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Results for “mutation analysis”

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PubMed1996

Mutational analysis of a transmembrane segment in a bacterial chemoreceptor.

Trg is a member of a family of receptors that mediates chemotaxis by Escherichia coli. Its transmembrane domain is a loose four-helix bundle consisting of two helices from each of the two identical subunits. This domain mediates transmembrane signaling through a conformational change in which the second transmembrane segment (TM2) is thought to move relative to TM1, but mutational analysis of TM2 by cysteine scanning had identified only a few positions at which substitutions perturbed function or induced signaling. Thus, we performed mutational analysis by random mutagenesis and screening. Among 42 single-residue substitutions in TM2 that detectably altered function, 16 had drastic effects on receptor activity. These substitutions defined a helical face of TM2. This functionally important surface was directed into the protein interior of the transmembrane domain, where TM2 faces the helices or the other subunit. The functionally perturbing substitutions did not appear to cause general disruption of receptor structure but rather had more specific effects, altering aspects of transmembrane signaling. An in vivo assay of signaling identified some substitutions that reduced and others that induced signaling. These two classes were distributed along adjacent helical faces in a pattern that strongly supports the notion that conformational signaling involves movement between TM2 and TM1 and that signaling is optimal when stable interactions are maintained across the interface between the homologous helices in the transmembrane domain. Our mutational analysis also revealed a striking tolerance of the chemoreceptor for substitutions, including charged residues, usually considered to be disruptive of transmembrane segments.

Amino Acid Sequence↗

PubMed1999

DGGE method for the mutational analysis of the coding and proximal promoter regions of the Alzheimer's disease presenilin-1 gene: two novel mutations.

Many different mutations that cause Alzheimer's disease (AD) have been found in the presenilin-1 gene (PSEN1) and are associated with the most aggressive forms of the disease. With the aim of screening for PSEN1 genetic variations, we developed a method based on denaturing gradient gel electrophoresis (DGGE) that allows the mutational analysis of all the coding exons and the proximal promoter of PSEN1 using only four DGGE gels. The analysis by this methodology of a sample of 58 early-onset AD (EOAD) patients nonselected for family history resulted in finding four genetic variants within the PSEN1 coding region, two of which are novel mutations (M233L and A409T), whereas the other two have been reported previously (L282R and E318G). We also found a novel mutation within the PSEN1 proximal promoter (-280 C-->G) that, interestingly, provokes significant changes in the transcriptional activity of the gene in cell lines of neuronal and astrocytic, but not hepatic origin. These data strongly suggest that the region around -280 of PSEN1 promoter contains a regulatory element that controls its transcription specifically in neural cells.

Adult↗

PubMed2003

Mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel mutations.

Epidermolysis bullosa simplex is a group of blistering skin disorders caused by defects in one of the keratin genes, KRT5 and KRT14. Previously reported KRT5 and KRT14 mutations are clustered in several hotspots, namely the rod ends of the 1A and 2B domains and in the non-helical linker region L12. Therefore, genomic KRT5 and KRT14 mutation analysis was initially limited to these hotspots. In this study we describe the screening of nine EBS patients for mutations in the hotspots. In two patients, with the Koebner and the Weber-Cockayne subtypes of epidermolysis bullosa simplex respectively, we could, however, not identify any mutation in one of the hotspot domains of KRT5 and KRT14. Therefore, it appeared to be necessary to screen the entire genes for mutations. For KRT5, a complete genomic mutation detection system was previously described. We now developed a complete genomic mutation detection system for KRT14. For the amplification of the KRT14 genes, we make use of restriction sites to exempt the keratin 14 pseudogene sequence from polymerase chain reaction amplification. Using the complete genomic mutation detection system for both KRT5 and KRT14, we identified four novel KRT5 mutations (IVS1-1G>C, K404E, A438D, E475K), two of which are outside the KRT5 hotspot domains, and three novel KRT14 mutations (IVS4+1G>A, L408M, L130P).

Amino Acid Substitution↗

PubMed1992

The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.

Amino Acid Sequence↗

PubMed2006

Dysferlin expression in monocytes: a source of mRNA for mutation analysis.

Dysferlin protein is expressed in peripheral blood monocytes. The genomic analysis of the DYSF gene has proved to be time consuming because it has 55 exons. We designed a mutational screening strategy based on cDNA from monocytes to find out whether the mutational analysis could be performed in mRNA from a source less invasive than the muscle biopsy. We studied 34 patients from 23 families diagnosed with dysferlinopathy. The diagnosis was based on clinical findings and on the absence of protein expression using either immunohistochemistry or Western blot of skeletal muscle and/or monocytes. We identified 28 different mutations, 13 of which were novel. The DYSF mutations in both alleles were found in 30 patients and only in one allele in four. The results were confirmed using genomic DNA in 26/34 patients. This is the first report to furnish evidence of reliable mutational analysis using monocytes cDNA and constitutes a good alternative to genomic DNA analysis.

DNA Mutational Analysis↗

PubMed2006

Medullary cystic kidney disease type 1: mutational analysis in 37 genes based on haplotype sharing.

Medullary cystic kidney disease type 1 (MCKD1) is an autosomal dominant, tubulo-interstitial nephropathy that causes renal salt wasting and end-stage renal failure in the fourth to seventh decade of life. MCKD1 was localized to chromosome 1q21. We demonstrated haplotype sharing and confirmed the telomeric border by a recombination of D1S2624 in a Belgian kindred. Since the causative gene has been elusive, high resolution haplotype analysis was performed in 16 kindreds. Clinical data and blood samples of 257 individuals (including 75 affected individuals) from 26 different kindreds were collected. Within the defined critical region mutational analysis of 37 genes (374 exons) in 23 MCKD1 patients was performed. In addition, for nine kindreds RT-PCR analysis for the sequenced genes was done to screen for mutations activating cryptic splice sites. We found consistency with the haplotype sharing hypothesis in an additional nine kindreds, detecting three different haplotype subsets shared within a region of 1.19 Mb. Mutational analysis of all 37 positional candidate genes revealed sequence variations in 3 different genes, AK000210, CCT3, and SCAMP3, that were segregating in each affected kindred and were not found in 96 healthy individuals, indicating, that a single responsible gene causing MCKD1 remains elusive. This may point to involvement of different genes within the MCKD1 critical region.

Chromosome Mapping↗

PubMed2002

Mutation analysis of the G4.5 gene in patients with isolated left ventricular noncompaction.

Mutations in the gene G4.5, originally associated with Barth syndrome, have been reported to result in a wide spectrum of severe infantile X-linked cardiomyopathies. The purpose of this study was to investigate patients with isolated left ventricular noncompaction (LVNC) for disease-causing mutations in G4.5. In 27 patients including 10 families with isolated LVNC, mutation analysis of G4.5 was performed using single-strand DNA conformation polymorphism (SSCP) analysis and DNA sequencing. A novel splice acceptor site mutation of intron 8 of G4.5 was identified in a family with severe infantile X-linked LVNC without the usual findings of Barth syndrome. This mutation results in deletion of exon 9 from the mRNA, and is predicted to significantly disrupt the protein product. Genotype-phenotype correlation of G4.5 mutations in all 38 cases reported in the literature to date revealed that there was no correlation between location or type of mutation and either cardiac phenotype or disease severity. We suggest that males presenting with cardiomyopathy, particularly during infancy, even in the absence of the typical signs of Barth syndrome, should be evaluated for mutations in G4.5.

Acyltransferases↗

PubMed1993

Mutational analysis of patients with adenomatous polyposis: identical inactivating mutations in unrelated individuals.

Samples of constitutional DNA from 60 unrelated patients with adenomatous polyposis coli (APC) were examined for mutations in the APC gene. Five inactivating mutations were observed among 12 individuals with APC; all were different from the six inactivating mutations previously reported in this panel of patients. The newly discovered mutations included single-nucleotide substitutions leading to stop codons and small deletions leading to frameshifts. Two of the mutations were observed in multiple APC families and in sporadic cases of APC; allele-specific PCR primers were designed for detecting mutations at these common sites. No missense mutations that segregated with the disease were found.

Adenomatous Polyposis Coli↗

PubMed2005

Photosensitive Smith-Lemli-Opitz syndrome is not caused by a single gene mutation: analysis of the gene encoding 7-dehydrocholesterol reductase in five U.K. families.

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome characterized by a disorder in cholesterol metabolism. SLOS is caused by mutations in the DHCR7 gene which encodes 7-dehydrocholesterol reductase, an enzyme that catalyses the final step in cholesterol biosynthesis. We have previously established the clinical and photobiological features of the photosensitivity that is frequently a feature of SLOS. OBJECTIVES: In this study, we have performed mutational analysis of the DHCR7 gene in individuals from five families with SLOS. In each family, one member was affected by severe photosensitivity as a manifestation of SLOS. METHODS: Fifteen samples (including family controls) were screened using polymerase chain reaction amplification and direct automated sequencing. RESULTS: Six different DHCR7 mutations were identified of which five were single point mutations that caused missense amino acid substitutions (P51H, T93M, L99P, E448K and R450L). The other was a splice site mutation (G-->C in splice acceptor site) affecting the intron 8-exon 9 splice junction (IVS8-1 G-->C). This splice site mutation and four of the five missense mutations have been previously published as causal in SLOS but the P51H is a novel mutation which has not previously been reported. CONCLUSIONS: This is the first study in which DHCR7 gene mutational analysis has been performed on SLOS subjects with severe photosensitivity and indicates that no single mutation is responsible for the photosensitivity which characterizes this disorder.

Adolescent↗

PubMed2002

Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes.

Steroid 21-hydroxylase deficiency, the primary cause of congenital adrenal hyperplasia, is caused by defects of the CYP21A2 gene. As a complement to hormonal measurements, mutation analysis of CYP21A2 is an important tool in the diagnosis of steroid 21-hydroxylase deficiency. Contemporary mutation-detection protocols based on the polymerase chain reaction often depend on the assumption that no more than one CYP21A2 gene is present on each chromosome 6. We describe three haplotypes with two CYP21A2 genes on the same chromosome, with defects typical of salt-losing steroid 21-hydroxylase deficiency in one of those genes, but not necessarily in the other. The frequency of these haplotypes in the general population is 6/365 (1.6%), so they are no less common than other haplotypes that indeed carry steroid 21-hydroxylase deficiency. Chromosomes that carry two CYP21A2 genes therefore represent a significant pitfall in the molecular diagnosis of steroid 21-hydroxylase deficiency. We recommend that, whenever CYP21A2 mutation analysis of an individual who is not a known carrier of steroid 21-hydroxylase deficiency is performed, the overall structure of the CYP21/ C4 region (the RCCX area) is determined by haplotyping to avoid erroneous assignment of carrier status.

Adrenal Hyperplasia, Congenital↗

PubMed2005

LRRK2 gene in Parkinson disease: mutation analysis and case control association study.

BACKGROUND: In addition to the four well-confirmed genes linked to early-onset Parkinson disease (PD) (SNCA, PARKIN, DJ-1, and PINK1), mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have recently been identified in families with autosomal dominant late-onset PD. OBJECTIVE: To perform mutation analysis of LRRK2 in probands of families showing dominant inheritance of PD and to conduct a case control association study to test the hypothesis that common coding variations might be associated with increased susceptibility to PD. METHODS: All 51 LRRK2 coding exons were sequenced in 23 probands and the mutation frequencies were evaluated in 180 neurologically normal control subjects. For the association study the authors genotyped four coding LRRK2 polymorphisms in 250 normal control subjects and 121 patients with PD (predominantly white patients of Canadian origin), 84% of whom had age at onset before 50 years and 42% had a positive family history. RESULTS: The authors identified three probands with heterozygous LRRK2 mutations: two of them have the known G2019S substitution and one proband has a novel I1371V substitution. Mutation analysis of a large family demonstrated complete segregation of the G2019S with PD. However, there was no association between PD and any of the four polymorphisms at the allelic or genotypic levels (p > 0.17). Furthermore, the authors did not detect a modifying effect for any genotype or of APOE genotypes upon the age at onset in the PD group (p > 0.20). CONCLUSIONS: The results support the prior suggestion that LRRK2 mutations cause PD. The disease in the families reported here presents a phenotype indistinguishable from typical PD. All three families demonstrate a very variable age at onset that is not explained by APOE genotypes. The common coding variations in the LRRK2 gene neither constitute strong PD risk factors nor modify the age at onset; however, the possibility of a modest risk effect remains to be assessed in large datasets.

Adult↗

PubMed2003

Mutational analysis of BRCA1 and BRCA2 in Mediterranean Spanish women with early-onset breast cancer: identification of three novel pathogenic mutations.

In Spain, the contribution of BRCA mutations to the population incidence of early-onset breast cancer was unknown. We carried out a mutational analysis of the BRCA1 and BRCA2 genes in 124 Spanish women diagnosed with breast cancer before the age 41 and who were not selected for a family history of this disease. The genetic study was performed by PCR-SSCP analysis and DNA sequencing. We identified 6 pathogenic BRCA mutations in 7 unrelated probands (5.6%; 95% CI=2.3% to 11.3%): 1 BRCA1 (c.2080delA) and 5 BRCA2 (p.Y3006X, p.Q1994X, c.9204_9217del14, c.9254_9258del5 and c.295+2T>C). Three out of 6 mutations were novel (BRCA2 p.Y3006X, c.9204_9217del14, and c.295+2T>C), and two further mutations had not been previously found in Spain (BRCA1 c.2080delA and BRCA2 p.Q1994X). The one remaining (BRCA2 c.9254_9258del5) was detected in two probands of our sample. Additionally, we identified two new missense mutations: BRCA1 p.P1812A and BRCA2 p.G2044A. Our data support the notion that Spaniards represent a heterogeneous population with its own spectrum of BRCA mutations, some of which appear as founding mutations. We categorized patients into familial or non-familial groups on the basis of her family history of breast/ovarian cancer; this analysis indicated that among Spanish women with early-onset breast cancer, an even moderate family history is a good predictor of being a BRCA mutation carrier.

Adult↗

PubMed1998

Mutation analysis of the RSK2 gene in Coffin-Lowry patients: extensive allelic heterogeneity and a high rate of de novo mutations.

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. By using a positional cloning approach, we have recently shown that mutations in the gene coding for the RSK2 serine-threonine protein kinase are responsible for this syndrome. To facilitate mutational analysis, we have now determined the genomic structure of the human RSK2 gene. The open reading frame of the RSK2 coding region is split into 22 exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single-strand conformation polymorphism analysis. We screened 37 patients with clinical features suggestive of CLS. Twenty-five nucleotide changes predicted to be disease-causing mutations were identified, including eight splice-site alterations, seven nonsense mutations, five frameshift mutations, and five missense mutations. Twenty-three of them were novel mutations. Coupled with previously reported mutations, these findings bring the total of different RSK2 mutations to 34. These are distributed throughout the RSK2 gene, with no clustering, and all but two, which have been found in two independent patients, are unique. A very high (68%) rate of de novo mutations was observed. It is noteworthy also that three mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms. No obvious correlation was observed between the position or type of the RSK2 mutations and the severity or particular clinical features of CLS.

Abnormalities, Multiple↗

PubMed2004

Mutational analysis of carbamoylphosphate synthetase I deficiency in three Japanese patients.

We describe the results of mutational analysis of the carbamoylphosphate synthetase I (CPSI) gene in three nonconsanguineous patients with CPSI deficiency. Compound heterozygotes of 3422T/G (V1141G) plus 3784C/T (R1262X), 1528delG (510-514 ARQLX) plus 2752T/C (S918P), and 2549G/A (R850H) plus 2797delT (L933X) were identified through genomic analysis; however, the 2797delT (L933X) mutation was not detected in cDNA analysis using biopsied liver, suggesting that mRNA expression rom this mutant allele is absent or markedly low.

Adolescent↗

PubMed2002

Application of mutation analysis for the previously uncertain cases of adult-onset type II citrullinemia (CTLN2) and their clinical profiles.

Type II citrullinemia (CTLN2) is characterized by a deficiency of argininosuccinate synthetase (ASS) in the liver. Mutation analysis of the SLC25A13 gene, which is responsible for CTLN2, provides a rapid and accurate diagnosis. We describe clinical, biochemical and histologic features of two patients, whose diagnosis was finally made by mutation analysis. They initially presented with symptoms related to hyperammonemia at 16 to 22 years of age. A patient had shown mental retardation and growth failure from early childhood. Laboratory findings including amino acids, were characteristic, such as elevated citrulline, arginine, and lysine concentrations, but definitive diagnosis had not been made. The patients died of liver cirrhosis and hepatoma at 31 and 34 years old, respectively. Fatty change in the hepatocytes was commonly observed in the autopsied specimens. ASS activity was decreased in the liver of both patients, and a concomitant decrease of arginase activity was found in one case. Investigation for the SLC25A13 mutation revealed that one patient was homozygous for IVS11 + 1G>A, and the other was compound heterozygote (851del4/S225X). Comparison of genetic, enzymatic and biochemical data among various cases of CTLN2 will be essential to understand the real nature of the disease.

Adult↗

PubMed1998

Transcriptional map of 170-kb region at chromosome 11p15.5: identification and mutational analysis of the BWR1A gene reveals the presence of mutations in tumor samples.

Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci D11S601 and D11S679 resulted in the identification of six transcriptional units. Three genes, hNAP2, CDKN1C, and KVLQT1, are well characterized, whereas three genes are novel. The three additional genes were designated BWR1A, BWR1B, and BWR1C. Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress, BWR1C cDNA was described as IPL [Qian, N., Franck, D., O'Keefe, D., Dao, D. , Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997) Hum. Mol. Genet. 6, 2021-2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith-Wiedemann syndrome samples that resulted in the identification of genetic alterations in the BWR1A gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that BWR1A may play a role in tumorigenesis.

Beckwith-Wiedemann Syndrome↗

PubMed2000

Gene structure of the human receptor tyrosine kinase RON and mutation analysis in lung cancer samples.

The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other malignancies. It encodes a receptor tyrosine kinase (RTK) closely related to MET, whose mutations are associated with neoplasia. We investigated whether RON might be involved in the development or progression of lung cancer. We first determined the exon-intron structure of the gene by direct sequencing of RON cosmid DNA and PCR products containing intronic sequences, and then developed primers suitable for mutation analysis by the single-strand conformation polymorphism (SSCP) method. Twenty coding exons were characterized, all but the first one small (average size: 170 bp), a feature shared with other RTK genes. We performed SSCP analysis of RON in small and non-small cell lung cancer samples, upon detection of its expression in a sample of lung cancer cell lines. A mutation (T915C: L296P) was found in an adenocarcinoma specimen. Several single nucleotide polymorphisms were also found. The panel of intron-anchored primers developed in this work will be useful for mutation analysis of the RON gene in different types of human tumors.

Animals↗

PubMed2006

BRAF mutation analysis in fine needle aspiration (FNA) cytology of the thyroid.

BRAF mutations have been detected in 30% to 80% of papillary thyroid carcinomas (PTC). Several detection methods for BRAF mutation have been reported, but a direct comparison between different assay methods has not been previously reported. In this study, we examined the diagnostic utility of BRAF (T1799A) mutation in 71 cases of thyroid fine needle aspiration specimens using 4 different methods, including direct sequencing, Colorimetric Mutector Assay, real-time LightCycler polymerase chain reaction (LC PCR) with fluorescence resonance energy transfer probes, and an allele-specific LC PCR with CYBR green 1. BRAF mutation was detected in 31 of 58 cases of PTC, but not in 13 cases of non-PTC lesions. The 4 assay methods used in this study were sensitive, reliable, and comparable with each other (100% of specificity and 53.5% of sensitivity). PTC harboring BRAF mutation had higher extrathyroidal invasion and/or lymph node metastasis than PTC with wild-type BRAF. BRAF mutation analysis should be useful for the clinical diagnosis of PTC in cases of indeterminate fine needle aspiration specimen, because of the high degree of specificity. Our results indicate that there is similar sensitivity for the four detection methods. However, the allele-specific LC PCR with CYBR green 1 method is most rapid, easier to perform, and least expensive technique, and it can be readily performed in most molecular diagnostic laboratories.

Adult↗