DNA Bank Research Articles

Dan Roden: learn, apply, evolve.

As a scientist, Dan Roden grew up as clinical electrophysiology did. Widely recognized for his research into mechanisms behind abnormal heart rhythms and drug responses, Roden was on the front lines in the 1980s and 1990s as new antiarrhythmic drugs were tested, the ability to use catheters to study drug actions evolved, and the specialty of clinical electrophysiology arose. From his first publication, appearing in the New England Journal of Medicine in 1980,1 Roden set a tone for a career focused on variable action of antiarrhythmic therapies and genetic determinants of that variation. Much of his current work focuses on individual cardiac ion channel mutations and their role in variable responses to drug therapy, and applications of genomics to healthcare. Among his achievements, Roden is credited with developing the idea of reduced repolarization reserve leading to acquired long QT intervals and arrhythmias2–4 and was one of the first to suggest that early afterdepolarizations cause long QT-related arrhythmias.5,6 As leader of Vanderbilt University’s pharmacogenomics project PREDICT (Pharmacogenomic Resource for Enhanced Decisions in Care & Treatment)—which pre-emptively enhances individuals’ electronic medical records with personalized information about genetic variants that could guide drug therapy—Roden and colleagues7 have demonstrated multiple advantages of preemptive genotyping. Meanwhile Vanderbilt’s massive BioVU DNA bank, which includes ≈175 000 human DNA samples and which Roden pioneered and directs, is similarly showing the power of incorporating genomic data into electronic medical records, for research purposes.7–11 Roden, who initially flirted with a future in journalism, described to Circulation Research some of his interests beginning as a child of immigrants in Montreal and leading to not just 1 career as an investigator, but a series of interlinked careers—in clinical pharmacology, cellular electrophysiology, molecular genetics, and population science. The paradigm for his progress, …

The effect of age on DNA concentration from whole saliva: implications for the standard isolation method.

Adequate quantity and quality of the DNA isolated from saliva samples are crucial for ensuring successful genotyping rates in genetic studies. However, there is little information about these issues when saliva samples are collected from children. The objectives of this study were to assess whether there are differences in DNA quality or quantity isolated from saliva samples of children at different ages and adolescents compared to adults and, if so, to establish a modified protocol to improve and standardize DNA isolation from saliva samples of children. Saliva samples were collected with Oragene DNA Sample Collection Kit from 41 healthy subjects including children of different ages, adolescents, and adults. Quantity and quality of isolated DNA were determined spectrophotometrically. DNA concentration and age were positively correlated (r = 0.676, P < 0.001). A high percentage of samples from children below 12 years yielded DNA concentrations <100 ng/µL and DNA quality a260/a280 ratios of <1.8. Modifying the standard DNA isolation method raised DNA quantity and quality in these critical samples. Age determines, at least in part, the high variability observed in the concentration of DNA isolated from saliva samples. This fact should be taken into account for a better standardization of the DNA isolation to ensure DNA banking in large-scale genetic studies involving children.

Donor and Recipient Angiotensin II Type 1 Receptor Polymorphisms and Haplotypes and Allograft Outcome in Renal Transplantation.

We investigated the association between single nucleotide polymorphisms and haplotypes of the Angiotensin II Type 1 receptor (AGTR1), and long term kidney allograft outcome. Three thousand adult, deceased donor kidney transplant recipients and paired donors (transplanted 1988 to 2010) were randomly selected from Caucasian recipient and donor pairs in the Collaborative Transplant Study DNA bank. DNA specimens were genotyped for 6 single nucleotide polymorphisms (SNPs) of the AGTR1. The selected polymorphisms were tag SNPs within the two haplotype blocks (1 and 2) of the AGTR1 gene (haplotype 1: rs2933249, rs10935724, rs4681443; haplotype 2: rs718858, rs1492099, rs12721331). The primary outcome was 10-year allograft survival (Kaplan-Meier survival censored for death). We analysed outcomes by individual recipient and donor genotypes and haplotypes (wild-type versus remainder) as well as groups (presence or absence of minor allele), according to the recipient and donor AGTR1 genotype and haplotype. The selected sample was representative of the DNA bank (recipient age ≥18 years, Caucasian recipient and donor and initially on a calcineurin inhibitor). The mean transplant year was 1996. 437 (15%) of recipients were retransplants. Overall, median (interquartile range) follow up time was 7.5 (3.3-13.0) years and the mean 10-year death censored graft survival rate was 69.5%. The recipient mean (standard deviation, SD) age was 48 (13) yrs with 38% female. The donor mean (SD) age was 41 (17) yrs with 40% female. There were no significant (p<0.05) associations between donor or recipient AGTR1 SNPs and recipient allograft outcome. Similarly, neither haplotype of the donor nor recipient was significantly associated with recipient allograft outcome. When grouping the donor and recipient for individual AGTR1 SNP (p=0.29-0.97) or haplotype (p=0.68, p=0.93), again there was no significant association with the primary outcome. These individual SNPs or haplotypes of the AGTR1, whether in the donor, recipient or any combination of donor-recipient, were not associated with 10-year allograft outcome in the recipient following deceased donor kidney transplantation.

THU0477 Genetic Risk Factors for Fibromyalgia Associated Symptoms/Syndromes and Disease Severity

BackgroundFibromyalgia (FM) is a condition characterized by chronic widespread pain associated to multiple symptoms, including fatigue, sleep disturbances, cognitive dysfunction, and depressive episodes. In recent years, many candidate gene association...

78 Oct Findings from the European Multi-centre Prestige Stent Thrombosis Study

BackgroundCoronary stent thrombosis (ST) remains the ‘Achilles Heel’ of percutaneous coronary intervention (PCI). Although relatively rare, it is associated with a high rate of acute myocardial infarction and death.A specific...

Association Between rs2200733 and rs7193343 Genetic Variants and Atrial Fibrillation in a Spanish Population, and Meta-analysis of Previous Studies

Introduction and objectivesThe objectives of this study were to analyze the association between two genetic variants (rs2200733 and rs7193343) in a Spanish population and the risk of developing atrial fibrillation, and to carry out a systematic review and meta-analysis of these associations. MethodsWe performed a case-control study involving 257 case patients with atrial fibrillation and 379 controls. The case patients were individuals who had donated samples to the Spanish National DNA Bank; the controls were participating in a population-based cross-sectional study. Genotyping was carried out using a TaqMan assay. We conducted a systematic literature search in which 2 independent reviewers extracted the necessary information. The study involved a meta-analysis, a heterogeneity analysis, and a meta-regression analysis to identify the variables that explain the heterogeneity across studies. ResultsIn our population, the presence of atrial fibrillation was found to be associated with rs2200733 (odds ratio = 1.87; 95% confidence interval, 1.30-2.70), but not with rs7193343 (odds ratio = 1.18; 95% confidence interval, 0.80-1.73). In the meta-analysis, we observed an association between atrial fibrillation and both variants: odds ratio = 1.71 (95% confidence interval, 1.54-1.90) for rs2200733 and odds ratio = 1.18 (95% confidence interval, 1.11-1.25) for rs7193343. We observed heterogeneity among the studies dealing with the association between rs2200733 and atrial fibrillation, partially related to the study design, and the strength of association was greater in case-control studies (odds ratio = 1.83) than in cohort studies (odds ratio = 1.41). ConclusionsVariants rs2200733 and rs7193343 are associated with a higher risk of atrial fibrillation. Case-control studies tend to overestimate the strength of association between these genetic variants and atrial fibrillation.

Asociación de los polimorfismos rs2200733 y rs7193343 con la fibrilación auricular en población española y metanálisis de la evidencia existente

Introduction and objectivesThe objectives of this study were to analyze the association between two genetic variants (rs2200733 and rs7193343) in a Spanish population and the risk of developing atrial fibrillation, and to carry out a systematic review and meta-analysis of these associations. MethodsWe performed a case-control study involving 257 case patients with atrial fibrillation and 379 controls. The case patients were individuals who had donated samples to the Spanish National DNA Bank; the controls were participating in a population-based cross-sectional study. Genotyping was carried out using a TaqMan assay. We conducted a systematic literature search in which 2 independent reviewers extracted the necessary information. The study involved a meta-analysis, a heterogeneity analysis, and a meta-regression analysis to identify the variables that explain the heterogeneity across studies. ResultsIn our population, the presence of atrial fibrillation was found to be associated with rs2200733 (odds ratio = 1.87; 95% confidence interval, 1.30-2.70), but not with rs7193343 (odds ratio = 1.18; 95% confidence interval, 0.80-1.73). In the meta-analysis, we observed an association between atrial fibrillation and both variants: odds ratio = 1.71 (95% confidence interval, 1.54-1.90) for rs2200733 and odds ratio = 1.18 (95% confidence interval, 1.11-1.25) for rs7193343. We observed heterogeneity among the studies dealing with the association between rs2200733 and atrial fibrillation, partially related to the study design, and the strength of association was greater in case-control studies (odds ratio = 1.83) than in cohort studies (odds ratio = 1.41). ConclusionsVariants rs2200733 and rs7193343 are associated with a higher risk of atrial fibrillation. Case-control studies tend to overestimate the strength of association between these genetic variants and atrial fibrillation.Full English text available from:www.revespcardiol.org/en

Evaluation &validation of a highly multiplexed LATE-PCR single-tube assay for M(X)DR-TB

Background: In 2006, the World Health Organization (WHO) and the Stop TB partnership called for strengthening of diagnostic services and highlighted the need for the development of rapid diagnostics to fight the tuberculosis pandemic. In 2011, WHO estimated that approximately 630,000 (5.3%) of the 12 million TB cases had multiple drug resistant (MDR)-TB, while more than 80 countries have reported cases of extremely drug-resistant (XDR)TB. Only a small fraction of reported cases (<20%) were correctly diagnosed and even fewer were treated according to WHO standards. In response the WHO endorsed the Genotype® MTBDRplus (version 1.0) line probe assay (LPA) in 2008 and theXpert®MTB/RIF assay in 2010. But these tests only provide evidence for resistance to isoniazid and/or rifampicin. There continues to be a critical need for a more comprehensive convenient diagnostic technology. The highly multiplexed LATE-PCR assay for M(X)DR-TB described here was designed to meet that need. Our goal was to firmly establish that a highly multiplexed Linear-After-the-Exponential (LATE) PCR single closed-tube assay can simultaneously detect and distinguish multiple mutations in multiple gene targets that are known to confer resistance to isoniazid, rifampicin, ethambutol, ofloxacin, amikacin, kanamycin and capreomycin. Methods & Materials: In this initial study, DNA from clinical isolates with different rpoB, katG, embB, inhA promoter, gyrB, gyrA and rrs genotypes were selected from a DNA bank housed at Stellenbosch University. Each DNA samples was amplified and the singled-strandedDNAproductswere scanned formutations at endpoint using the same mixture of Lights-On/Lights-Off Probes. The resulting fluorescent signatures were compared to that of H37Rv, a pan-susceptible “wildtype” strain. Results: Each clinical isolate harbouring a uniquemutation had its own, highly reproducible fluorescent signature distinct from that of H37Rv, as well as all other isolates with differentmutations. Conclusion: This study achieved the intended transfer of the Brandeis University technology to Stellenbosch University. This study also demonstrates that this single tube multiplexed assay can simultaneously distinguish the different mutations that confer resistance to rifampicin, isoniazid, ethambutol, fluoroquinolones, aminoglycosides and ethionamide in less than three hours. (Supported by NIH Grant R01 A1099532)

Novelties in Catasetum (Orchidaceae) in the State of Rondônia, Brazil

AbstractThe State of Rondônia is rich in species of Catasetum that are still undervalued, which presents a major challenge in the preparation of a map that shows the distribution and all occurrences of the species of the genus. As a result of our studies, four new records are presented for the State of Rondônia and a new natural hybrid is described and illustrated. Samples of all species were collected for DNA extraction (aliquots kept in the Total DNA Bank of Plants UFPR – UPCB‐DNA) and vouchers deposited at the herbaria UFMT and the RB. (© 2014 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Influence of Demographic Characteristics of Participants on Consent to Genomic Research into Congenital Heart Disease

Background: The enrollment of sick children and their families in genomics studies calls for a comprehensive view of the consent process. Few studies have searched for correlations between the demographic characteristics of participants (age, gender, parental lineage) or their level of participation (affected children, parents, or other relatives), on the one hand, and patterns of consent to specific pediatric research procedures, on the other (DNA banking, use of cardiac tissue, disclosure of a cardiac condition, creation of cell lines, recall of a participant).

The importance of biobanking in molecular taxonomy, with proposed definitions for vouchers in a molecular context

The importance of biobanking in molecular taxonomy, with proposed definitions for vouchers in a molecular context

15: Genetic predisposition to adverse neurodevelopmental outcome after early preterm birth: a validation analysis

Validate genetic risk loci associated with adverse neurodevelopment after early preterm birth. We previously conducted a large candidate gene association study in a cohort of 1013 extremely low birth weight infants (<1000 gms). The case-control analysis utilized samples in the NICHD Neonatal Research Network’s DNA Bank and evaluated 1634 SNPs in 145 genes in hypothesized causal pathways, with emphasis on inflammation, angiogenesis, and brain development. Cases were children who died by age 1 or who were diagnosed with CP or neurodevelopmental delay (Bayley II MDI or PDI <70) by 18-22 months. Controls were survivors with normal neurodevelopment. The outcomes of CP, combined CP or death, mental delay (MDI<70), and motor delay (PDI<70) were evaluated. Twenty-five SNPs with P<0.01 for one or more outcomes in the previous analysis were selected for validation in this analysis. Validation samples were derived from an RCT of magnesium sulfate before anticipated early preterm birth (<32 weeks) for prevention of cerebral palsy (CP). Case/control definitions were equivalent to the primary cohort, with the exception that neurodevelopmental outcomes were evaluated at 24 months. As in the primary analysis, four outcomes were evaluated. Cases and controls were matched for race and infant sex; covariates included gestational age at birth, small for gestational age, maternal education level, treatment group, and antenatal corticosteroids. Significance in the validation cohort was defined as P<0.05. The validation cohort included 364 infants, 170 cases and 192 controls. Three genetic loci from the primary analysis were significantly associated with the outcomes CP, CP/death and mental delay after early preterm birth in the validation analysis (Table). Genetic loci involved in inflammation and brain development are associated with CP, CP/death, and mental delay after early preterm birth in primary and validation genetic analyses.Tabled 1 Open table in a new tab

Donor Genotypes For Interleukin-17A Gene Single Nucleotide Polymorphisms (SNPs) Allow Anticipation Of Complications After HLA-Identical Allogeneic Stem Cell Transplantation (allo-SCT)

Introduction Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in in the promoter region of cytokine genes have shown to alter their expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 17 (IL-17) is secreted by CD4+ T-cells and has been implicated in the pathogenesis of various autoimmune diseases but its importance in SCT is not well-known. Objective To analyse the influence of IL-17A SNP genotypes on the risk and severity of GvHD and other complications after HLA-identical allo-SCT. Patients and Methods Genomic DNA obtained from peripheral blood samples belonging to 546 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). Genotyping of the polymorphisms of interest, rs8193036 (-737C&gt;T), rs2275913 (-197G&gt;A), rs3819024 (-444A&gt;G), rs4711998 (-877A&gt;G), were performed by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Results Genotype frequencies are shown in Table 2 and the association between IL-17A genotypes and complications after allo-SCT are shown in Table 3. Patients transplanted from donors harboring genotype CC for the SNP rs8193036 show increased risk of grade III-IV acute GvHD (7/26 vs 47/397, p=0.035) and of grade II-IV acute GvHD (13/26 vs 133/409, p=0.048). Patients transplanted from donors harboring allele A in the SNP rs4711998 show increased risk of extensive chronic GvHD (53/161 vs 43/177, p=0.045). Relapse rate was not related with IL-17A SNP genotypes. Finally a higher risk of toxicity-related mortality (TRM) was observed in patients transplanted from donors harboring allele A for SNP rs2275913 (78/293 vs 46/227, p=0.048), donors harboring allele G for SNP rs3819024 (78/279 vs 46/242, p=0.011) and donors harboring allele A for SNP rs4711998 (68/250 vs 55/229, p=0.044). Conclusions IL-17A SNP genotyping might be useful to anticipate complications after sibling HLA-identical allo-SCT and, therefore, to improve the clinical management of transplanted patients. This results further support the idea of a genetic predisposition to certain complications after allo-SCT. Paper presented on behalf of the GvHD/Immunotherapy committee of the Spanish Group for Hematopoietic Transplantation (GETH). Disclosures: No relevant conflicts of interest to declare.

Haplotype analysis of DFNB8/10 locus reveals contribution of TMPRSS3 mutations in Pakistani deaf population

TMPRSS3 mutations are associated with non-syndromic recessive deafness (DFNB8/10). To evaluate the frequency of TMPRSS3 mutation in Pakistani population, highly consanguineous families were enrolled. A group of five consanguineous families without any history of associated environmental causes were found to be linked to DFNB8/10 locus. To correlate haplotypes and to evaluate founder affect 17 other families linked to DFNB8/10 were provided by NCEMB DNA bank. Haploytpe analysis revealed that out of 22 families, haplotypes of 8 families (42 %) were found similar to PKDF003 and PKDF311 having 207delC mutation, 5 (26 %) families had haplotypes similar to PKDF040 and 4 families (15.7 %) shared halpotypes similar to PKDF064, having C407R (1219T>C) and C194F (518G>T) mutations, respectively. Interestingly, PKDF321 and PKDF337 (10.5 %) showed different haplotypes and might harbor novel mutation. Taken together, these data imply that Punjab region is more affected by TMPRSS3 mutation, and the founder-effect mutation might be traced back to Punjab region.

Familial adenomatous polyposis of the colon

Familial adenomatous polyposis (FAP) is a well-defined autosomal dominant predisposition to the development of polyposis in the colon and rectum at unusually early ages. The first symptoms of FAP are diarrhea and blood in the stool. Weight loss and weaknesses occur after the development of advanced tumour. The incidence of the FAP disorder is one per 10000 newborns. There are high levels of heterogeneity with regard to the number and timing of the occurrence of polyps. The classical form of FAP is characterized by the presence of more than 100 polyps, which appear in the second decade of life. The average time of occurrence of polyps is 15 years. The earliest symptoms of polyposis have been observed in a three-year-old child. The polyps are characterized by large potential for the development towards malignant tumour. Malignancy can occur from late childhood onwards. Attenuated adenomatous polyposis coli is characterized by a more benign course of disease in contrast to classical FAP. The occurrence of FAP is associated with mutations in the APC tumour suppressor gene, which was described in 1991. The APC gene is located on chromosome 5q21 and is involved in cell proliferation control. A recessive form of adenomatous polyposis is caused by mutations in the base excision repair gene - MUTYH gene. The MUTYH gene is involved in repairing DNA lesions as a result of oxidative DNA damage. MUTYH associated polyposis (MAP) is a predisposition to the development of polyps of the colon but the number of polyps is lower in comparison to classical FAP. The high risks of cancer observed in these two diseases make them important medical issues. Molecular studies of colonic polyposis have been performed in Poland for over fifteen years. A DNA Bank for Polish FAP patients was established at the Institute of Human Genetics in Poznan in which DNA samples from 600 FAP families have been collected.

TEN YEARS OF NEUROGENETIC REQUESTS: TO TEST OR NOT TO TEST?

BackgroundClinical neurology has benefitted significantly from advances in molecular genetics with the discovery of disease–associated genes leading to the clinical application of genetic testing. With an ever increasing arsenal of...

MOLECULAR GENETIC STUDIES OF NEURODEGENERATIVE DISEASE

IntroductionLarge scale genetic studies such as genome–wide association studies (GWAS) in Parkinson's disease (PD) have revealed genetic susceptibility factors and continue to offer new insights both into the genetics of...

The Design and Conduct of a Community‐Based Registry and Biorepository: A Focus on Cardiometabolic Health in Latinos

Latinos are disproportionately impacted by obesity and type 2 diabetes but remain underrepresented in biomedical research. Therefore, the purpose of this project was to develop a research registry and biorepository to examine cardiometabolic disease risk in the Latino community of Phoenix, Arizona. The overarching goal was to establish the research infrastructure that would encourage transdisciplinary research regarding the biocultural mechanisms of obesity-related health disparities and facilitate access to this research for the Latino community. Prior to recruitment, key stakeholders from the local Latino community were engaged to develop a broad rapport within the community and seek advice regarding recruitment, enrollment, and follow-up. Self-identified community-dwelling Latinos underwent a comprehensive cardiometabolic health assessment that included anthropometrics, a fasting laboratory panel, and a 2-hour oral glucose tolerance test with measures of insulin and glucose to estimate insulin action and secretion. Separate consent was requested for future contact and banking of serum, DNA, and RNA. Research collaborations were sought out based on the cultural and metabolic profile of participants, faculty research agendas, and the potential for generating hypotheses. A total of 667 participants (20.4% children, and 79.6% adults) were enrolled with 97% consenting to the registry and 94% to banking of samples. The prevalence of overweight/obesity was 50% in children and 81% in adults. Nearly 20% of children and more than 45% of the adults exhibited some degree of hyperglycemia. To date, more than 15 research projects have been supported through this infrastructure and have included projects on the molecular biology of insulin resistance to the sociocultural determinants of health behaviors and outcomes. The high prevalence of obesity and cardiometabolic disease risk factors coupled with the overwhelming majority of participants consenting to be re-contacted, highlights the importance of supporting research infrastructure to generate hypotheses about obesity-related health in Latinos. Future studies that stem from the initial project will likely advance the limited understanding regarding the biocultural determinants of health disparities in the Latino community.

Creation of a DNA bank from patients with infertility: a powerful resource for advancing the biology of reproduction

Creation of a DNA bank from patients with infertility: a powerful resource for advancing the biology of reproduction

A Large Animal Model for CNGB1 Autosomal Recessive Retinitis Pigmentosa

Retinal dystrophies in dogs are invaluable models of human disease. Progressive retinal atrophy (PRA) is the canine equivalent of retinitis pigmentosa (RP). Similar to RP, PRA is a genetically heterogenous condition. We investigated PRA in the Papillon breed of dog using homozygosity mapping and haplotype construction of single nucleotide polymorphisms within a small family group to identify potential positional candidate genes. Based on the phenotypic similarities between the PRA-affected Papillons, mouse models and human patients, CNGB1 was selected as the most promising positional candidate gene. CNGB1 was sequenced and a complex mutation consisting of the combination of a one basepair deletion and a 6 basepair insertion was identified in exon 26 (c.2387delA;2389_2390insAGCTAC) leading to a frameshift and premature stop codon. Immunohistochemistry (IHC) of pre-degenerate retinal sections from a young affected dog showed absence of labeling using a C-terminal CNGB1 antibody. Whereas an antibody directed against the N-terminus of the protein, which also recognizes the glutamic acid rich proteins arising from alternative splicing of the CNGB1 transcript (upstream of the premature stop codon), labeled rod outer segments. CNGB1 combines with CNGA1 to form the rod cyclic nucleotide gated channel and previous studies have shown the requirement of CNGB1 for normal targeting of CNGA1 to the rod outer segment. In keeping with these previous observations, IHC showed a lack of detectable CNGA1 protein in the rod outer segments of the affected dog. A population study did not identify the CNGB1 mutation in PRA-affected dogs in other breeds and documented that the CNGB1 mutation accounts for ∼70% of cases of Papillon PRA in our PRA-affected canine DNA bank. CNGB1 mutations are one cause of autosomal recessive RP making the CNGB1 mutant dog a valuable large animal model of the condition.