cause of cryptosporidiosis in birds. In addition, recent reports have described the parasite as an etiologic agent of cryptosporidiosis in both immunocompetent and immunocompromised humans. Therefore, it is important to genetically characterize isolates of C. meleagridis from different hosts and geographic areas, and to develop molecular tools to differentiate isolates from various hosts or areas. In this study, a total of 11 isolates of Cryptosporidium meleagridis from both human and avian hosts were examined at three genetic loci: the small-subunit rRNA, 60-kDa glycoprotein precursor, and 70-kDa heat shock protein genes. Two genotypes of C. meleagridis were seen at the small-subunit rRNA locus. These differed from each other by the presence or lack of a heterogeneous copy of the gene and an ATT repeat. The 60-kDa glycoprotein precursor gene divided these eleven isolates of C. meleagridis into six genotypes with high sequence diversity between groups. The highest genetic heterogeneity, however, was seen at the 70-kDa heat shock protein locus, and was primarily present at the 3' end of the gene. This heterogeneity separated eight isolates of C. meleagridis into six genotypes. These data could be useful in the development of molecular tools to promote understanding of the transmission of C. meleagridis in humans.
In the present study we characterized the frequencies of two polymorphisms within the MHC-linked heat shock protein (HSP) 70 genes in patients with insulin-dependent diabetes mellitus (IDDM) (n = 114) and healthy control individuals (n = 110). Significant differences in genotype and allelic frequencies were observed for both polymorphisms between randomly selected patients and controls. However, for the HSP70-2 polymorphisms this was solely due to linkage disequilibrium with DR3. The rate HSP70-Hom 2-allele was significantly more frequent in controls than in patients. It showed strong association with certain tumour necrosis factor (TNF) (class III) and HLA-B and -A (class I) alleles independent of HLA-DQ and -DR alleles. By typing 257 individuals from 55 IDDM multiple-case families two extended MHC-haplotypes, including class II-, TNF- and class I-markers, carrying the rare HSP70-Hom allele were defined. One was only transmitted to diabetic offspring, whereas the other was only transmitted to unaffected offspring. The functional implication of the polymorphism in the heat shock-inducible HSP70-2 gene was analysed by studying HSP70-2 mRNA expression after heat shock in peripheral blood mononuclear cells from individuals with different HSP70-2 genotypes. Preliminary data showed that individuals homozygous for the PstI 8.5-kb allele consistently had slightly lower expression than heterozygous and 9.0-kb homozygous individuals.
Allelic distributions and frequencies of five Candida albicans microsatellite loci have been determined for strains isolated from the bloodstream and obtained through active population-based surveillance in two U.S. metropolitan areas between 1998 and 2000. These data were compared to data for isolates obtained from two other U.S. regions in 1992 to 1993. In a majority of pairwise combinations between sites, no evidence was seen for shifts in microsatellite allelic frequencies. One to three alleles were highly predominant and correlated with major genotypes. These data both support the concepts of allelic stability and genetic equilibria and suggest that, in the United States, strains of C. albicans isolated from the bloodstream may form a defined, genetically homogeneous population across geographical distance and time.
The National Tuberculosis Genotyping and Surveillance Network was established in 1996 to perform a 5-year, prospective study of the usefulness of genotyping Mycobacterium tuberculosis isolates to tuberculosis control programs. Seven sentinel sites identified all new cases of tuberculosis, collected information on patients and contacts, and obtained patient isolates. Seven genotyping laboratories performed DNA fingerprinting analysis by the international standard IS6110 method. BioImage Whole Band Analyzer software was used to analyze patterns, and distinct patterns were assigned unique designations. Isolates with six or fewer bands on IS6110 patterns were also spoligotyped. Patient data and genotyping designations were entered in a relational database and merged with selected variables from the national surveillance database. In two related databases, we compiled the results of routine contact investigations and the results of investigations of the relationships of patients who had isolates with matching genotypes. We describe the methods used in the study.
Two children in a family with five siblings were investigated because of low levels of fibroblast arylsulfatase A activity. Neither child had metachromatic leukodystrophy (MLD) and they were diagnosed as having benign pseudo arylsulfatase A deficiency trait (PD). Analysis of arylsulfatase A subunit profiles in fibroblasts provided data for genotype assignments for each family member. Father and mother were assigned an n/pd and n/mld phenotype, respectively. The low enzyme siblings were both assigned pd/mld; two of the three normal enzyme siblings were assigned an n/n phenotype and one an n/mld.
The cagA 3' region shows marked variation among Helicobacter pylori strains. Two segments of 102 bp and 57 bp are reportedly responsible for this variation. We analysed the cagA 3' region in 70 H. pylori strains using polymerase chain reaction and sequencing. We found that another segment, namely beta segment, was also involved in the variation of this region. The beta segment was 105 bp long and located between the aforementioned two segments. Six genotypes were identified based on the structure of the cagA 3' region. No relationship was found between these genotypes and the clinical outcomes or vacA genotypes. The numbers of tyrosine phosphorylation sites within the cagA 3' region varied among strains, but this was not related to the cagA genotypes. Our data suggest that the cagA 3' region is significantly variable. It appears that the variation of the cagA 3' region might contribute to the modification of virulence.
Aggravation of anemia in chronic renal failure patients by angiotensin-converting enzyme inhibitors (ACEIs) has been attributed to the inhibition of angiotensin II which facilitates erythropoietin(Epo) production. This study was aimed at evaluating whether ACEIs aggravate anemia in maintenance hemodialysis patients and to investigate the influence of ACE gene polymorphism on erythropoiesis in these patients. Ninety-one hemodialysis patients were divided into 2 groups, based on whether or not they were administered ACEIs, into the ACEI group(n = 24) and the non-ACEI group(n = 67), and comparisons were made of the doses of recombinant human Epo(rHuEpo) administered, the hematocrit(Hct) and the plasma Epo concentrations. Among the patients in the non-ACEI group, only 17 did not receive rHuEpo, while all of the patients in the ACEI group received rHuEpo. The average dose of rHuEpo was 102.7 +/- 45.4 IU/kg/week in the ACEI group and 57.8 +/- 55 IU/kg/week in the non-ACEI group and the difference between the two groups was statistically significant. A statistically significant difference in the Hct was also observed between the two groups: the mean Hct in the ACEI group was 28.7 +/- 2.9% while that in the non-ACEI group was 31.1 +/- 3.7%. The plasma Epo concentrations were significantly lower in the ACEI group than in the non-ACEI group. No significant differences in the rHuEpo dose and Hct were observed between the three ACE genotype classes in either the ACEI or the non-ACEI group, however, there was a significant difference among the three genotypes in the non-ACEI group in regard to the plasma Epo concentrations; patients with the DD genotype had higher concentrations than those with the DI or II genotypes. These data suggest that anemia in maintenance hemodialysis patients is worsened by ACEIs as a result of the suppression of Epo production. Although it has been suggested that the endogenous Epo concentrations in maintenance hemodialysis patients are associated with ACE gene polymorphism, no significant influence of the ACE genotype on the rHuEpo dose or Hct was evident. Therefore, it is possible that exacerbation of anemia by ACEIs in the patients receiving rHuEpo is a result of an inhibited bone marrow response to Epo.
Locating quantitative trait loci (QTL) in mammalian systems has proven difficult due to the lack of genetic control and reproducibility, as well as the expense of maintaining sufficiently large populations for genotyping and phenotyping. In plants, populations of recombinant inbred lines (progeny bred to homozygosity from a single cross) do not have these problems. Methods developed to identify QTL in a recombinant inbred soybean population provide a basis for analysis of a suitable mammalian population, such as Portuguese water dogs in the United States. The more than 6,000 dogs have accurate pedigrees, available phenotypic data and samples for genotyping, as well as interesting quantitative trait variation. The computer program Georgie allows us to choose large subpopulations with desirable characteristics such as high degrees of consanguinity that capture some of the benefits of recombinant inbred lines in plants. Computer simulations extending methods developed for simpler plant populations indicate that QTL with realistic effects can be identified from such subpopulations. Currently we are developing markers and collecting phenotypic and genotypic data from this population to begin the process of unraveling the genetic basis of quantitative traits in dogs.