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At least 253 records · Page 14Linked to original sources
PubMed2003

Pseudomigraine with lymphocytic pleocytosis: a calcium channelopathy? Clinical description of 10 cases and genetic analysis of the familial hemiplegic migraine gene CACNA1A.

OBJECTIVE: To report the clinical findings of 10 patients diagnosed with pseudomigraine with lymphocytic pleocytosis and the results of mutational analysis of the CACNA1A gene in 8 of these patients. BACKGROUND: Pseudomigraine with lymphocytic pleocytosis, also referred to as headache with neurologic deficits and cerebrospinal fluid lymphocytosis (HaNDL), is characterized by episodic transient neurologic dysfunction associated with moderate to severe headache and cerebrospinal fluid lymphocytic pleocytosis. Episodes are recurrent and the condition is self-limiting. The etiology of this sporadic condition remains unknown, but the episodic nature and its ability to be triggered by angiography is somewhat reminiscent of the phenotypic features of familial hemiplegic migraine, a condition caused by mutations in the CACNA1A gene. DESIGN/METHODS: Utilizing retrospective chart review, we describe the clinical features of pseudomigraine with lymphocytic pleocytosis in 10 patients. Whole blood was taken from 8 patients (2 were lost to follow-up) and used for DNA testing. The CACNA1A gene was screened for mutations using heteroduplex analysis and direct DNA sequencing. RESULTS: Clinical features of pseudomigraine with lymphocytic pleocytosis included transient episodes of weakness, sensory and visual symptoms, aphasia, and confusion lasting minutes up to 4 hours. Sensory symptoms, typically affecting the face and arm, were the most common presentation. Localization of symptoms did not conform to vascular territories. Headache was typically throbbing and most often bilateral. Genetic analysis did not identify any mutations in the CACNA1A gene. CONCLUSIONS: Similarities between familial hemiplegic migraine and pseudomigraine with lymphocytic pleocytosis include recurrent headache with reversible neurologic deficit, cerebrospinal fluid lymphocytic pleocytosis, and triggers such as angiography. Even so, heteroduplex analysis and DNA sequencing failed to identify any sporadic mutations or shared polymorphisms in the exons or the intron/exon boundaries of the CACNA1A gene. These results do not support a role of the CACNA1A gene in the etiology of pseudomigraine with lymphocytic pleocytosis.

Adult↗
PubMed1998

Comprehensive mutational analysis of the TSC1 gene: observations on frequency of mutation, associated features, and nonpenetrance.

We performed a comprehensive analysis for mutations in the TSC1 gene using Southern blot analysis, and SSCP and heteroduplex analysis of amplified exons in 13 families with genetic linkage to the TSC1 region, 22 small families without linkage information, and 126 sporadic patients. 17 unique mutations were identified in 21 patients. Mutations were found in 7/13 (54%) TSC1-linked families, 1/22 (5%) small families without linkage, and 13 of 126 (10%) sporadic cases. The mutations were all chain-terminating, with 14 small deletions, 1 small insertion, and 6 nonsense mutations. In families with mutations, all individuals carrying a mutation met formal diagnostic criteria for TSC, apart from a 3-year-old girl who had inherited a deletion mutation, and who had no seizures, normal intelligence, normal abdominal ultrasound, and hypomelanotic macules only on physical exam. We assessed the incidence and severity of mental retardation in the 13 sporadic patients with TSC1 mutations versus the entire sporadic cohort, and found no significant difference. The observations indicate that TSC1 mutations are all inactivating, suggest that TSC1 disease occurs in only 15-20% of the sporadic TSC population, and demonstrate that presymptomatic TSC does occur.

Adolescent↗
PubMed1997

Detection of novel LAMC2 mutations in Herlitz junctional epidermolysis Bullosa.

BACKGROUND: Laminin 5, an anchoring filament attachment protein within the lamina lucida of the basement membrane zone involved in the pathogenesis of junctional epidermolysis bullosa (JEB), consists of three polypeptide subunits, the alpha 3, beta 3, and gamma 2 chains which are encoded by the LAMA3, LAMB3, and LAMC2 genes, respectively. To facilitate identification of pathogenetic mutations in LAMC2, a strategy based on direct amplification of genomic DNA by PCR or mRNA by RT-PCR, followed by heteroduplex analysis of the PCR products, was developed. MATERIALS AND METHODS: Primer pairs for amplification of the complete cDNA as well as the 23 individual exons in the genomic DNA, which encode the entire gamma 2 chain of laminin 5, were established. The primers for amplification of exons from genomic DNA were positioned at least 24 bp away from the intron-exon borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of the entire coding sequence of LAMC2 mRNA were used. The amplified sequences were scanned for pathogenetic mutations and sequence variations in JEB patients and unrelated control individuals by heteroduplex analysis by means of conformation sensitive gel electrophoresis (CSGE). RESULTS: Utilizing the strategy developed in this study, we identified pathogenetic mutations in three patients with the Herlitz (lethal) variant of JEB, and eight intragenic normal polymorphisms, which are useful for linkage analysis, in the LAMC2 gene. CONCLUSIONS: The methodology described in this study is capable of detecting single-base substitutions or small insertions and deletions in the LAMC2 gene. Demonstration of mutations in this gene in JEB patients further emphasizes the role of laminin 5 in providing integrity to the cutaneous basement membrane zone.

Epidermolysis Bullosa, Junctional↗
PubMed1977

Characterization of hybrid plasmids carrying individual ribosomal ribonucleic acid transcription units of Escherichia coli.

We have screened the strains with ColE1 hybrid plasmids constructed by Clarke and Carbon (Cell 9:91-99, 1976) for the presence of ribosomal ribonucleic acid (rRNA) genes on the plasmids and identified 16 strains whose plasmids carry rRNA genes. The structures of these 16 plasmids were compared by heteroduplex analysis, and the plasmids were classified into six groups on the basis of their chromosomal origins. Homology with known transducing-phage deoxyribonucleic acids and genetic mapping have assigned locations on the Escherichia coli chromosome to three of the six groups. These are rrnB near rif at 88 min, rrnC near ilvE at 83 min, and rrnD near aroE at 71 min. A fourth group is probably rrnA at 85 min (T. Ikemura and M. Nomura, Cell, 11:779-793, 1977). We conclude that the minimum number of rRNA transcription units per haploid chromosomes is seven, that is, the six groups identified in this work plus a known operon (rrnE near metA at 89 min) that we failed to find among the hybrid plasmids. This heteroduplex analysis also suggests that there are only two kinds of rRNA operons with respect to their spacer region; three of the six rRNA operon groups studied here have one kind, whereas the remaining three have the other kind.

Bacterial Proteins↗
PubMed2000

Mutations at the ataxia-telangiectasia locus and clinical phenotypes of A-T patients.

Mutations at the ataxia-telangiectasia (A-T) locus on chromosome band 11q22 cause a distinctive autosomal recessive syndrome in homozygotes and predispose heterozygotes to cancer, ischemic heart disease, and early mortality. PCR amplification from genomic DNA and automated sequencing of the entire coding region (66 exons) and splice junctions detected 77 mutations (85%) in 90 A-T chromosomes. Heteroduplex analysis detected another 42 mutations at the A-T locus. Out of a total of 71 unique mutations, 50 were found only in a single family, and 51 had not been reported previously. Most (58/71, 82%) mutations were frameshift and nonsense mutations that are predicted to cause truncation of the A-T protein; the less common mutation types were missense (9/71, 13%), splicing (3/71, 4%) and one in-frame deletion, 2546 3 (1/71, 1%). The mean survival and height distribution of 134 A-T patients correlated significantly with the specific mutations present in the patients. Patients homozygous for a single truncating mutation, typically near the N-terminal end of the gene, or heterozygous for the in-frame deletion 2546 3, were shorter and had significantly shorter survival than those heterozygous for a splice site or missense mutation, or heterozygous for two truncating mutations. Alterations of the length or amino acid composition of the A-T gene product affect the A-T clinical phenotype in different ways. Mutation analysis at the A-T locus may help estimate the prognosis of A-T patients.

Adolescent↗
PubMed2005

Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.

We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.

Animals↗
PubMed1979

Evoluation of Drosophila mitochondrial DNAs. Analysis of heteroduplex molecules.

We have mapped the single block of non-homologous sequences and measured the extent and distribution of base-pair substitutions within the homologous sequences in Drosophila melanogaster: Drosophila virilis heteroduplex mitochondrial DNAs (mtDNAs). Of the 4.8 kilobases long, unusually (A + T)-rich region in D. melanogaster mtDNA, only 0.5 kilobases can react with related, but not identical sequences in D. virilis mtDNA, while the rest (4.3 kilobases in the long arm of a heteroduplex loop) is replaced by a shorter, non-homologous region (1.0 kilobases in the short arm of the loop). No additional heterologous regions are evident. Homologous sequences have accumulated on the average 15.5% base-pair changes. Regionally, these substitutions are relatively uniformly distributed (14.5--16.5%) except for a single, more conserved region (10--13%), which presumably represents the ribosomal cistrons. The lack of general sequence stability suggests that the invariant topographic organization of the nucleotide sequence, previously recognized among Drosophila mtDNAs, is under more stringent selection than the sequence per se.

Animals↗
PubMed2003

Rapid detection methods for five HGO gene mutations causing alkaptonuria.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO) activity. The disease is characterized by homogentisic aciduria, ochronosis and ochronotic arthritis. AKU shows a very low prevalence (1:250 000), in most ethnic groups. Altogether 43 HGO mutations have been identified in approximately 100 patients. In Slovakia, however, the incidence of this disorder rises up to 1:19 000, and 10 different AKU mutations have been identified in this relatively small country. Here, we report detection methods developed for rapid identification of five HGO mutations. PCR primers were designed enabling detection of mutations IVS5 + 1G-->A, R58fs, and V300G by restriction digestion of amplification-created restriction sites (ACRS). Mutation G152fs is readily identified by heteroduplex analysis, and G161R by amplification refractory mutation system (ARMS) PCR.

Alkaptonuria↗
PubMed1979

Genome of avian myelocytomatosis virus MC29: analysis by heteroduplex mapping.

The virion RNA of avian myelocytoma virus MC29 was hybridized to full genome length DNA of the Prague strain of Rous sarcoma virus and analyzed by heteroduplex mapping in the electron microscopy. The results show that MC29 specific sequences for which there are no homologous counterparts in the Rous sarcoma virus genome make up a contiguous stretch of RNA about 1.8 kilobases long. These sequences are located approximately in the middle of the genome, replacing the 3' half of the gag gene, the entire pol gene, and the 5' portion of the env gene, which are absent from MC29. This MC29 specific genetic substitution may contain information for the leukemogenic transformation of the host cell.

Avian Leukosis Virus↗
PubMed1999

Molecular genetic study of autosomal dominant retinitis pigmentosa in Lithuanian patients.

Lithuanian patients with visual problems were clinically examined for retinitis pigmentosa (RP). A total of 33 unrelated families with autosomal dominant RP (adRP) were identified. Screening for mutations in the rhodopsin (RHO) and peripherin/RDS (RDS) genes was performed using DNA heteroduplex analysis. Direct DNA sequencing in the cases of heteroduplex formation showed the presence of the following mutations and polymorphisms in 14 adRP patients: RHO gene - Lys248Arg (1 case), and Pro347Leu (2 cases); RDS gene - Glu304Gln (12 cases), Lys310Arg (5 cases), and Gly338Asp (12 cases). The presence of these mutations (except Lys248Arg in the RHO gene) was confirmed by relevant restriction enzyme digestion. The frequency of the RDS gene mutations Glu304Gln and Gly338Asp was estimated to be 36.4%, while mutation Lys310Arg was less frequent (15.2%). These 3 RDS gene mutations appear to be polypeptide polymorphisms not related to adRP.

Exons↗
PubMed2001

Comprehensive mutation screening in a cystic fibrosis center.

OBJECTIVES AND BACKGROUND: The identities of a cystic fibrosis (CF) patient's CFTR mutations can influence therapeutic strategies, but because >800 CFTR mutations exist, cost-effective, comprehensive screening requires a multistage approach. Single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) can be an important part of mutation detection, but must be calibrated within each laboratory. The sensitivity of a combined commercial-SSCP/HA approach to genotyping in a large, ethnically diverse US center CF population has not been established. STUDY DESIGN: We screened all 27 CFTR exons in 10 human participants who had an unequivocal CF diagnosis including a positive sweat chloride test and at least 1 unknown allele after commercial testing for the 70 most common mutations by SSCP/HA. These participants were compared with 7 participants who had negative sweat tests but at least 1 other CF-like symptom meriting complete genotyping. RESULTS: For the 10 CF participants, we detected 11 of 16 unknown alleles (69%) and all 4 of the known alleles (100%), for an overall rate of 75% inpatients not fully genotyped by conventional 70 mutation screen. For 7 participants with negative sweat tests, we confirmed 1 identified mutation in 14 alleles and detected 3 additional mutations. Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, DeltaF508, 451-458Delta8 bp, 5T, 663DeltaT, exon 13 frameshift, 1261+1G-->A and 3272-26A-->G). Three of these mutations were novel (G970D, L1093P, and 451-458Delta8 bp(1)). Thirteen other changes were detected, including the novel changes 1812-3 ins T, 4096-278 ins T, 4096-265 ins TG, and 4096-180 T-->G. CONCLUSION: When combined with the 70 mutation Genzyme test, SSCP/HA analysis allows for detection of >95% of the mutations in an ethnically heterogeneous CF center population. We discuss 5 possible explanations that could account for the few remaining undetected mutations.

Cystic Fibrosis↗
PubMed1995

Rapid genotype analysis in type 2B von Willebrand's disease using a universal heteroduplex generator.

A new diagnostic technique based on DNA heteroduplex analysis has been used to identify specific point mutations in the von Willebrand's factor (vWF) gene of patients with von Willebrand's disease type 2B. Molecular analysis in these patients has shown previously that their mutations are clustered in a short region of sequence in exon 28 of the vWF gene. The principle of the method involves heteroduplex formation between amplified genomic sequence containing the defect and an exon 28 vWF gene universal heteroduplex generator (UHG). The UHG is a synthetic vWF gene exon 28 homologue which contains a number of sequence mismatches designed to generate allele specific heteroduplexes for each type 2B mutation. Individual mutant genotypes are identified by characteristic banding patterns following polyacrylamide minigel electrophoresis. The technique is rapid, simple, inexpensive, and is ideally suited for adoption by non-specialist haematology laboratories for screening purposes.

Base Sequence↗
PubMed2000

Detection of sequence variability of the collagen type IIalpha 1 3' variable number of tandem repeat.

The variable number of tandem repeat (VNTR) 3' of the collagen type II (COL2A1) gene has been shown to be highly variable with a complex molecular structure. In a previous pilot experiment we observed discordance between methods to genotype this informative marker. To further investigate the extent and molecular nature of this discordance, we genotyped a random sample of 207 Caucasian individuals with two genotyping methods and sequenced new alleles. We compared single-strand (SS) analysis, which is based on detection of size differences between the different alleles, and heteroduplex analysis (HA), which is sensitive to both size and sequence differences. Overall, 26% of discordance between the two methods was detected. Approximately two thirds of this discordance was caused by subdivision of SS-alleles 13R1 and 14R2 into HA-alleles 4A + 4B and 3B + 3C, respectively. Sequence analysis of the COL2A1 VNTR alleles 4B and 3C showed that these alleles differed in sequence, but not in size, from already described SS-alleles, which explains why they escape detection by SS. The 4B allele is a frequent allele in the population (14%) and is, therefore, important to distinguish in association studies. We conclude that HA is a reliable method when the described optimized electrophoretic conditions are used. HA is a sensitive genotyping method to document allelic diversity at this locus, which can distinguish more alleles compared to the SS method.

Aged↗
PubMed2001

Increased sensitivity of melanocytes to oxidative stress and abnormal expression of tyrosinase-related protein in vitiligo.

BACKGROUND: Vitiligo is a depigmenting disease of the skin, which may derive from programmed melanocyte death or destruction due to inherent sensitivity to oxidative stress arising from either toxic intermediates of melanin, a melanocyte-specific protein, or other sources. Tyrosinase-related protein (TRP) -1 has been shown to be involved not only in melanin biosynthesis but also in the prevention of premature melanocyte death in animals. OBJECTIVES: To clarify the biological role of human TRP-1 in melanocyte survival. METHODS: Cultured melanocyte strains from an active advancing border of vitiligo were established and studied. RESULTS: The established 'vitiligo melanocytes' showed large perikaryon and stubby dendrites. They showed early cell death when exposed to oxidative stress (ultraviolet B) and increased and abnormal immunostaining and immunoprecipitation by antibodies against human and mouse TRP-1, indicating an altered synthesis and processing of TRP-1. In pulse-chase and sequential immunoprecipitation experiments, vitiligo melanocytes revealed abnormal protein-protein interaction with calnexin, a melanogenesis-associated chaperone, suggesting altered folding and maturation of nascent TRP-1 polypeptides. Northern blot analysis indicated a decreased expression of TRP-1 mRNA, but heteroduplex analysis and verification of the mutation at the carboxy terminus of TRP-1 by restriction enzyme analysis did not show any abnormality. CONCLUSIONS: Our study suggests that the early cell death of vitiligo melanocytes is related to their increased sensitivity to oxidative stress, which may arise from complex processes of abnormal synthesis and processing of TRP-1 and its interaction with calnexin.

Adult↗
PubMed2000

A de novo glycine substitution mutation in the collagenous domain of COL7A1 in dominant dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a hereditary mechanobullous disorder characterized by fragility of the skin and mucous membrane due to abnormalities of anchoring fibrils. Both dominant and recessive DEB have been shown to be caused by mutations in COL7A1, the gene encoding type VII collagen which is the major component of anchoring fibrils. De novo mutation in dominant DEB is rare. In this study, we report a novel de novo glycine substitution mutation in COL7A1 in a Chinese female patient presenting with mild DEB. In search of the mutation, we scanned the entire COL7A1 using polymerase chain reaction (PCR) amplification of all exons of COL7A1, followed by heteroduplex analysis and direct sequencing of the PCR products that exhibited heteroduplex pattern. A G-to-A transition at nucleotide position 6082 within exon 73 of COL7Al was detected. The mutation converted a glycine to an arginine (G2028R) within the triple-helical domain of type VII collagen. It was confirmed that the mutation was present only in the proband. Haplotype analyses suggested that the case arose as a de novo occurrence of autosomal dominant DEB.

Alleles↗
PubMed1999

A novel truncating mutation of cytochrome P4501B1 (CYP1B1) gene in primary infantile glaucoma.

PURPOSE: To report a novel mutation of the CYP1B1 gene in a Japanese patient with primary infantile glaucoma. METHODS: DNA was extracted from leukocytes of six unrelated patients with primary infantile glaucoma. The coding regions of the CYP1B1 gene were amplified by polymerase chain reaction, examined by agarose gel separation and heteroduplex methods, and directly sequenced. RESULTS: One of the patients with primary infantile glaucoma had a mutation of the CYP1B1 gene, with an abnormal shift in agarose gel separation and heteroduplex analysis. Direct sequencing disclosed a homozygous insertion of guanine at nucleotide 1620 of exon 3, which produced a frameshift leading to premature termination of amino acid translation. The patient was male and had sporadic, classic primary infantile glaucoma. None of the other five patients with infantile glaucoma, the 30 patients with primary adult-onset glaucoma, or the 70 healthy control subjects showed any abnormalities in the CYP1B1 gene. CONCLUSIONS: This is the first report of CYP1B1 gene mutation in primary infantile glaucoma from the Eastern world. CYP1B1 gene mutation for primary infantile glaucoma spreads worldwide, but its prevalence may have ethnic or geographic differences.

Aryl Hydrocarbon Hydroxylases↗
PubMed2000

A novel deletion mutation within the carboxyl terminus of the copper-transporting ATPase gene causes Wilson disease.

In patients with Wilson disease (WD), an autosomal recessive disorder, toxic accumulation of copper results in fatal liver disease and irreversible neuronal degeneration. ATP7B, the gene mutated in WD, contains 21 exons and encodes a copper-transporting ATPase. In this study, all exons of the ATP7B gene of nine WD patients were screened for alterations by conventional mutation detection enhancement (MDE) heteroduplex analysis, followed by direct sequencing of the regions that showed heteroduplex formation. For the first time, a novel deletion mutation (4193delC) in exon 21, causing a frameshift leading to premature truncation of the protein was detected in four of nine patients. The 4193delC removes several signals within the carboxyl terminal domain that may disrupt trafficking of ATP7B protein through trans-Golgi network at the cellular level.

Adenosine Triphosphatases↗