Biomedical subjects

Cathrine Jespersgaard

Publications and source records attributed to Cathrine Jespersgaard.

6 recordsLinked to original sources

PubMed2006

Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics.

Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.

Acrylamides↗

PubMed2005

Genetic and environmental factors in monozygotic twins with Crohn's disease and their first-degree relatives: a case report.

BACKGROUND/AIMS: Familial Crohn's disease has shown concordance concerning location and clinical type of the disease especially among monozygotic twins. Susceptibility to Crohn's disease is both based on genetic and environmental factors. We investigated polymorphisms of CARD15, TLR4, and OCTN, and environmental factors in a monozygotic twin pair with Crohn's disease and their first-degree relatives. METHODS: 22-year-old monozygotic female twins with ileocolonic Crohn's disease and their healthy brother and parents were examined. DNA samples from patients and relatives were genotyped for CARD15, TLR4,and OCTN polymorphisms. ASCA and p-ANCA analyses were performed. Additionally, patients and relatives filled out a questionnaire concerning multiple environmental factors. RESULTS: Both twins presented in the same year with identical Vienna Classification phenotypes: stenotic behavior (B2) and localization in terminal ileum and colon (L3). Both carried a CARD15 R702W variant, but had normal alleles in TLR4 and OCTN. They were smokers since the age of 15, used oral contraceptives and had undergone appendectomy. The healthy father and brother were CARD15 R702W positive, were non-smokers and had not undergone appendectomy. CONCLUSION: This case report is the first to describe complete concordance in CARD15 status, phenotypic appearance, and smoking, appendectomy and oral contraceptive use in a pair of monozygotic twins with CD.

Adult↗

PubMed2005

Mutations in the genes KCND2 and KCND3 encoding the ion channels Kv4.2 and Kv4.3, conducting the cardiac fast transient outward current (ITO,f), are not a frequent cause of long QT syndrome.

BACKGROUND: Long QT syndrome (LQTS) is a hereditary cardiac arrhythmogenic disorder characterized by prolongation of the QT interval in the electrocardiogram, torsades de pointes arrhythmia, and syncopes and sudden death. LQTS is caused by mutations in ion channel genes. However, only in half of the families is it possible to identify mutations in one of the seven known LQTS genes, why further genetic heterogeneity is expected. The genes KCND2 and KCND3, encoding the alpha-subunits of the voltage-gated potassium channels Kv4.2 and Kv4.3 conducting the fast transient outward current (I(TO,f)) of the cardiac action potential (AP) in the myocardium, have been associated with prolongation of AP duration and QT prolongation in murine models. METHODS: KCND2 and KCND3 were examined for mutations using single-strand conformation polymorphism (SSCP) analysis in 43 unrelated LQTS patients, where mutations in the coding regions of known LQTS genes had been excluded. RESULTS: Seven single nucleotide polymorphismsm (SNPs) were found, two exonic SNPs in KCND2 and three exonic and two intronic in KCND3. None of the five exonic SNPs had coding effect. All seven SNPs are considered normal variants. CONCLUSION: The data suggest that mutations in KCND2 and KCND3 are not a frequent cause of long QT syndrome.

Action Potentials↗

PubMed2005

Disease concordance, zygosity, and NOD2/CARD15 status: follow-up of a population-based cohort of Danish twins with inflammatory bowel disease.

OBJECTIVES: A Danish cohort of twins with inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), has previously been collected. The aim of the present study was to reassess this cohort in order to compare clinical characteristics in concordant versus discordant twin pairs, test twin zygosity genetically, follow-up on disease concordance, and examine NOD2/CARD15 genetic status. METHODS: The Danish cohort is one of two population-based cohorts worldwide and consists of 103 twin pairs. After median 13 yr of follow-up, all twins were contacted and hospital files were scrutinized to reassess disease concordance and obtain phenotype data. DNA was obtained from 123 twins for analysis of zygosity and prevalence of the three common NOD2/CARD15 mutations. RESULTS: Zygosity tested genetically was consistent with the former assessment based on questionnaires. The proband concordance for CD remained fairly stable: 63.6% among monozygotic (MZ) twins and 3.6% among dizygotic (DZ) twins. Clinical characteristics were similar in twins from concordant versus discordant pairs. Forty-four percent of patients with CD were positive for >or=1 mutant allele of NOD2/CARD15 compared to 2% of UC patients (p < 0.001) and 19% of healthy twins (p= 0.02). The allele mutation frequency was 43% among the healthy twins to patients with CD versus 9% among twins to UC patients (p= 0.01). CONCLUSIONS: Previous questionnaire assessment of twin zygosity was confirmed by genetic test. Concordance for CD remained quite stable and was significantly higher among MZ than DZ twins. A high NOD2/CARD15 mutation frequency was observed both among CD twins and their healthy siblings.

Adult↗

PubMed2003

High-throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method.

Capillary array electrophoresis (CAE) is a novel technique, which allows for high throughput analysis of DNA fragments. When screening for mutations in whole populations or large patient groups it is necessary to have robust and well-characterized setups for high throughput analysis. For large-scale mutation screening, we have developed procedures for single strand conformation polymorphism (SSCP) assays using CAE (CAE-SSCP) whereby we may increase both the sensitivity and the throughput compared to conventional SSCP analysis. In this study we have validated CAE-SSCP by 1) comparing detection by slab-gel based SSCP with CAE-SSCP of mutations in the MYH7, MYL2, and MYL3 genes encoding sarcomere proteins from patients suffering from hypertrophic cardiomyopathy; and 2) by constructing a series of 185 mutants having substitution mutations, as well as insertion/deletion mutations, or some combinations of these, in different sequence contexts in four exons and different positions relative to the end of the amplicon (three from the KCNQ1 gene, encoding a cardiac potassium channel, and one from the TNNI3 gene encoding cardiac troponin I). The method identified 181 out of 185 mutations (98%), and the data suggest that the position of mutation in the fragment had no effect on the sensitivity. Analysis of the specificity of the method showed that only very few mutants could not be distinguished from each other and there were no false positives.

Automation↗

PubMed2003

Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening.

The generation of the draft human genome sequence has created new possibilities for diagnosis, prevention, and treatment of human disease. One consequence of these new possibilities is an increasing need for methods and technology that can be used for high-throughput screening for mutations in large DNA sample materials. In recent years, a number of mutation screening methods have emerged that are based on the analysis of sequence-dependent changes in the conformation of single- and double-stranded DNA using capillary electrophoresis. Common features of these methods are high sensitivity and reproducibility as well as the possibility for automation and massive parallelization. Thus, at present they are among the most attractive technologies for high-throughput mutation screening. This review describes the recent advances in capillary electrophoresis-based single strand conformation polymorphism (CE-SSCP) for detection of unknown mutations, and assesses its practical usability for high-throughput mutation screening based on the available literature. In addition, future prospects are outlined in light of the recent advances in microchip-based capillary electrophoresis.

DNA Mutational Analysis↗