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Lars Allan Larsen

Publications and source records attributed to Lars Allan Larsen.

15 recordsLinked to original sources
PubMed2006

[Screening for fragile X syndrome. International experiences].

Fragile X syndrome (FXS) is the most prevalent cause of inherited mental retardation, and calculations show that there are approximately 700 non-diagnosed cases in Denmark. Since the disease is severe, screening for FXS should be considered in order to improve genetic counselling. International experience indicates that efforts should initially be on active case finding among persons with learning difficulties and subsequently on cascade screening to identify carriers. Prenatal screening programmes may be an option when reliable high-throughput diagnostic methods are available.

Child↗
PubMed2006

Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA.

Several methods have been developed for amplification of RNA, making it possible to use cDNA microarrays for analysis of samples limited in amount of total RNA. The most widely used amplification protocol, the Eberwine method, amplifies RNA in a linear manner through in vitro transcription (IVT). However, when starting material is limited to nanogram amounts of total RNA, several rounds of amplification are necessary, making this method both expensive and labor-intensive. Amplification by PCR is robust and is able to amplify extremely limiting material. However, it is possible that the nonlinear nature of PCR could result in reduced reproducibility of the amplification compared with IVT. We have evaluated two methods that use a combination of PCR and IVT for amplification of nanogram amounts of total RNA. We have compared microarray results obtained by these methods with results obtained by two established methods: indirect labeling of 20 microg total RNA and Eberwine amplification of 1 microg total RNA. Starting from as little as 5 ng of total RNA, both methods yielded results in concordance with the Eberwine method.

Animals↗
PubMed2006

Hedgehog signaling in small-cell lung cancer: frequent in vivo but a rare event in vitro.

The hedgehog (HH) signaling pathway plays multiple roles during embryonic development and increasing evidence suggests that this embryonic pathway is involved in development and progression of several human cancers including those of the brain, skin, lung and gastrointestinal tract. To investigate HH signaling activity in small-cell lung cancer (SCLC), we have performed gene expression analysis on members of the HH pathway on a panel of 20 SCLC cell lines. Sonic hedgehog (SHH) expression was detected in only DMS79 and GLC16 and only DMS114 expressed detectable protein levels of GLI1, one of the key transcription factors of the pathway. Involvement of HH signaling in SCLC proliferation was investigated in a subset of cell lines using the HH signaling inhibitor cyclopamine or small interfering RNA (siRNA) against GLI1. Cells expressing GLI1 responded only weakly to both cyclopamine and RNA interference, suggesting that HH signaling plays only a minor role in the growth of SCLC cell lines. To investigate HH pathway activity in vivo, GLI1 immunohistochemistry was performed on SCLC tumors. Interestingly, GLI1 was expressed in most SCLC tumors studied, indicating that HH signaling is important for in vivo growth of SCLC but establishment of cell lines from SCLC tumors may lead to loss of expression of key HH pathway members. Thus, the data support the idea that the HH pathway may be a therapeutic target in SCLC. However, the data also suggest that the SCLC cells can circumvent the apparent in vivo requirement of HH signaling.

Carcinoma, Small Cell↗
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

High-throughput single-strand conformation polymorphism analysis on a microfabricated capillary array electrophoresis device.

A high-density 384-lane microfabricated capillary array electrophoresis device is evaluated for high-throughput single-strand conformation polymorphism (SSCP) analysis. A delayed back bias direct electrokinetic injection scheme is used to provide better than 10-bp resolution with an 8.0-cm effective separation length. Separation of a HaeIII digest of PhiX174 yielded theoretical plate numbers of 4.0 x 10(6). Using 5% PDMA containing 10% glycerol and 15% urea, 21 single-nucleotide polymorphisms (SNPs) from HFE, MYL2, MYL3, and MYH7 genes associated with hereditary hemochromatosis (HHC) and hereditary hypertrophic cardiomyopathy (HCM) are discriminated at two running temperatures (25 degrees C and 40 degrees C), providing 100% sensitivity. The data in this study demonstrate that the 384-lane microCAE device provides the resolution and detection sensitivity required for SSCP analysis, showing its potential for ultrahigh-throughput mutation detection.

Cardiomyopathies↗
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

One third of Danish hypertrophic cardiomyopathy patients with MYH7 mutations have mutations [corrected] in MYH7 rod region.

Familial hypertrophic cardiomyopathy (FHC) is, in most cases, a disease of the sarcomere, caused by a mutation in one of 10 known sarcomere disease genes. More than 266 mutations have been identified since 1989. The FHC disease gene first characterized MYH7, encodes the cardiac beta-myosin heavy chain, and contains more than 115 of these mutations. However, in most studies, only the region encoding the globular head and the hinge region of the mature cardiac beta-myosin heavy chain have been investigated. Furthermore, most studies carries out screening for mutations in the most prevalent disease genes, and discontinues screening when an apparent disease-associated mutation has been identified. The aim of the present study was to screen for mutations in the rod region of the MYH7 gene in all probands of the cohort, regardless of the known genetic status of the proband. Three disease-causing mutations were identified in the rod region in four probands using capillary electrophoresis single-strand conformation polymorphism as a screening method. All mutations were novel: N1327K, R1712W, and E1753K. Two of the probands had already been shown to carry other FHC-associated mutations. In conclusion, we show that in the Danish cohort we find one third of all MYH7 mutations in the rod-encoding region and we find that two of the patients carrying these mutations also carry mutations in other FHC disease genes stressing the need for a complete screening of all known disease genes in FHC-patients.

Amino Acid Substitution↗
PubMed2004

Does KCNE5 play a role in long QT syndrome?

BACKGROUND: Long QT syndrome [LQTS] is a congenital cardiac disease characterised by prolonged QTC-time, syncopes and sudden cardiac death. LQTS is caused by mutations in genes coding for ion channels involved in the action potential. KCNE5 codes for a novel beta-subunit of the ion channel conducting the delayed rectifier repolarizing current IKs. As KCNE5 is expressed in the human heart and suppresses the IKs current in heterologous systems, it is a candidate gene that may be mutated in LQTS families where no causative mutations in known LQTS associated genes have been found. We examined whether this was the case. METHODS: Genomic DNA from LQTS patients [n=88] and normal controls [n=90] was screened for mutations in KCNE5 by endonuclease-enhanced single strand conformation polymorphism analysis [EE-SSCP], and DNA sequencing of aberrant conformers. Mutations in other LQTS associated ion channels were excluded by SSCP. RESULTS: No mutations were found in the coding region of the KCNE5 gene in LQTS patients. One polymorphism, a T-to-C transition at nucleotide 97, causing an amino acid polymorphism P33S, was present in 16 persons, nine heterozygotes and seven homozygotes. The T-allele frequency was 0.13 in LQTS patients and 0.10 in controls.

Action Potentials↗
PubMed2004

GJB2 and GJB6 mutations in 165 Danish patients showing non-syndromic hearing impairment.

Thirty-two genes causing non-syndromic hearing impairment (NSHI) have been cloned, including GJB2 and GJB6 encoding the gap junction subunits connexin 26 and connexin 30, respectively. One mutation in GJB2, 35delG, accounts for a large percentage of GJB2 hearing impairment in Southern Europe whereas a considerably lower frequency has been reported from Northern European populations. Recently, a 342-kb deletion implicating GJB6 was found in 22 out of 44 NSHI patients of Spanish origin with only one mutated allele of GJB2. We report the first study of GJB2 and GJB6 mutations in Danish patients with NSHI. We tested 165 individuals and found GJB2 mutations in 16 individuals. The deletion implicating GJB6 was found in two individuals out of 9 heterozygous for GJB2 mutation. Furthermore, we screened 509 unselected samples from the Danish newborn population for the 35delG mutation in GJB2. We found 9 samples heterozygous for 35delG and 11 samples heterozygous for mutations leading to amino acid variants in GJB2 protein. In conclusion, our data are in accordance with results from other Northern European populations. Furthermore, our data on the GJB6 deletion suggest that routine screening for this deletion could help to explain hearing impairment in some Northern European NSHI patients heterozygous for a mutation in GJB2.

Adolescent↗
PubMed2004

Identification of a novel EYA1 splice-site mutation in a Danish branchio-oto-renal syndrome family.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by variable clinical manifestations including branchial fistulae, preauricular pits, ear malformations, hearing impairment, and renal anomalies. BOR is caused by mutations in the genes EYA1 and SIX1. A Danish BOR family with five affected individuals in three generations was analyzed for mutations in all 17 exons of EYA1 using direct sequencing of polymerase chain reaction (PCR) amplified genomic DNA. A novel splice-site mutation (IVS9+1 G>C) was detected in all affected family members but not in unaffected family members or in 96 controls. We conclude that this mutation is causing BOR in the family, most likely as a result of haploinsufficiency or an abnormal protein product caused by aberrant splicing of EYA1 mRNA.

Branchio-Oto-Renal Syndrome↗
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↗
PubMed2003

Outcome of clinical versus genetic family screening in hypertrophic cardiomyopathy with focus on cardiac beta-myosin gene mutations.

OBJECTIVE: Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in genes encoding cardiac sarcomere proteins. Although available, genetic analyses are generally not used clinically. In the present study, we evaluated the outcome of clinical vs. genetic screening of family members with specific focus on mutations in the cardiac beta-myosin heavy chain (MYH7) gene. METHODS: A consecutive cohort of 68 FHC probands and their families (395 persons) of Danish origin was evaluated including patient- and family histories, physical examinations, electrocardiogram and echocardiography. Mutation screening was performed by a combination of single strand conformation/heteroduplex analysis and direct sequencing. RESULTS: Eight different MYH7 gene mutations were identified in nine (13%) families (96 persons). In eight (89%) of the families, major cardiac events had occurred. Myectomy or percutaneous septal alcohol ablation had been performed in a higher number of MYH7 probands i.e. in five of nine (56%) as compared to 10 of 59 (17%) (P<0.05) non-MYH7 mutation probands. Neither echocardiographic nor ECG findings were useful to distinguish MYH7 from non-MYH7 probands. Between adult MYH7 mutation-carriers (n=38) and their non-carrier relatives (n=39), low sensitivity and specificity of the clinical diagnostic criteria tested were observed and minor clinical diagnostic criteria alone were not useful for identification of mutation carriers. By genetic screening of relatives with no or only minor hypertrophy on echocardiography, i.e. a priori possible mutation-carriers normally recommended clinical follow-up-the diagnosis was excluded in 52 (83%) persons. In addition, six relatives with secondary hypertrophy were identified as non-carriers. CONCLUSION: Neither echocardiographic nor ECG findings were useful to distinguish MYH7 from non-MYH7 probands. Extension of screening to include genetic analyses offered a marked diagnostic advantage as compared to clinical screening alone in FHC families.

Adolescent↗
PubMed2003

The Hedgehog signaling pathway--implications for drug targets in cancer and neurodegenerative disorders.

The Hedgehog (Hh) pathway is a highly conserved signaling cascade involved in many developmental processes. Among others, these include patterning of the ventral neural tube and establishment of left-right asymmetry of the embryo. Additionally, the pathway regulates the development of numerous tissues and cell types. Mutations in elements of the pathway are associated with congenital diseases and defects, and ectopic Hh signaling activity is implicated in the development of a number of neoplasms. While little is known of Hh signaling function in the adult organism, a role of the pathway in maintenance of adult organs and cell types, including several neuronal subtypes in the central nervous system, is beginning to emerge. Elements of the Hh pathway are therefore potential drug targets for the treatment of cancers and degenerative diseases like Parkinson's disease, and the recent isolation of synthetic molecules capable of modulating the activity of the Hh cascade through a direct interaction with elements of the pathway is promising.

Animals↗