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At least 235 records · Page 13Linked to original sources
PubMed2006

[Mutation screening and prenatal diagnosis of hidrotic ectodermal dysplasia in a Chinese family].

OBJECTIVE: To analyze the mutations in Cx30 gene in a Chinese family with hidrotic ectodermal dysplasia (HED) and to make prenatal diagnosis on the embryo which has been pregnant for 5 months. METHODS: A family including 2 affected and 4 unaffected individuals was collected, and their informed consents were obtained. The affected woman had a five-month pregnancy. An 884 bp fragment containing the whole GJB6 coding sequence was amplified by PCR and the products were bi-direction sequenced directly. The mutation was further confirmed with restriction endoenzyme digesting. On the base of successful gene diagnosis, the following detection procedure on the pregnant baby was performed. First the whole coding region of Cx30 was amplified using primers Cx30-F and Cx30-R and the PCR products were digested by Hae II. Then the PCR products were cloned into pUCm-T vector. Blue-white blot screening method and PCR-restriction endoenzyme digesting technique were used to identify the correct clones. The mutant allele clone was sequenced to confirmed the mutation. RESULTS: A heterozygous missense mutation 263C --> T in the Cx30 gene was detected in the affected little girl and her affected mother, which led to an amino acid substitution (A88V) in the second transmembrane domain of GJB6. The mutation was confirmed by Hae II digestion. A88V mutant allele cannot be cut while the wild normal allele can be cut into two fragments, 520 and 278 bp. The result of analyse on the five-month pregnancy show the embryo carried the A88V mutation too. So the embryo will be a patient. CONCLUSION: An A88V missense mutation in the Cx30 gene can also cause HED in Chinese Han population. Based on the gene diagnosis, prenatal diagnosis can be played using bi-direction sequencing and confirmed with restriction endoenzyme digesting.

Adult↗
PubMed2005

Mutation screening of the TP53 gene by temporal temperature gradient gel electrophoresis.

A protocol for detection of mutations in the TP53 gene using temporal temperature gradient gel electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) and eliminates some of the problems. The result is a rapid and sensitive screening technique that is robust and easily set up in smaller laboratory environments.

DNA↗
PubMed1996

Myosin VIIA mutation screening in 189 Usher syndrome type 1 patients.

Usher syndrome type 1b (USH1B) is an autosomal recessive disorder characterized by congenital profound hearing loss, vestibular abnormalities, and retinitis pigmentosa. The disorder has recently been shown to be caused by mutations in the myosin VIIa gene (MYO7A) located on 11q14. In the current study, a panel of 189 genetically independent Usher I cases were screened for the presence of mutations in the N-terminal coding portion of the motor domain of MYO7A by heteroduplex analysis of 14 exons. Twenty-three mutations were found segregating with the disease in 20 families. Of the 23 mutations, 13 were unique, and 2 of the 13 unique mutations (Arg212His and Arg212Cys) accounted for the greatest percentage of observed mutant alleles (8/23, 31%). Six of the 13 mutations caused premature stop codons, 6 caused changes in the amino acid sequence of the myosin VIIa protein, and 1 resulted in a splicing defect. Three patients were homozygotes or compound heterozygotes for mutant alleles; these three cases were Tyr333Stop/Tyr333Stop, Arg212His-Arg302His/Arg212His-Arg302His, and IVS13nt-8c-->g/Glu450Gln. All the other USH1B mutations observed were simple heterozygotes, and it is presumed that the mutation on the other allele is present in the unscreened regions of the gene. None of the mutations reported here were observed in 96 unrelated control samples, although several polymorphisms were detected. These results add three patients to single case reported previously where mutations have been found in both alleles and raises the total number of unique mutations in MYO7A to 16.

Alleles↗
PubMed1993

Mutational screening of the Wilms's tumour gene, WT1, in males with genital abnormalities.

Several lines of evidence suggest that the Wilms's tumour susceptibility gene, WT1, has an important role in genital as well as kidney development. WT1 is expressed in developing kidney and genital tissues. Furthermore, mutations in WT1 have been detected in patients with the Denys-Drash syndrome (DDS), which is characterised by nephropathy, genital abnormalities, and Wilms's tumour. It is possible that WT1 mutations may cause genital abnormalities in the absence of kidney dysfunction. We tested this hypothesis by screening the WT1 gene for mutation in 12 46,XY patients with various forms of genital abnormality. Using single strand conformation polymorphism (SSCP) we did not detect any WT1 mutations in these patients. However, in addition to the 12 patients, three DDS patients were also analysed using SSCP, and in all three cases heterozygous WT1 mutations were found which would be predicted to disrupt the DNA binding activity of WT1 protein. These results support the notion that DDS results from a dominant WT1 mutation. However, WT1 mutations are unlikely to be a common cause of male genital abnormalities when these are not associated with kidney abnormalities.

Base Sequence↗
PubMed2003

Mutation screening and association analysis of the parkin gene in Parkinson's disease patients from South-West China.

Various deletions and point mutations in the Parkin gene have been strongly associated with Parkinson's disease (PD) and parkinsonism, especially when the onset occurs at a young age. In this study, we screened 25 "early-onset" (<49 years at onset) and 91 later-onset PD patients from a Han Chinese population from South-West China for deletions and mutations in the Parkin gene. We found no deletions or point mutations in exons 1-12 of the Parkin gene using direct sequence analysis and only detected the common Ser167Asn polymorphism. We analysed Ser167Asn in 116 patients with sporadic PD and 124 controls, matched for age and gender. There were significant differences in allele and genotype frequency between PD patients, with the 167Asn allele more common in cases than controls (46.6 vs. 35.1%; chi(2) = 6.54, p = 0.011, odds ratio = 1.61, 95% confidence interval, CI, 1.10-2.37), as was the 167Asn genotype (17.3 vs. 11.3%; p = 0.04). The frequency of the 167Ser genotype was significantly lower in PD patients than in controls when compared with that of the other two genotypes combined (chi(2) = 7.84, p = 0.005, odds ratio = 0.46, 95% CI 0.25 - 0.82). No significant differences in the frequencies of the allele and genotypes were found between patients classified into two groups according to symptoms at onset (chi(2) = 0.191, p = 0.66, odds ratio = 1.12, 95% CI 0.65-1.95; chi(2) = 0.24, p = 0.887) or age of onset (p = 0.787). In summary, homozygous deletion mutations and point mutations in exons 1-12 of the Parkin gene were not detected in this Han Chinese population, although we cannot exclude compound heterozygous deletions. In addition, our study suggests that the variant 167Asn increases the risk of developing PD.

Adult↗
PubMed1994

Mutation screening by a combination of biotin-SSCP and direct sequencing.

We have developed a mutation detection strategy that combines single strand conformational polymorphism (SSCP) analysis of one strand of a double-stranded amplification product with direct sequencing of the other. Using this strategy, which we find economical of both time and resources, we have identified a G to A transition, which substitutes a serine for glycine residue at position 862 in the major helix of the alpha 1 chain of Type I collagen. We use this mutation, which causes a lethal form of osteogenesis imperfecta, to illustrate the technique.

Amino Acid Sequence↗
PubMed1996

Mutation screening of MSH2 and MLH1 mRNA in hereditary non-polyposis colon cancer syndrome.

Germline mutations in four human mismatch repair genes (MSH2, MLH1, PMS1, and PMS2) have been reported to cause hereditary non-polyposis colon cancer syndrome (HNPCC). The identification of germline mutations in HNPCC kindreds allows precise diagnosis and accurate predictive testing. To investigate further the genetic epidemiology of HNPCC and the nature and frequency of germline mutations in this disorder, we studied 17 English HNPCC kindreds for germline mutations in MSH2 and MLH1. A previous genetic linkage study had suggested that most English HNPCC families will have mutations in one of these genes. Mutation analysis was performed in a three step process. (1) mRNA extracted from lymphoblastoid cell lines was analysed for gross rearrangements, (2) the in vitro transcription-translation (IVTT) assay was then performed to detect protein truncating mutations, and (3) partial cDNA sequencing of MSH2 or MLH1 was undertaken in families (n = 6) linked to MSH2 or MLH1 but without a detectable mutation. Seven different germline mutations were identified in eight of 17 (47%) kindreds (five in MSH2 and three in MLH1). In three cases there was a deletion of a single exon in MSH2 mRNA, three mutations resulted in a truncated protein product, and two missense mutations were identified by direct sequencing. Six mutations were novel. No precise correlation between genotype and phenotype was observed, although a MSH2 missense (Thr905Arg) mutation was associated with a susceptibility to multiple colorectal polyps. Age related risks for colorectal and uterine cancer were similar for MSH2 and MLH1 mutations.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli.

Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.

Base Sequence↗
PubMed2002

Mutation screen of the GABA(A) receptor gamma 2 subunit gene in Chinese patients with childhood absence epilepsy.

Childhood absence epilepsy (CAE) is considered to be a genetic disease, the genes responsible for which have not yet been identified. To investigate whether or not GABA(A) receptor gamma 2 subunit gene (GABRG2) is the susceptibility gene for CAE in the Chinese population, we screened 68 CAE patients of Han ethnicity from North China for mutations in the nine exons of GABRG2. Although we found no mutation in the exons of GABRG2, we did identify two single nucleotide polymorphisms (SNPs) in exon 3 and exon 5. Using the two SNPs as markers, we carried out a transmission/disequilibrium test (TDT) in 68 trios with CAE. TDT results showed that there were no significant discrepancies between the CAE patients and 'internal controls' in allele frequencies of the two SNPs. We postulate that the GABRG2 gene might be neither a susceptibility gene for CAE nor in linkage disequilibrium with disease-predisposing sites in the Chinese population.

Adolescent↗
PubMed2002

Mutation screening and LD mapping in the VCFS deleted region of chromosome 22q11 in schizophrenia using a novel DNA pooling approach.

We examined whether variation within six genes from the VCFS critical region at 22q11 (DGSC, Stk22A1, DGSI, Gscl, Slc25A1 and Znf74) confers susceptibility to schizophrenia. We screened the exons and flanking intronic sequence of each gene for mutations in 14 individuals with DSM-IV schizophrenia using DHPLC. All polymorphisms identified were characterised and genotyped in a sample of 184 schizophrenics and matched controls, using novel DNA pooling methods. Of the polymorphisms identified, 17 were located within exons, six were within coding sequence, and two were non-synonymous. Pooled genotyping revealed no differences in the allele frequencies for any polymorphism between cases and controls that met our pre-defined criterion (P < or = 0.1). In a complementary approach we also attempted to define the location of a schizophrenia susceptibility locus more precisely by performing association mapping using seven microsatellites spanning the VCFS region with an average inter-marker distance of 450 kb. Conventional chi(2) analysis of genotypes in 368 cases and 368 controls revealed that none of the markers was significantly associated (P < 0.05) with schizophrenia. However, evidence for significant association (P = 0.003) was obtained for D22S944 when alleles were combined. TDT analysis of D22S944 genotyped in a further 278 cases of schizophrenia and their parents failed to find any overall allele-wise significant transmission disequilibrium (chi(2) = 18.3, P = 0.17). However, individual analysis of the alleles revealed that allele 12 was excessively non-transmitted and that this almost reached significance when corrected for multiple alleles (chi(2) = 7.35, P = 0.006, P = 0.078 corrected for 13 alleles).

Chromosome Deletion↗
PubMed2002

Mutation screening of the fibrillin-1 (FBN1) gene in 76 unrelated patients with Marfan syndrome or Marfanoid features leads to the identification of 11 novel and three previously reported mutations.

Mutations in the gene encoding fibrillin-1 (FBN1) cause Marfan syndrome (MFS) and other related connective tissue disorders. In this study we performed SSCP to analyze all 65 exons of the FBN1 gene in 76 patients presenting with classical MFS or related phenotypes. We report 7 missense mutations, 3 splice site alterations, one indel mutation, one nonsense mutation and two mutations causing frameshifts: a 16bp deletion and a single nucleotide insertion. 5 of the missense mutations (Y1101C, C1806Y, T1908I, G1919D, C2251R) occur in calcium-binding Epidermal Growth Factor-like (EGFcb) domains of exons 26, 43, 46 and 55, respectively. One missense mutation (V449I) substitutes a valine residue in the non-calcium-binding epidermal growth factor like domain (EGFncb) of exon 11. One missense mutation (G880S) affects the "hybrid" motif in exon 21 by replacing glycine to serine. The 3 splice site mutations detected are: IVS1-1G>A in intron 1, IVS38-1G>A in intron 38 and IVS46+5G>A in intron 46. C628delinsK was identified in exon 15 leading to the substitution of a conserved cysteine residue. Furthermore two frameshift mutations were found in exon 15 (1904-1919del ) and exon 63 (8025insC) leading to premature termination codons (PTCs) in exon 17 and 64 respectively. Finally we identified a nonsense mutation (R429X) located in the proline rich domain in exon 10 of the FBN1 gene. Y1101C, IVS46+5G>A and R429X have been reported before.

Adolescent↗
PubMed2006

Mutational screening of FOXO3A and FOXO1A in women with premature ovarian failure.

FOXO3A and FOXO1A are excellent candidate genes for the development of premature ovarian failure and have not been analyzed previously in POF patients. Potentially causal mutations in FOXO3A (2/90; 2.2%) and FOXO1A (1/90; 1.1%) were identified in POF patients; however, the pathological role of these mutations will be determined only by screening increased numbers of patients and controls, or by functional studies.

DNA Mutational Analysis↗
PubMed2001

Mutation screening of the Wolfram syndrome gene in psychiatric patients.

Wolfram syndrome, a rare autosomal recessive neurodegenerative disorder, was originally described as a combination of familial juvenile-onset diabetes mellitus and optic atrophy. It was later demonstrated that Wolfram syndrome patients were highly prone to psychiatric disorders. Mutations in exon 8 of the Wolfram syndrome gene account for 88% of the patients with Wolfram syndrome. To examine whether the gene responsible for causing Wolfram syndrome is involved in psychiatric disorders, we screened exon 8 of the Wolfram syndrome gene for mutations in 119 patients with schizophrenia, one patient with schizoaffective disorder, 12 patients with bipolar disorder and 15 patients with major depression, using sequence analysis. In Wolfram syndrome patients, this gene has been shown to have primarily nonsense or frameshift mutations, which would result in a premature truncation of the protein. None of the psychiatric patients screened in this study carried these types of mutations. We identified, however, 24 new variations whose significance remains to be determined.

Bipolar Disorder↗
PubMed2005

OPA3 mutation screening in patients with unexplained 3-methylglutaconic aciduria.

We have screened 13 patients with neurological abnormalities and 3-methylglutaconic aciduria (3MGA) for mutations in the OPA3 gene, which are known to be the cause of Costeff syndrome (optic atrophy, chorea and spasticity; type III 3MGA). We aimed to explore whether mutations in the OPA3 gene are present in patients with 3MGA but without classic Costeff syndrome. OPA3 mutations (IVS1-1G>C) were identified in 2 patients with the classic phenotype of type III 3MGA, but not in the other 11 patients with differing non-Costeff phenotypes associated with developmental delay and neurological signs and symptoms as described. We identified a previously described sequence variation in the OPA3 gene (c.231T>C) in 12/13 patients. The alteration was homozygous in 8/12 and heterozygous in 4/12. This alteration was also found in 60 of 98 normal control alleles. In a single patient, a novel sequence variation in the 5' UTR was identified, (c.-38A>G). Our studies suggest that the c.231T>C sequence variation is of no clinical significance, whereas the significance of the 5' UTR sequence variation remains open to speculation. Our study of the OPA3 gene in patients with 3MGA without Costeff syndrome suggests that mutations in OPA3 are not a common cause of 3MGA in the absence of signs of Costeff syndrome.

5' Untranslated Regions↗
PubMed1999

Reduction in hMSH2 mRNA levels by premature translation termination: implications for mutation screening in hereditary nonpolyposis colorectal cancer.

We have used single-strand conformational polymorphism (SSCP) and heteroduplex analysis to examine DNA from 50 colorectal carcinoma patients coming from families meeting the Amsterdam criteria for hereditary nonpolyposis colorectal cancer (HNPCC) or having developed colorectal carcinoma at age 45 or younger. We identified mutations in 12 of these patients, with seven of these being novel mutations. We examined four of the truncating mutations using in vitro transcription and translation (IVTT) assays and determined that the mutation causing an in-frame deletion of exon 5 could easily be detected by the IVTT assay, but the three mutations resulting in premature translation termination were not detected because the steady-state levels of the mutant allele transcripts are too low. Our findings suggest that some but not all mutant hMSH2 alleles have significantly lower steady-state mRNA levels than the normal allele. Under ideal circumstances, where lymphoblastoid cell lines are available for RNA extraction, IVTT may be useful for detecting truncating mutations. However, our data suggest that caution should be taken in using IVTT in routine screening of clinical samples for truncating HNPCC mutations, as many mutations may go undetected.

Colorectal Neoplasms, Hereditary Nonpolyposis↗
PubMed2001

MECP2 mutation screening in Swedish classical Rett syndrome females.

Rett syndrome (RS) is a neurodevelopmental disorder almost exclusively affecting females. We have studied the mutation spectrum of the responsible gene MECP2, encoding methyl-CpG-binding protein 2 (MeCP2), in 16 sporadic classical RS females from Sweden. In 13 of 16 patients (81%) we detected nonsense or missense mutations in the coding parts of MECP2. This mutation rate is in agreement with other reports (65-80%). In all, 12 different mutations and one polymorphism were found; three of the mutations have not been reported previously. The missense mutations were restricted to highly conserved regions of the gene. None of the mutations was detected in parents; thus, they had probably arisen de novo. In contrast, two normal variants, one intron deletion and one silent mutation, were seen singly only in two patients' mothers; neither has been reported previously. One patient showed two different mutations closely located, i.e. 802C > T (R268W) together with 808C > T (R270X). Another patient had a mutation in the stop codon 1459T > C (X487R), leading to a gene product prolonged with 27 amino acids. In conclusion, our results indicate that the majority of Swedish RS patients (81%) have mutations in MECP2 that are sporadic cases with de novo mutations. Moreover, both missense and nonsense mutations occur, but in different parts of the gene, probably reflecting the function of the domains in MeCP2. This study has improved our ability to offer these families an early confirmation of Rett diagnoses.

Child↗
PubMed2003

A novel semiquantitative polymerase chain reaction/enzyme digestion-based method for detection of large scale deletions/conversions of the CYP21 gene and mutation screening in Turkish families with 21-hydroxylase deficiency.

21-Hydroxylase deficiency is a recessively inherited disorder resulting from mutations in the CYP21 gene. The CYP21 gene is located along with the CYP21P pseudogene in the human leukocyte antigen major histocompatibility complex region on chromosome 6. Molecular diagnosis is difficult due to the 98% similarity of CYP21 and CYP21P genes and the fact that almost all frequently reported mutations reside on the pseudogene. Allele-specific PCR for the 8 most frequently reported point mutations was performed in 31 Turkish families with at least a single 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L, 0%; IVS2 (AS,A/C-G,-13), 22.5%; G110delta8nt, 3.2%; I172N, 11.4%; exon 6 cluster (I236N, V237E, M239K), 3.2%; V281L, 0%; Q318X, 8%; and R356W, 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, and the allele frequencies were 9.6% and 22.5%, respectively. Sequence analysis of the gene, performed on patients with only 1 mutated allele, revealed 2 missense mutations (R339H and P435S). A novel semiquantitative PCR/enzyme digestion-based method for the detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.

Adrenal Hyperplasia, Congenital↗
PubMed2006

Mutation screening in the human epsilon-globin gene using single-strand conformation polymorphism analysis.

The human epsilon-globin gene is necessary for primitive human erythropoiesis in the yolk sac. Herein we report a non-radioactive single-strand conformation polymorphism (SSCP) approach to screen the human epsilon-globin gene and its regulatory regions for possible mutations and single-nucleotide polymorphisms in normal adult subjects, in order to determine those genomic regions, which are not necessary for its proper regulation and function. We identified no sequence variations apart from the expected 5'epsilon /HincII polymorphism in the fragments analyzed, suggesting that genomic alterations in the epsilon-globin gene are most likely incompatible with normal erythropoiesis and proper embryonic development.

DNA Mutational Analysis↗