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Results for “mutational analysis”

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PubMed2005

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Alanine↗

PubMed2005

Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.

Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.

Adolescent↗

PubMed1993

Sequencing of double-stranded polymerase chain reaction products for mutation analysis.

This report describes a reproducible, straightforward approach to sequencing double-stranded DNA products from the polymerase chain reaction (PCR) for analysis of mutations. The sequencing protocol is a modification of that published by Kretz (Kretz et al., 1989) and has been successful in the hands of a number of investigators working on diverse projects. Following this procedure, PCR DNA products generated from bacterial sources (including pBR322 and F' derivatives), as well as cDNA and genomic DNA from both hamster and human cell lines, have been sequenced with equal success. Close attention to the molar ratio of nucleotides to double-stranded DNA template present during the labeling reaction ensures best results.

Animals↗

PubMed1993

Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis.

The product of the vpr open reading frame of human immunodeficiency virus type 1 (HIV-1) is a 15-kDa, arginine-rich protein that is present in virions in molar quantities equivalent to that of Gag. We report here the results of our investigations into the mechanism by which Vpr is incorporated into virions during assembly in infected cells. For these studies we used an expression vector encoding a Vpr molecule fused at its amino terminus to a nine-amino-acid peptide from influenza virus hemagglutinin. The tagged Vpr expression vector and a vpr mutant HIV-1 provirus were used to cotransfect COS cells, and the resulting virions were tested for the presence of the tagged protein on immunoblots probed with monoclonal antibody against the hemagglutinin peptide. The COS-produced virions were found to contain readily detectable amounts of tagged Vpr and smaller amounts of a putative tagged Vpr dimer. Infectivity of the particles was not altered by incorporation of tagged Vpr. Our results using this system in combination with mutant HIV-1 proviruses suggested that incorporation of Vpr into virions requires the carboxy-terminal Gag protein of HIV-1 (p6) but not gp160, Pol, or genomic viral RNA. In addition, analysis of mutated, tagged Vpr molecules suggested that amino acids near the carboxy terminus (amino acids 84 to 94) are required for incorporation of Vpr into HIV-1 virions. The single cysteine residue near the carboxy terminus was required for production of a stable protein. Arginine residues tested were not important for incorporation or stability of tagged Vpr. These results suggested a novel strategy for blocking HIV-1 replication.

Amino Acid Sequence↗

PubMed2002

Mutation analysis of the MPZ and PMP22 genes in Croatian patients.

We used single-strand conformation polymorphism analysis for mutational screening in two candidate genes, MPZ and PMP22, which have an important role in the pathogenesis of Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies. A novel Ser8Ser polymorphism was found in exon 1 of the MPZ gene in two heterozygous subjects, in a father with mild CMT2 phenotype and his daughter with normal clinical data. Thr118Met polymorphism was found in exon 5 of the PMP22 gene. The patient heterozygous for 118Met allele had CMT1 disease. We can conclude that the occurrence of the 118Met allele does not usually cause CMT1 and that it is not a clinically relevant disease marker.

Charcot-Marie-Tooth Disease↗

PubMed2006

Probing the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) by mutational analysis.

Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

Amines↗

PubMed1998

A rapid method for DNA extraction from fine-needle aspiration biopsies of thyroid tumors, and subsequent RET mutation analysis.

The presence of germline and somatic mutations in the RET proto-oncogene (RET) in patients with hereditary and sporadic medullary thyroid carcinoma (MTC) may have important ramifications for patient management. It has been suggested that the presence of the mutation ATG-->ACG at codon 918 in sporadic MTC is associated with a worse prognosis, and hence the need for more aggressive surgical, or other, treatment. Since fine-needle aspiration biopsy (FNAB) is an important component of the preoperative management of patients with thyroid tumors, we developed a rapid (20-min) method that enables extraction of DNA from FNABs for RET mutation analysis. The method allows preoperative genetic analysis to aid in management of patients with MTC, and may be applied to other tumor types where a preoperative mutation analysis based on polymerase chain reaction amplification is desired.

Biopsy, Needle↗

PubMed2005

Autosomal dominant hereditary spastic paraplegia: DHPLC-based mutation analysis of SPG4 reveals eleven novel mutations.

We set up a new denaturing high-performance liquid chromatography (DHPLC)-based protocol to screen patients with autosomal dominant hereditary spastic paraplegia (AD-HSP) for mutations in SPG4. Six patients had a complicated form and 49 a pure HSP phenotype. We also analyzed 19 unrelated patients presenting with an HSP phenotype (pure in 17 and complicated in two subjects) but no clear family history, as such patients may be cases of dominant inheritance with low penetrance. The overall frequency of SPG4 mutations in our study of HSP (in which prior linkage data were unavailable) was 32.4%, rising to 46.9% when only pure AD-HSP patients were considered. This figure falls well within the range reported in different populations. Rather as expected, the clinical data available for the patients did not support an easy genotype-phenotype correlation. Moreover, the clinical picture was not influenced by the length of the predicted residual gene product. As well as identifying novel variants in SPG4, this study constitutes the molecular characterization of the largest cohort of Italian AD-HSP patients studied to date. In addition, it provided an efficient, cost-effective, and reliable detection protocol for mutational screening of SPG4, which might facilitate medical genetic counseling.

Adenosine Triphosphatases↗

PubMed1998

Higher plants light harvesting proteins. Structure and function as revealed by mutation analysis of either protein or chromophore moieties.

Mutation analysis of higher plants light harvesting proteins has been prevented for a long time by the lack of a suitable expression system providing chromophores essential for the folding of these membrane-intrinsic pigment-protein complexes. Early work on in vitro reconstitution of the major light harvesting complex of photosystem II (LHCII) indicated an alternative way to mutation analysis of these proteins. A new procedure for in vitro refolding of the four light harvesting complexes of photosystem II, namely CP24, CP29, CP26 and LHCII yields recombinant pigment-proteins indistinguishable from the native proteins isolated from leaves. This method allows both the performing of single point mutations on protein sequence and the exchange of the chromophores bound to the protein scaffold. We review here recent results obtained by this method on the pigment-binding properties, on the chlorophyll-binding residues, on the identification of proton-binding sites and on the role of xanthophylls in the regulation of light harvesting function.

Amino Acid Sequence↗

PubMed1993

Multiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells.

We report here the development of multiplex in vitro DNA amplification and solid-phase direct exon sequencing for the analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. 18 representative HPRT-deficient mutants, derived either spontaneously, or after exposure to UV light or ionizing radiation, were analyzed. All 9 hprt exons were simultaneously amplified via the polymerase chain reaction (PCR) for rapid deletion detection. 5 mutants involve single- or multiple-exon deletions. Altered multiplex PCR patterns were detected in mutants Bsp-040, Bsp-065 and BGR-606. Subsequent direct sequence analysis reveals that Bsp-040 and Bsp-065 carry a 52-bp and a 13-bp intragenic DNA deletion in exon 3, respectively. BGR-606 contains a 223-bp insertion accompanied by a 10-bp deletion of intron sequence within exon 4 fragment. Other subtle DNA alterations identified by direct exon sequence analysis include single-base substitutions, small deletions and insertions, and RNA splicing mutations.

Animals↗

PubMed2006

Megalencephalic leukoencephalopathy with subcortical cysts: an update and extended mutation analysis of MLC1.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive cerebral white matter disorder in children. This disease is histopathologically characterized by myelin splitting and intramyelinic vacuole formation. MLC is caused by mutations in the gene MLC1, which encodes a novel protein, MLC1. Since the first report, 50 mutations in this gene have been found. Mutations occur throughout the entire coding region and include all different types: 11 splice-site mutations; one nonsense mutation; 24 missense mutations; and 14 deletions and insertions. Until now, six polymorphisms within the coding sequence of MLC1 had been reported. In about 20% of the patients with a typical clinical and MRI picture, no mutations in the MLC1 gene are found. Several of the families, in which no mutations are found, also do not show linkage with the MLC1 locus, which suggests a second gene involved in MLC. The absence of mutations may also be the consequence of performing standard mutation analysis that can miss heterozygous deletions, mutations in the promoter, 3' and 5' untranslated regions (UTRs), and intron mutations, which may influence the amino acid composition of the end product. In this work we describe 13 novel mutations, including those found with extended mutation analysis on MLC patients. This study shows that extended mutation analysis is a valuable tool to identify at least some of the missing mutations. Therefore, we suggest extended mutation analysis for the MLC1 gene, if no mutations are found during standard analysis.

Base Sequence↗

PubMed1995

DNA-based mutation analysis of Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinaemia.

The identification of the BTK (Bruton's tyrosine kinase) gene defective in human immunoglobulin deficiency X-linked agammaglobulinaemia (XLA) and characterisation of BTK exon-intron boundaries has now allowed the analysis of mutations and polymorphisms at the level of genomic DNA. Using Southern blot analysis and the polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) assay, amplifying all 19 exons and the putative promoter region with a single annealling temperature, mutations have been identified in 19 out of 24 unrelated patients diagnosed as having XLA. Apart from a large deletion involving exon 19, nine missense (F25S, R288W, 1370M, M509V, R525P, N526K, R562W, A582V and G594R), two nonsense (E277X and R525X), five frameshift and two splice site mutations have been found affecting most coding exons and all major enzyme domains. No mutations or polymorphisms were detected in the putative promoter region. A single nucleotide deletion located in the last exon, resulting in a truncation of the eight C-terminal residues of Btk and a typical XLA phenotype, indicates structural and/or functional importance of Btk helix I in the catalytic domain. Although allelic heterogeneity at the BTK locus may partly explain clinical variability in families with XLA, compensatory and redundant mechanisms involved in B-cell development must play a role in the phenotypic diversity of the disease.

Adolescent↗

PubMed2001

Sensitive detection of p53 mutation: analysis by direct sequencing and multisequence analysis.

In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mutational screening methods often are restricted to the core domain and, therefore, do not detect all mutations. We improved existing sequencing-based mutation analysis methods consisting of direct sequencing of all exons of the p53 gene and RNA. Multisequence analysis software was developed and applied to increase the sensitivity of mutation identification. Multisequence analysis compares a large number of sequences and identifies profiles with additional small peaks, potentially indicating a mutation. Concordance between blood and tumor sequences indicates polymorphism, whereas discordance indicates a mutation. We could detect mutations with a limit of approximately 5% to 10% mutated DNA. In our ongoing studies, we applied sequencing-based mutation analysis for more than 50 patients with head and neck squamous cell carcinoma and identified mutations in more than 95% of all tumors. We encountered some differences between the previously published reference p53 sequence and all our sequences and identified polymorphism in six regions.

Base Sequence↗

PubMed2003

Mutational analysis of 206 families with cavernous malformations.

OBJECT: A gene contributing to the autosomal-dominant cerebral cavernous malformation (CCM) phenotype, KRIT1 (an acronym for Krev Interaction Trapped 1), has been identified through linkage analysis and mutation screening. The authors collected blood samples from 68 patients with familial CCM and 138 patients with apparently sporadic CCM as well as from their families, in an effort to characterize the prevalence and spectrum of disease-causing sequence variants in the KRIT1 gene. METHODS: The authors used single-strand conformational polymorphism analysis to identify genomic variants in KRIT1, which were sequenced to determine the specific mutation. Among 43 Hispanic-American kindreds who immigrated to the southwestern US from northern Mexico, 31 share an identical founder mutation. This Q455X mutation is found in 18 (86%) of 21 persons with a positive family history and in 13 (59%) of 22 persons with apparently sporadic CCM. This mutation was not found among 13 persons with CCM who were recruited from Mexico. These findings establish the key role of a recent founder mutation in Hispanic persons with CCM who live in the US. Although nearly all Hispanic families in the US in which there are multiple CCM cases linked to the CCM1 locus, only 13 of 25 non-Hispanic CCM-carrying families have displayed evidence of linkage to the CCM1 locus. Among these 13 families, the authors identified eight independent mutations in nine kindreds. They identified four additional mutations among 22 familial CCM kindreds with no linkage information, bringing the total number of independent mutations to 12. Inherited KRIT1 mutations were not detected among 103 non-Hispanic persons in whom a family history of CCM was rigorously excluded. CONCLUSIONS: All mutations were nonsense mutations, frame-shift mutations predicting premature termination, or splice-site mutations located throughout the KRIT1 gene, suggesting that these are genetic loss-of-function mutations. These genetic findings, in conjunction with the clinical phenotype, are consistent with a two-hit model for the occurrence of CCM.

Codon↗

PubMed2003

Clinical usefulness of KRAS mutational analysis in the diagnosis of pancreatic adenocarcinoma by means of endosonography-guided fine-needle aspiration biopsy.

AIM: To establish the usefulness of KRAS mutational analysis in the diagnosis of pancreatic adenocarcinoma by comparing this technique with conventional cytology in aspirates obtained by endosonography-guided fine-needle aspiration. METHODS: All consecutive patients with pancreatic focal lesions undergoing endosonography-guided fine-needle aspiration were included. Samples were obtained with the concurrence of an attendant cytopathologist. Detection of codon-12 KRAS mutations was performed by the restriction fragment length polymorphism-polymerase chain reaction method. The effectiveness of conventional cytology, KRAS mutational analysis and their combination was established with respect to the definitive diagnosis. A cost-effectiveness analysis was also performed. RESULTS: Thirty-three patients had pancreatic adenocarcinoma and 24 patients had other lesions. A total of 136 samples was obtained. In patients in whom specimens were adequate (93% for cytology; 100% for mutational analysis), the specificity of both techniques was 100%, whereas the sensitivity favoured cytology (97% vs. 73%). When inadequate samples were considered as misdiagnosed, a combination of both techniques reached the highest overall accuracy (cytology, 91%; mutational analysis, 84%; combination of both, 98%). CONCLUSIONS: Cytology from aspirates obtained by endosonography-guided fine-needle aspiration is the most precise single technique for the diagnosis of pancreatic adenocarcinoma. However, when adequate specimens are not available to reach a cytological diagnosis, the addition of KRAS mutational analysis represents the best strategy.

Adenocarcinoma↗

PubMed2005

[Molecular genetic analysis of the BIGH3 gene in lattice type I (Biber-Haab-Dimmer) and granular type II (Avellino) corneal dystrophy: is indirect mutation analysis for hot spots recommended?].

BACKGROUND: Mutations of the BIGH3 gene were delineated as the underlying gene defect for corneal dystrophy Lattice Type I (CDL1) and corneal dystrophy Avellino type (CDA) in families with different regional provenance. Missense mutations in exon 4 with single base pair substitution which result in amino acid alterations Arg124Cys (CDL1) and ARG124His are described as hot spots. We report on histopathological and molecular genetic investigations in 2 German families and a single patient with CDL1 and CDA. METHOD: In 3 affected family members and 1 unaffected family member and in one single patient with CDL1 and in 3 affected family members and 1 unaffected family member of a family with CDA mutation analysis in exon 4 of BIGH3 gene by direct sequencing of genomic DNA from peripheral blood was performed. Histopathological examination of corneal tissue of both index patients was performed after penetrating keratoplasty. RESULTS: We revealed a heterozygous single base pair substitution 417C-->T in family A and patient B (CDL1) and a heterozygous single base pair substitution 418G-->A in family C (CDA). In all index patient's diagnosis was confirmed by histopathological examination of corneal tissue. The sequencing results were confirmed by restriction digestion with HpyCH4V (NEB; CDL1) restriction endonuclease site and AvaII (NEB; CDA) restriction endonuclease site. The heterozygous 417C-->T transition in family A and patient B alters the amino acid sequence from Arg124Cys while the heterozygous 418G-->A transition in family C alters the amino acid sequence from Arg124His in the keratoepithelin. COMMENT: Codon 124 of the BIGH3 gene appears as a mutation hot spot also in German families with CDL1 and CDA. Indirect mutation analysis with restriction digestion is suggested as first step investigation in families with relevant corneal dystrophies. Direct sequencing of all exons is recommended as a second step if there are no results in restriction digestion.

Adult↗

PubMed1995

Diagnostic utility of K-ras mutational analysis on bile obtained by endoscopic retrograde cholangiopancreatography.

BACKGROUND: Because K-ras oncogene mutations are present in more than 90% of pancreaticobiliary tumors, we tested prospectively whether K-ras mutational analysis of bile samples obtained during ERCP was superior to conventional bile cytology for diagnosis of malignancy. METHODS: Bile samples from 20 patients undergoing ERCP for evaluation of jaundice were examined by both cytologic study and the polymerase chain reaction for the presence of K-ras oncogene mutations. RESULTS: Polymerase chain reaction products were amplified from the bile of 8 of 12 patients with malignancy and 3 of 8 with benign disease; K-ras oncogene mutations were present in 4 of 8 polymerase chain reaction products from malignant samples but absent in all 3 from benign samples. No cytologic results were positive for malignancy. CONCLUSIONS: Bile obtained during ERCP can yield positive results in K-ras mutational analysis, even when results of conventional bile cytology are negative. In this study, K-ras mutational analysis had a sensitivity of 33% (4 of 12), a specificity of 100%, and a positive predictive value of 100% for diagnosis of malignancy.

Bile↗

PubMed2002

Mutational analysis of the GNAS1 exons encoding the stimulatory G protein in five patients with pseudohypoparathyroidism type 1a.

We analyzed the GNAS1 gene in five patients with pseudohypoparathyroidism type 1a (PHP1a) by performing polymerase chain reaction, followed by sequencing all 13 exons of the gene, single-stranded conformational polymorphism (SSCP) or heteroduplex analysis (HD). Three novel mutations were discovered: (1) a de novo 3 bp insertion of CTG in codon 47 of exon 1; (2) a missense mutation 1103T in exon 4; and (3) a de novo mutation of Arg280Gly in exon 10. Two other mutations, previously described in the literature, include: (1) a de novo 4 bp deletion (deltaGACT) involving codons 189 and 190 in exon 7, and (2) a deletion of a cytosine nucleotide at codon 115 in exon 5. We conclude that mutational analysis of the GNAS1 gene is a strong supportive tool for the diagnosis of PHP1a, and is a useful adjunct to the synthetic parathyroid hormone infusion test for PTH resistance.

Adolescent↗