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2.
Prenat Diagn ; 21(10): 806-12, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11746119

ABSTRACT

Feto-specific markers are necessary for genetic diagnostics on fetal cells isolated from maternal blood. Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to identify mRNA species preferentially expressed in trophoblast-enriched primary cell cultures compared to female peripheral mononuclear blood cells (FPMBCs). We obtained 15 different cDNA clones expressed predominantly in placenta cells using a commercial kit with oligo-(dT) anchor primers and arbitrary upstream primers for differential display analysis of total RNA. Reverse Northern dot-blot analysis and semi-quantitative RT-PCR confirmed the different expression levels. Seven mRNA species were exclusively expressed in the placenta: the mRNAs of epsilon-globin, a renowned marker of fetal erythroblasts, early placenta insulin-like peptide (EPIL), Yes-associated protein (YAP65) and osteopontin were expressed at high levels, whereas the mRNAs of PP14, JM27 protein, and an unidentified expressed sequence tag (EST) were moderately expressed. A further eight mRNAs with low expression in FPMBCs and higher expression in first trimester placenta were identified. Some mRNAs were expressed in a trophoblast cell line, e.g. YAP65, whereas others were not, e.g. EPIL. The new mRNA markers may be used to construct DNA/RNA probes to identify fetal cells in maternal circulation by hybridization techniques, or to identify proteins selectively expressed in fetal cells and amenable to immunocytochemical detection.


Subject(s)
Adaptor Proteins, Signal Transducing , Biomarkers/blood , Cell Separation/methods , Fetal Blood/cytology , Growth Substances , Intercellular Signaling Peptides and Proteins , RNA, Messenger/blood , Actins/genetics , Aldehyde Reductase/genetics , Blotting, Northern , Carrier Proteins/genetics , Cells, Cultured , Chorionic Gonadotropin/genetics , Female , Fetal Blood/chemistry , Fibrillins , Globins/genetics , Humans , Leukocytes, Mononuclear , Microfilament Proteins/genetics , Osteopontin , Phosphoproteins/genetics , Pregnancy , Pregnancy Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/genetics , Transcription Factors , Trophoblasts/cytology , YAP-Signaling Proteins
3.
Pharmacogenomics ; 2(4): 387-99, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11722288

ABSTRACT

Screening of large sample materials for the presence of known or unknown mutations is a key element in pharmacogenomics. Although automated DNA sequencing has developed rapidly during the last decade, the technology is not well suited for projects involving analysis of hundreds of thousands of mutations. Consequently, a number of methods for high-throughput mutation screening have been developed. DNA microarrays and high-density oligonucleotide chips have proven to be well suited for parallel hybridisation-based analysis of hundreds or thousands of known mutations. Methods based on detection using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) have been developed. MALDI-TOF MS detection is limited to analysis of small DNA fragments but has a large potential for high-throughput single nucleotide polymorphism (SNP) analysis, due to a very fast analysis time and possibilities for automation. Currently, the best suited methods for high-throughput screening for unknown mutations are probably methods like single strand conformation polymorphism (SSCP) analysis or conformation sensitive gel electrophoresis (CSGE), combined with capillary array electrophoresis or denaturing high-performance liquid chromatography. This is due to a relatively short analysis time, potential for automation and a high sensitivity. The recent development of capillary array electrophoresis chips suggests that the analysis time for some of these methods may be reduced by one order of magnitude in the near future.


Subject(s)
DNA Mutational Analysis/methods , Chromatography, High Pressure Liquid , Electrophoresis, Capillary , Humans , Oligonucleotide Array Sequence Analysis , Polymorphism, Single Nucleotide , Polymorphism, Single-Stranded Conformational , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
4.
Hum Mutat ; 18(5): 451-7, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11668638

ABSTRACT

The rapid progress in the isolation of genes associated with human disease has resulted in an increasing demand for mutation screening methods. The molecular diagnosis of the long QT syndrome (LQTS), a cardiac disorder characterized by prolongation of the QT(c) interval in the ECG, syncopes, and sudden death, requires mutation screening of all exons in at least five genes, encoding cardiac Na(+) and K(+) channel subunits. A method for automated dideoxy fingerprinting (ddF) using capillary array electrophoresis (CAE) was developed and the efficiency of the method was tested by analyzing 24 DNA samples with mutations in one of the genes KCNQ1 and KCNH2, which are involved in 50% of LQTS cases. One of these mutations, 362insQK in KCNQ1, is novel. The sensitivity was 100% using a single electrophoresis temperature of 18 degrees C or 25 degrees C. However, analysis of the samples in both the sense and anti-sense direction were required for high sensitivity. Analysis in a single direction resulted in a decrease of the sensitivity to 74% and 70%, respectively. The throughput of the ddF method, if performed with a 16 capillary CAE instrument, is 288 samples per seven hr if each sample is analyzed on both strands.


Subject(s)
Cation Transport Proteins , DNA Fingerprinting/methods , DNA-Binding Proteins , Dideoxynucleosides/metabolism , Electrophoresis, Capillary/methods , Genetic Testing/methods , Mutation/genetics , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Trans-Activators , Automation/methods , DNA Mutational Analysis/methods , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels , Exons/genetics , Humans , Injections , Introns/genetics , KCNQ Potassium Channels , KCNQ1 Potassium Channel , Long QT Syndrome/genetics , Polymorphism, Single-Stranded Conformational , Reproducibility of Results , Sensitivity and Specificity , Temperature , Transcriptional Regulator ERG
5.
Eur J Hum Genet ; 9(9): 724-7, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11571563

ABSTRACT

Fragile X syndrome, the most common form of inherited mental retardation, is caused by expansion of a (CGG)(n) repeat located in the FMR1 gene. The molecular factors involved in the mutation process from stable (CGG)(n) alleles towards unstable alleles are largely unknown, although family transmission studies and population studies have suggested that loss of AGG interruptions in the (CGG)(n) repeat is essential. We have analysed the AGG interspersion pattern of the FMR1 (CGG)(n) repeat and the haplotype distribution of closely located microsatellite markers DXS548 and FRAXAC1, in three circumarctic populations: Norwegians, Nenets and Saami. The data confirm the conservation, reported in all human populations studied so far, of an AGG interruption for each 9-10 CGG and support the stabilising effect of AGG interruptions. The data also indicate the existence of chromosomes of Asian origin in the Saami and Nenets population, thereby confirming a genetic relationship between Northern Europe and Asia. DXS548-FRAXAC1 haplotype frequencies were compared between 24 Norwegian fragile X males and 119 normal males. Significant linkage disequilibrium were found between the fragile X mutation and haplotype 6-4 and between normal (CGG)(n) alleles and haplotype 7-3.


Subject(s)
Alleles , Haplotypes/genetics , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Trinucleotide Repeats/genetics , Asia , DNA/genetics , Europe , Fragile X Mental Retardation Protein , Fragile X Syndrome/genetics , Gene Frequency , Genetics, Population , Humans , Microsatellite Repeats
6.
Clin Chem ; 47(8): 1390-5, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11468227

ABSTRACT

BACKGROUND: The voltage-gated, rapid-delayed rectifier current (I(Kr)) is important for repolarization of the heart, and mutations in the genes coding for the K+-ion channel conducting this current, i.e., KCNH2 for the alpha-subunit HERG and KCNE2 for the beta-subunit MiRP1, cause acquired and congenital long Q-T syndrome (LQTS) and other cardiac arrhythmias. METHODS: We developed a robust single-strand conformation polymorphism-heteroduplex screening analysis, with identical thermocycling conditions for all PCR reactions, covering all of the coding exons in KCNH2 and KCNE2. The method was used to screen 40 unrelated LQTS patients. RESULTS: Eleven mutations, of which six were novel, were found in KCNH2. Interestingly, six mutations were found in the region of the gene coding for the Per-Arnt-Sim (PAS) and PAS-S1 regions of the HERG protein, stressing the need to examine the entire gene when screening for mutations. No mutations were found in KCNE2, suggesting that direct involvement of MiRP1 in LQTS is rare. Furthermore, four novel single-nucleotide polymorphisms (SNPs) and one amino acid polymorphism (R1047L) were identified in KCNH2, and one novel SNP and one previously known amino acid polymorphism (T8A) were found in KCNE2. CONCLUSIONS: The potential role of rare polymorphisms in the HERG/MiRP1 K+-channel should be clarified with respect to drug interactions and susceptibility to arrhythmia and sudden death.


Subject(s)
Cation Transport Proteins , DNA-Binding Proteins , Long QT Syndrome/genetics , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Trans-Activators , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels , Heteroduplex Analysis , Humans , Long QT Syndrome/congenital , Mutation , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Potassium Channels/blood , Transcriptional Regulator ERG
8.
Mol Biochem Parasitol ; 111(1): 123-30, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11087922

ABSTRACT

Genetic variation of the glutamate-rich protein (GLURP) of Plasmodium falciparum was analysed in 29 field isolates and 15 laboratory lines of diverse geographical origin, by DNA sequencing of the non-repetitive 5'-region (R0) of the glurp gene. Polymorphism with respect to the length of the GLURP R2 repeat region was also analysed by a polymerase chain reaction method. As reference, the nucleotide sequence of the highly polymorphic 3'-region of the circumsporozoite protein gene was determined in the same isolates. It was found that even in the presence of variations in the GLURP R2 repeat region, immunodominant parts of the GLURP R0 region are surprisingly well conserved and the conservation is most pronounced in isolates from locations with very high malaria transmission. This might indicate that the R0 structure plays an important role in the parasite.


Subject(s)
Conserved Sequence , Genes, Protozoan , Genetic Variation , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Animals , Base Sequence , DNA, Protozoan/genetics , Immunodominant Epitopes/chemistry , Immunodominant Epitopes/genetics , Immunodominant Epitopes/immunology , Molecular Sequence Data , Plasmodium falciparum/chemistry , Plasmodium falciparum/immunology , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Repetitive Sequences, Nucleic Acid , Sequence Analysis, DNA
9.
Comb Chem High Throughput Screen ; 3(5): 393-409, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11032956

ABSTRACT

Molecular diagnosis of complex inherited disorders, population screening of genetic diseases, studies of the genetic basis of variable drug response (pharmacogenetics) as well as discovery and investigation of new drug targets (pharmacogenomics) involve screening for mutations in multiple DNA samples. Furthermore, the development of a third generation of the human genome map, based on single nucleotide polymorphisms (SNPs), requires screening for allelic variants through all of the three billion basepairs in the human genome. Thus, the need for high throughput mutation screening methods is great and is rapidly increasing. Traditional methods for mutation screening often involve slab-gel electrophoresis analyses which are laborious and difficult to automate. However, recent developments in capillary electrophoresis systems for DNA fragment analysis have made fully automated mutation screening possible and have dramatically increased the possible sample throughput. This review describes the recent advances in capillary electrophoresis of DNA and summarize the various methods for mutation screening based on this technique.


Subject(s)
DNA Mutational Analysis/methods , Electrophoresis, Capillary/methods , Automation , DNA Fingerprinting/methods , Electrophoresis, Capillary/instrumentation , Heteroduplex Analysis , Humans , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA/methods
10.
Int J Parasitol ; 30(7): 853-8, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10899531

ABSTRACT

The Toxoplasma gondii (TGR) genes constitute a family of non-coding sequences, three of which have been previously described as possible tools for typing of Toxoplasma gondii isolates. We obtained new isolates of T. gondii from domestic and wild animals, and used these to evaluate the possibility of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T. gondii isolates could be separated into seven groups. Sequencing the amplified products showed that at least 20 TGR sequences not hitherto described had been found, demonstrating that the TGR gene family comprises a large number of different yet highly homologous sequences. Each isolate had its own unique TGR sequence. The TGR gene family therefore seems a promising target for typing individual T. gondii isolates and for studying the genetic distance between two isolates, which can be used for tracing routes of infection.


Subject(s)
Phylogeny , Toxoplasma/classification , Toxoplasmosis, Animal/diagnosis , Animals , Base Sequence , Cats , Cloning, Molecular , DNA Primers/chemistry , DNA, Protozoan/chemistry , Denmark , Deoxyribonucleases, Type II Site-Specific/chemistry , Electrophoresis, Polyacrylamide Gel , Foxes , Lagomorpha , Molecular Sequence Data , Polymerase Chain Reaction/veterinary , Polymorphism, Restriction Fragment Length , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Sheep , Swine , Toxoplasma/chemistry , Toxoplasma/genetics
11.
Am J Med Genet ; 93(2): 99-106, 2000 Jul 17.
Article in English | MEDLINE | ID: mdl-10869110

ABSTRACT

The AGG interspersion pattern and flanking microsatellite markers and their association with instability of the FMR1 (CGG)(n) repeat, involved in the fragile X syndrome, were analyzed in DNA from filter-paper blood spots randomly collected from the Danish newborn population. Comparison of DXS548-FRAXAC1 haplotype frequencies in the normal population and among fragile X patients suggested strong linkage disequilibrium between normal alleles and haplotype 7-3 and between fragile X alleles and haplotype 2-1 and 6-4. Comparison of the AGG interspersion pattern in 143 alleles, ranging in size from 34-62 CGG, and their associated haplotypes indicates the existence of at least three mutational pathways from normal alleles toward fragile X alleles in the Danish population. Two subgroups of normal alleles, with internal sequences of (CGG)(10)AGG(CGG)(19) and (CGG)(9)AGG(CGG)(12) AGG(CGG)(9), possibly predisposed for expansion, were identified in the data set. When alleles larger than 34 CGG were investigated, comparing the length of 3' uninterrupted CGG triplets (uCGG), we found that alleles associated with haplotype 2-1 and 6-4 contain significantly longer stretches of uCGG than alleles associated with haplotype 7-3. Thus, the data support that (CGG)(n) instability is correlated to the length of uCGG.


Subject(s)
Alleles , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Trinucleotide Repeats/genetics , Base Sequence , Cohort Studies , DNA/chemistry , DNA/genetics , Denmark , Fragile X Mental Retardation Protein , Fragile X Syndrome/genetics , Genotype , Haplotypes , Humans , Infant, Newborn , Male , Microsatellite Repeats , Mutation , Sequence Analysis, DNA
12.
Clin Genet ; 57(2): 125-30, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10735633

ABSTRACT

In a four-generation family with long QT syndrome, syncopes and torsades de pointes ventricular tachycardia (TdP) were elicited by abrupt awakening in the early morning hours. The syndrome was associated with a novel KCNH2 missense mutation, G572R, causing the substitution of a glycine residue at position 572, at the end of the S5 transmembrane segment of the HERG K(+)-channel, with an arginine residue. This segment is involved in the channel pore and the mutation may cause a reduction in the rapidly activating delayed rectifier K+ current (Ikr), or changed gating properties of the ion channel, leading to prolonged cardiac repolarization. The electrocardiograms of affected persons showed prolonged QT interval and notched T waves. Despite treatment with atenolol, 200 mg twice daily, the proband still experienced TdP episodes. Three untreated relatives of the proband died suddenly, and unexpectedly, at 18, 32, and 57 years of age. The G572R mutation is thus associated with a high mortality rate, and the clinical presentation illustrates that some mutations may not be controllable by just beta-blockade.


Subject(s)
Cation Transport Proteins , DNA-Binding Proteins , Long QT Syndrome/genetics , Mutation, Missense , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Trans-Activators , Adolescent , Adult , Amino Acid Sequence , DNA Mutational Analysis , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels , Fatal Outcome , Female , Humans , Long QT Syndrome/diagnosis , Long QT Syndrome/therapy , Male , Molecular Sequence Data , Pedigree , Phenotype , Sequence Homology, Amino Acid , Transcriptional Regulator ERG
13.
Scand Cardiovasc J ; 34(6): 558-63, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11214007

ABSTRACT

OBJECTIVE: An examination of the genetic background and phenotypic presentation of familial hypertrophic cardiomyopathy (FHC) with respect to specific mutations in the MYH7-gene encoding the cardiac beta-myosin heavy chain. SETTINGS: Two families (n = 22) from a cohort of 67 families with FHC were studied at the National University Hospital, Rigshospitalet, Copenhagen. METHODS: Clinical, non-invasive examinations of all included family members followed by molecular genetic analysis including PCR-single strand conformation polymorphism/heteroduplex (SSCP/HD) analysis and sequencing of exon 3-23 of the MYH7-gene. RESULTS: We found FHC associated with a missense mutation in two families, i.e. a C > G transversion at position g10124 and a G > T transversion at position g10126 causing the change of a leucine residue at codon 390 to a valine residue. The mutation is located in the actin-binding region of the beta-myosin heavy chain. The leucine residue is evolutionarily conserved in vertebrate myosins. In the two families, the phenotypic presentations in the clinically affected were characterized by asymmetric septal hypertrophy (septum diameter 18.8 (5.0) mm (mean (SD)) with only minor involvement of the left ventricular free wall (posterior wall diameter 11.0 (2.2) mm). Furthermore, the left ventricular systolic and diastolic functions were well preserved, even at a high age. The symptomatic status of the clinically affected patients depended on the presence or absence of a concomitant left ventricular outflow tract gradient. CONCLUSIONS: We report a novel missense mutation associated with FHC caused by a double nucleotide transversion. The penetrance of the mutation was not complete, but in clinically affected patients the mutation gives rise to an echocardiographic phenotype, predominantly characterized by pronounced septal hypertrophy.


Subject(s)
Cardiomegaly/genetics , Cardiomyopathy, Hypertrophic/genetics , Mutation, Missense , Myosin Heavy Chains/genetics , Sarcomeres/genetics , Adolescent , Adult , Aged , Electrocardiography , Exons , Humans , Leucine/genetics , Middle Aged , Pedigree , Phenotype , Polymorphism, Single-Stranded Conformational , Valine/genetics
15.
Clin Chem ; 45(11): 1898-905, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10545058

ABSTRACT

BACKGROUND: Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs). METHODS: LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate. RESULTS: In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique. CONCLUSIONS: The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.


Subject(s)
Factor V/genetics , Oligonucleotide Probes , Alleles , Anthraquinones , Enzyme-Linked Immunosorbent Assay , Humans , Mutation , Oligonucleotide Probes/chemical synthesis , Polymerase Chain Reaction/methods , Prospective Studies , Reproducibility of Results , Sensitivity and Specificity , Ultraviolet Rays
16.
Eur J Hum Genet ; 7(7): 771-7, 1999.
Article in English | MEDLINE | ID: mdl-10573009

ABSTRACT

The fragile X syndrome is caused by the expansion of a polymorphic (CGG)n tract in the promoter region of the FMR1 gene. Apparently the incidence of fragile X syndrome is rare in the population of Greenland. In order to examine population-related factors involved in stability of the (CGG)n sequence, DNA samples obtained randomly from the Greenlandic population were analysed for size and AGG interspersion pattern of the FMR1 (CGG)n region and associated DXS548-FRAXAC1 haplotypes. In addition a large Greenland family with unstable transmission in the premutation range was analysed. The (CGG)n allele sizes in the Greenland population showed a narrow distribution similar to that reported for Asian populations. DNA sequencing of alleles with 36 CGG repeats revealed an AGG(CGG)6 insertion previously reported exclusively in Asian populations and a high frequency of alleles with a (CGG)10AGG(CGG)9AGG(CGG)9 or (CGG)9AGG(CGG)9AGG(CGG)6AGG(CGG)9 sequence pattern was found. Thus the data confirm the Asian origin of the Greenlandic (Eskimo) population and indicates that some (CGG)n alleles have remained stable for 15-30,000 years, since the population of the New World arrived from Asia via the Bering Strait.


Subject(s)
Alleles , Fragile X Syndrome/genetics , Genetics, Population , Inuit/genetics , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Trinucleotide Repeats/genetics , Asia , Asian People/genetics , Child , Child, Preschool , Family , Female , Fragile X Mental Retardation Protein , Fragile X Syndrome/ethnology , Gene Frequency , Greenland , Haplotypes , Humans , Infant, Newborn , Male , Pedigree , Sequence Analysis, DNA
17.
Eur J Hum Genet ; 7(6): 724-8, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10482963

ABSTRACT

We describe a Swedish family with the proband and his brother suffering from severe Romano-Ward syndome (RWS) associated with compound heterozygosity for two mutations in the KVLQT1 (also known as KCNQ1 and KCNA9) gene (R518X and A525T). The mutations were found to segregate as heterozygotes in the maternal and the paternal lineage, respectively. None of the heterozygotes exhibited clinical long QT syndrome (LQTS). No hearing defects were found in the proband. The data strongly indicates that the compound heterozygosity for R518X and A525T is the cause of an autosomal recessive form of RWS in this family. Our findings support the implication of a higher frequency of gene carriers than previously expected. We suggest that relatives of 'sporadic RWS' patients should be considered potential carriers, at risk of dying suddenly from drug-induced LQTS.


Subject(s)
Genes, Recessive , Heterozygote , Long QT Syndrome/genetics , Mutation , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Child, Preschool , DNA Mutational Analysis , Female , Humans , Infant, Newborn , KCNQ Potassium Channels , KCNQ1 Potassium Channel , Male , Mutation, Missense , Pedigree , Point Mutation , Polymorphism, Single-Stranded Conformational
18.
J Mol Cell Cardiol ; 31(4): 745-50, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10329202

ABSTRACT

Mutations in the cardiac beta -myosin heavy chain gene (MYH7), and other genes encoding cardiac sarcomere proteins may cause familial hypertrophic cardiomyopathy (F-HCM), an autosomal dominant disease, characterized by myocardial hypertrophy. We analysed the MYH7 gene in three generations of a family with one borderline and four clinically verified cases of hypertrophic cardiomyopathy, and identified a mutation in exon 7 changing the 190 arginine residue into a threonine residue. The mutation is located in the ATP-binding region of the myosin head and alters the charge in the F-helix close to the phosphate-binding P-loop. The mutation may thus interfere with the coupling between ATP-hydrolysis and the transition into mechanical energy. In conclusion, the novel Arg190Thr mutation in exon 7 of the MYH7 gene is associated with the development of symptomatic myocardial hypertrophy in adults.


Subject(s)
Cardiomyopathy, Hypertrophic/genetics , Mutation, Missense , Myosin Heavy Chains/genetics , Adenosine Triphosphate/metabolism , Adolescent , Adult , Arginine/chemistry , Base Sequence , Binding Sites/genetics , Cardiomyopathy, Hypertrophic/metabolism , Child , DNA Primers/genetics , Exons , Female , Humans , Male , Middle Aged , Myosin Heavy Chains/chemistry , Myosin Heavy Chains/metabolism , Pedigree , Phenotype
19.
Hum Mutat ; 13(4): 318-27, 1999.
Article in English | MEDLINE | ID: mdl-10220146

ABSTRACT

Genetic diagnosis of an inherited disease or cancer often involves analysis for unknown point mutations in several genes; therefore, rapid and automated techniques that can process a large number of samples are needed. We describe a method for high-throughput single-strand conformation polymorphism (SSCP) analysis using automated capillary electrophoresis. The operating temperature of a commercially available capillary electrophoresis instrument (ABI PRISM 310) was expanded by installation of a cheap in-house designed cooling system, thereby allowing us to perform automated SSCP analysis at 14-45 degrees C. We have used the method for detection of point mutations associated with the inherited cardiac disorders long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM). The sensitivity of the method was 100% when 34 different point mutations were analyzed, including two previously unpublished LQTS-associated mutations (F157C in KVLQT1 and G572R in HERG), as well as eight novel normal variants in HERG and MYH7. The analyzed polymerase chain reaction (PCR) fragments ranged in size from 166 to 1,223 bp. Seventeen different sequence contexts were analyzed. Three different electrophoresis temperatures were used to obtain 100% sensitivity. Two mutants could not be detected at temperatures greater than 20 degrees C. The method has a high resolution and good reproducibility and is very robust, making multiplex SSCP analysis and pattern-based identification of known allelic variants as single nucleotide polymorphisms (SNPs) possible. These possibilities, combined with automation and short analysis time, make the method suitable for high-throughput tasks, such as genetic screening.


Subject(s)
Alleles , DNA Mutational Analysis/methods , Electrophoresis, Capillary/methods , Polymorphism, Genetic , Polymorphism, Single-Stranded Conformational , Cardiomyopathy, Hypertrophic/genetics , DNA Mutational Analysis/instrumentation , Electrophoresis, Capillary/instrumentation , Humans , Long QT Syndrome/genetics , Point Mutation , Polymerase Chain Reaction , Sequence Analysis, DNA , Temperature
20.
Clin Chim Acta ; 280(1-2): 113-25, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10090529

ABSTRACT

Congenital long QT syndrome (LQTS) is characterised by prolongation of the QT interval on ECG and cardiac arrhythmias, syncopes and sudden death. A rapid and reliable genetic diagnosis of the disease may be of great importance for diagnosis and treatment of LQTS. Mutations in the KVLQT1 gene, encoding a potassium-channel subunit of importance for the depolarisation of cardiac myocytes, is believed to be associated with 50% of all LQTS cases. Our data confirms that KvLQT1 isoform 1 is encoded by 16 exons, and not 15, as reported previously. We have used genomic DNA sequences to design intronic PCR primers for amplification of 15 exons of KVLQT1 and optimised a non-radioactive single stranded conformation polymorphism/heteroduplex (SSCP/HD) method for detection of mutations in KVLQT1. The sensitivity of the method was 100% when it was tested on 15 in vitro constructed mutants. By multiplexing the PCR amplification of KVLQT1, it is possible to cover all 15 exons in four PCR reactions.


Subject(s)
Exons , Long QT Syndrome/genetics , Mutation , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Amino Acid Sequence , Base Sequence , DNA Primers , Humans , KCNQ Potassium Channels , KCNQ1 Potassium Channel , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational
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