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At least 397 records · Page 22Linked to original sources
PubMed2006

Mutation screening of BMP4, BMP7, HOXA4 and HOXB6 genes in Chinese patients with hypospadias.

Hypospadias, one of the most common congenital abnormalities of the male external genitalia with elusive etiology, are caused by a defect in the normal development of the urethra, foreskin and ventral aspect of the penis. Evidences indicate that BMP4 and BMP7, two of those major factors in a signaling cascade involved in controlling the embryonic urethral development, play central roles in the normal development of the urethra, and that HOXA4 and HOXB6 play important roles in the development of skin in various tissues at the time course of the urethral development. We directly sequenced all these exons and exon-intron boundaries of the four genes in 90 unrelated Chinese patients with hypospadias. Thirteen different heterozygous nucleotide variations were identified for the first time in the four genes in 14 of 90 cases. Of the 13 variations, eight are missense: c.619C>G (p.H207D), c.668G>A (p.R223H), c.751C>T (p.H251Y) in BMP4; c.907C>T (p.R303C) in BMP7; c.385G>T (p.G129C), c.869C>G (p.S290C) in HOXA4; c.124C>A (p.P42T), c.367T>C (p.C123R) in HOXB6. None of these variations were found in 380 control chromosomes. Amino-acid sequence alignments showed most of these changed amino acids are conserved across various vertebrate species. In a word, these findings, together with the indicated roles of the four genes, imply that it should not be random events for so many nucleotide variations found in the present study. Further functional studies are required to make the associations clear between these variants and hypospadias.

Amino Acid Sequence↗
PubMed2001

Mutation screening of the muscarinic M(2) and M(3) receptor genes in normal and asthmatic subjects.

1. Muscarinic receptors are important in the development of airway hyperresponsiveness, and dysfunction of these receptors has been suggested to be present in asthma. 2. The human muscarinic M(2) and M(3) receptor genes were screened for polymorphic variation using single-stranded conformation polymorphism (SSCP) analysis, complemented by direct fluorescent sequencing. Forty-six random DNA samples and 46 respiratory physician diagnosed asthmatic samples were used as a template for analysis. 3. Within the muscarinic M(2) receptor gene, we identified two degenerate single base substitutions (1197T-->C, Thr-->Thr and 976A-->C, Arg-->Arg) in one random and one asthmatic sample respectively. Analysis of the 3' UTR region revealed an additional 'A' at bp 1793 (c.f. ATG). This was present in all of 49 samples analysed by sequencing or BsmI digest, suggesting that the published sequence (GenBank Accession NO: M16404) is incorrect. A common 3' UTR polymorphism (T-->A) was found at bp 1696 (c.f. ATG) (allelic frequency=65%, n=60), but this does not alter transcription factor recognition sites. 4. We were unable to identify any polymorphic variation within the muscarinic M(3) coding region or the flanking regions investigated, using the methods described. 5. The coding regions for the human muscarinic M(2) and M(3) receptor genes are both highly conserved. These data suggest that polymorphic variation within these coding sequences is unlikely to account for inter-individual variability in response to methacholine or anticholinergic therapy. The potential functional significance of the muscarinic M(2) receptor 3' UTR polymorphism (bp 1696) remains to be determined.

3' Untranslated Regions↗
PubMed2007

Mutation screen of the GAD2 gene and association study of alcoholism in three populations.

Synaptic actions of gamma-amino butyric acid (GABA) have been implicated in many facets of ethanol's effects and risk for alcoholism. We examined whether variation in glutamate decarboxylase-2 (GAD2), a gene encoding for a major enzyme in the synthesis of GABA, contributes to risk of alcohol dependence (AD). We screened GAD2 for sequence variants using dHPLC in a population of 96 individuals. Several single nucleotide polymorphisms (SNPs), including four rare non-synonymous polymorphisms, were identified. Thirteen SNPs located in the GAD2 gene were genotyped in a sample of 113 Russian males with AD and 100 Russian male controls. These analyses revealed a modest association between the functional GAD2 -243 A > G SNP (rs2236418) and AD (allele P = 0.038, genotype P = 0.008). An additional sample of 138 Russian males with AD were genotyped for the GAD2 -243 A > G. These analyses supported an association of this polymorphism with AD (combined sample allele P = 0.038, genotype P = 0.0009). We extended these findings to additional populations: a sample of 538 college students assessed using the AUDIT and a sample of European-American (EA) AD subjects (n = 235) and controls (n = 310). Analyses in these populations did not support a role for GAD2 in alcoholism. In summary, the results of an extensive search for an association of GAD2 with AD suggest that variation in GAD2 is not a major risk factor for AD in EAs. The functional promoter GAD2 -243 A > G variant may influence risk for AD in some populations, or its role may be limited to susceptibility to severe AD.

Adult↗
PubMed2001

Mutational screen identifies critical amino acid residues of beta-actin mediating interaction between its folding intermediates and eukaryotic cytosolic chaperonin CCT.

The three-dimensional reconstruction of apo-CCT-alpha-actin by cryoelectron microscopy shows that actin binds either the CCTbeta-CCTdelta or the CCTepsilon-CCTdelta subunit pairs of the chaperonin in an open and apparently quasi-native conformation. The CCT-binding sites are seen located at the tips of the two arms of actin and these same regions of actin have been implicated in CCT binding through beta-actin peptide-array screening. Three main CCT binding regions exist: actin Sites I, II, and III, which are composed of loops that are surface-exposed in native actin. Sixty-eight amino acid residues on beta-actin have been screened by mutagenesis for effects on CCT interaction in quantitative in vitro translation assays in rabbit reticulocyte lysate. Actin seems to be folding cooperatively on chaperonin, since certain mutants discriminate CCT binding from processing. Actin Site II, located at the tip of actin subdomain 4, is the major determinant for CCT binding. Site II is composed of two anti-parallel extended beta-strands, with F200-T203 and D244 contributing substantially to the binding site. The substrate recognition chemistry of CCT thus seems different from that of Group I chaperonins and probably reflects the fact that it needs to be highly specific to enable capture and folding of the actins and tubulins.

Actins↗
PubMed1976

Spontaneous chromosome mutation and screening of mutator factors in Drosophila melanogaster.

(1) The marked inversion technique was used, and 38 major autosome sets of Drosophila melanogaster were extracted from populations of Jugoslavia, Taiwan and Japan. Spontaneous mutations were allowed to accumulate on the major chromosomes for 25 generations. Then the second chromosomes were tested to determine whether or not they were associated with such known mutator factors as the male crossing-over factor, SD, and the extrachromosomal element delta. Chromosome mutations were examined by salivary gland chromosome analysis, and viability mutations were done by the marked autosomal translocation method. (2) In 8 out of 38 major autosome lines, 5 inversions occurred in the second chromosomes, 4 inversions in the third chromosomes, and 1 reciprocal translocation between the second and the third chromosomes. Chromosome mutation rates were, therefore, 0.0063 per second chromosome per generation and 0.0053 per third chromosome per generation. Since there was no signficant difference in the rates, chromosome mutations seem to be occurring approximately equally in both major autosomes. (3) Lethal mutation rates were estimated to be 0.0097 per major autosome per generation. (4) Twenty-four second chromosome lines out of 37 demonstrated male crossing-over among the cinnabar and brown interval; the average frequencies were 0.0031 for all lines and 0.0034 when non-recombination lines were excluded. (5) One second chromosome exhibited delta retaining ability (ID), but no second chromosome carried SD.

Animals↗
PubMed2002

Determination of the genomic structure and mutation screening in schizophrenic individuals for five subunits of the N-methyl-D-aspartate glutamate receptor.

The glutamatergic system is the major excitatory neurotransmitter system in the CNS. Glutamate receptors, and in particular N-methyl-D-aspartate (NMDA) receptors, have been proposed as mediators of many common neuropsychiatric phenotypes including cognition, psychosis, and degeneration. We have reconstructed the genomic structure of all five genes encoding NMDA receptors in silico. We screened each for sequence variation and estimated the allele frequencies of all detected SNPs in pooled samples of 184 UK Caucasian schizophrenics and 184 UK Caucasian blood donor controls. Only a single non-synonymous polymorphism was found indicating extreme selection pressure. The rarity of non-synonymous changes suggests that such variants are unlikely to make a common contribution to common phenotypes. We found a further 26 polymorphisms within exonic or adjacent intronic sequences. The minor alleles of most of these have a relatively high frequency (63% above 0.2). These SNPs will therefore be suitable for studying neuropsychiatric phenotypes that are putatively related to NMDA dysfunction. Pooled analysis provided no support for association between any of the GRIN genes and schizophrenia.

Base Sequence↗
PubMed1996

Screening for mutations in exon 11 of the BRCA1 gene in 70 Italian breast and ovarian cancer patients by protein truncation test.

The most common mutations in the familial breast and ovarian cancer susceptibility gene BRCA1 are frameshift and nonsense mutations, which lead to the synthesis of truncated proteins. On this ground, we have analysed BRCA1 exon 11, which includes about 61% of coding region, in germline DNA from 70 Italian breast and/or ovarian cancer patients, using the protein truncation test (PTT). BRCA1 mutations were identified in nine of 29 (approximately 31%) patients with a family history of cancer and in three of 41 (approximately 7%) women with early-onset breast carcinomas, and were subsequently characterized by sequence analysis. In addition, BRCA1 mutations were also detected in six affected relatives of two positive index cases. The observed frequencies of mutations were not significantly different from those expected on the basis of the phenotypic characteristics of patients and their families, indicating that PTT is a rapid and sensitive method that can be used for a first BRCA1 mutational screening. The histological findings in BRCA1 mutated cases showed that eight of nine (approximately 89%) breast carcinomas were of grade III and nine of 9 (100%) ovarian carcinomas were of the endometrioid type (eight of grade III and one of grade II). This suggests that specific histological characteristics may represent additional criteria for selection of cases eligible to BRCA1 mutational analysis.

Adult↗
PubMed2005

Screening for mutations in CYP4V2 gene in Japanese patients with Bietti's crystalline corneoretinal dystrophy.

PURPOSE: To describe the clinical and genetic characteristics of six Japanese families with Bietti's crystalline corneoretinal dystrophy (BCD). DESIGN: Case reports and results of DNA analysis. METHODS: Mutation screening was performed on six unrelated patients with BCD by direct sequencing. The clinical features were characterized by the visual acuity, slit-lamp biomicroscopy, electroretinography, fluorescein angiography, and kinetic visual field testing. RESULTS: An identical IVS6 to 8delTCATACAGGTCATCGCG/insGC mutation in the CYP4V2 gene was identified in five of the patients with BCD; the sixth patient had a novel Trp340X mutation in the CYP4V2 gene. Three patients showed crystalline-like deposits at the limbus by specular microscopy. Ophthalmic findings of all patients had a rapid progression after age 50 years. CONCLUSIONS: Our findings suggest that the IVS6 to 8delTCATACAGGTCATCGCG/insGC mutation is a common mutation in Japanese patients with BCD. Although phenotypic variability was found, the natural course was almost the same in all of our patients.

Adult↗
PubMed2001

A screen for mutations in human homologues of mice exencephaly genes Tfap2alpha and Msx2 in patients with neural tube defects.

BACKGROUND: Very little is known about the identity of genetic factors involved in the complex etiology of nonsyndromic neural tube defects (NTD). Potential susceptibility genes have emerged from the vast number of mutant mouse strains displaying NTD. Reasonable candidates are the human homologues of mice exencephaly genes Tfap2alpha and Msx2, which are expressed in the developing neural tube. METHODS: A single-strand conformation analysis (SSCA) mutation screen of the coding sequences of TFAP2alpha and MSX2 was performed for 204 nonsyndromic NTD patients including cases of anencephaly (n = 10), encephalocele (n = 8), and spina bifida aperta, SBA (n = 183). A selected number of SBA patients was additionally tested for specific mutations in MTHFD, FRalpha, and PAX1 already shown to be related to NTD. RESULTS: Two TFAP2alpha point mutations in individual SBA patients were silent on the amino acid level (C308C, T396T). On nucleic acid level, these mutations change evolutionary conserved codons and thus may influence mRNA processing and translation efficiency. One SBA patient displayed an exonic 9-bp deletion in MSX2 leading to a shortened and possibly less functional protein. None of these mutations was found in 222 controls. Seven polymorphisms detected in TFAP2alpha and MSX2 were equally distributed in patients and controls. Patients with combined heterozygosity of an exonic MSX2 and an intronic TFAP2alpha polymorphism were at a slightly increased risk of NTD (OR 1.71; 95% CI 0.57-5.39). CONCLUSIONS: Although several new genetic variants were found in TFAP2 and MSX2, no statistically significant association was found between NTD cases and the new alleles or their combinations. Further studies are necessary to finally decide if these gene variants may have acted as susceptibility factors in our individual cases.

Alleles↗
PubMed2000

Screening for mutations in the peripheral myelin genes PMP22, MPZ and Cx32 (GJB1) in Russian Charcot-Marie-Tooth neuropathy patients.

Charcot-Marie-Tooth disease (CMT) and related inherited peripheral neuropathies, including Dejerine-Sottas syndrome, congenital hypomyelination, and hereditary neuropathy with liability to pressure palsies (HNPP), are caused by mutations in three myelin genes: PMP22, MPZ and Cx32 (GJB1). The most common mutations are the 1.5 Mb CMT1A tandem duplication on chromosome 17p11.2-p12 in CMT1 patients and the reciprocal 1.5 Mb deletion in HNPP patients. We performed a mutation screening in 174 unrelated CMT patients and three HNPP families of Russian origin. The unrelated CMT patients included 108 clinically and electrophysiologically diagnosed CMT1 cases, 32 CMT2 cases, and 34 cases with unspecified CMT. Fifty-nine CMT1A duplications were found, of which 58 belonged to the CMT1 patient group. We found twelve distinct mutations in Cx32, six mutations in MPZ, and two mutations in PMP22. Of these respectively, eight, five, and two lead to a CMT1 phenotype. Eight mutations (Cx32: Ile20Asn/Gly21Ser, Met34Lys, Leu90Val, and Phe193Leu; MPZ: Asp134Gly, Lys138Asn, and Thr139Asn; PMP22: ValSer25-26del) were not reported previously. Phenotype-genotype correlations were based on nerve conduction velocity studies and mutation type.

Adult↗
PubMed1995

Screening for known mutations in the LDL receptor gene causing familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is caused by defective low density lipoprotein (LDL) receptors and is characterized by hypercholesterolemia and premature coronary heart disease. Two strategies can be used to identify the mutation in the LDL receptor gene underlying FH. One strategy is to search for novel mutations by DNA sequencing with or without prior mutation screening. The other strategy is to screen for known mutations. In this study we employed the latter strategy to screen 75 unrelated, Norwegian FH subjects for 38 known mutations. Three of the 38 mutations were detected in our group of FH subjects. Two subjects had FH-Padova, one had FH-Cincinnati-2 and one had FH-Gujerat. When additional unrelated FH heterozygotes were screened for the three mutations, the gene frequencies were 1.3%, 1.0% and 3.0%, respectively. In addition to identifying known mutations we also detected a novel stop codon in codon 541 (S541X). We conclude that screening for known mutations in the LDL receptor gene should be used as a complementary strategy to screening for novel mutations in order to understand the molecular genetics of FH.

Base Sequence↗
PubMed2004

Genomewide two-generation screens for recessive mutations by ES cell mutagenesis.

Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genome-wide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)-mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.

Animals↗
PubMed2002

Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography.

AIM: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases on DHPLC resolution, and evaluate the sensitivity of DHPLC in the mutation screening of mitochondrial DNA (mtDNA). METHODS: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243G mutated fragment were used to analyze the UV detection limit and determine the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNA polymerases on resolution of DHPLC. Under the optimal conditions, we analyzed the mtDNA mutations from muscle tissues of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and screened blindly for variances in D-loop region of mtDNA from human gastric tumor specimen. RESULTS: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results obtained by analysis of RFLP with ApaI. We also identified 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ng PCR products containing a minimum of 5% mutant sequences could be detected by using DHPLC with UV detector. Moreover, Ampli-Taq Gold polymerase was equally as good as the proofreading DNA polymerase (e.g., Pfu) in eliminating the false positive produced by Taq DNA polymerases. CONCLUSION: DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase.

Case-Control Studies↗
PubMedDate not supplied

Genomic semi-automated cycle sequencing as a sensitive screening technique for p53 mutations in frozen tumor samples.

Single-strand conformational polymorphism (SSCP) is the most widely used method for p53 gene mutation screening. Nucleotide sequence analysis is considered more sensitive for detection of mutations. We established a genomic semi-automated cycle sequencing protocol suitable for p53 gene mutation screening. The technique was applied to 44 SSCP-negative frozen ovarian cancer samples: Eleven mutations (11/44, 25%) were found, 6 point missense mutations, 3 silent point mutations, 1 nonsense mutation and 1 single-base deletion. Heterozygous mutations were readily detectable. Genomic semi-automated cycle sequencing is a sensitive, time-effective screening method requiring only small amounts of tumor tissue.

Amino Acid Substitution↗