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PubMed1991

Mutational analysis of the interaction between CD4 and class II MHC: class II antigens contact CD4 on a surface opposite the gp120-binding site.

Using functional and adhesion assays, we have studied the ability of 30 human CD4 mutants to interact with class II major histocompatibility complex (MHC) molecules and also with gp120 from human immunodeficiency virus. The mutants cover the four domains (D1-D4) of CD4 and include several single-site substitutions. Analysis of the results, in the context of the CD4 crystal structure, shows that mutations that affect the interaction with class II MHC molecules are located on three exposed loops from CD4 domains 1 and 2. The specifically implicated residues, 19, 89, and 165, are separated from one another by 9 A, 24 A, and 24 A on one face of the CD4 molecule. Moreover, the class II binding site does not include residues 43 to 49 of the CD4 molecule, a region on an opposite face known to be involved in the binding of gp120.

Antibodies, Monoclonal↗
PubMed2003

Mutational analysis of forkhead transcriptional factor 2 (FOXL2) in Korean patients with blepharophimosis-ptosis-epicanthus inversus syndrome.

We screened for mutations in the forkhead transcription factor gene, FOXL2, in Korean patients with sporadic or familial blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) and direct sequencing. Five of nine BPES families and three of seven sporadic cases were detected to have FOXL2 mutations. We identified four types of FOXL2 mutations, two of which are novel. A new 14 bp deletion (939-952del14) causing a frameshift from G235W and the extension of the predicted protein to 527 amino acids was detected in a BPES family patient. In addition, a novel 845C > A transversion, resulting in a nonsense mutation (S203X), was found in a sporadic case of BPES. The previously reported in-frame 30 bp duplication (909-938dup30) was the most common mutation and was found in eight patients of four BPES families and one sporadic case. A known 17 bp duplication (1080-1096dup17) was observed in a sporadic BPES case. We were unable to find a causal mutation in four BPES families and four sporadic cases. These results suggest that in a fraction of BPES patients, the genetic defect might be associated with a mutation in the non-coding region of the FOXL2 gene or in other genes.

Adolescent↗
PubMed1997

Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region.

We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA. The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.

DNA Mutational Analysis↗
PubMed2001

Mutational analysis of INI1 in sporadic human brain tumors.

The INI1/SMARCB1/hSNF5 gene on chromosome 22 is frequently mutated in rhabdoid tumors. An association of INI1 mutations with allelic losses on chromosome 22 supports a classical tumor suppressor mechanism. Several brain tumor entities including astrocytomas, glioblastomas and ependymomas are characterized by allelic losses on chromosome 22. In the present study we examined a series of 200 brain tumors by Single-strand conformation polymorphism analysis and direct sequencing for point mutations in INI1. In addition, all tumors were analyzed for homozygous deletions spanning both exons 3 and 8 of INI1. No mutations or homozygous deletions were detected in astrocytomas, glioblastomas, oligodendroglial tumors, neurinomas or medulloblastomas. However, a point mutation could be identified in the single case of plexus carcinoma. Our data suggest that INI1 mutations are involved in the pathogenesis of plexus carcinoma; however, INI1 alterations are not a frequent event in the majority of brain tumor entities.

Base Sequence↗
PubMed2002

Mitochondrial DNA mutation analysis in human skin fibroblasts from fetal, young, and old donors.

Multibase deletions in mitochondrial DNA (mtDNA) have been shown to accumulate with age in several tissues, including skin, whereas point mutations have only recently been demonstrated to increase during aging, with several specific mutations occurring at high levels (up to 50%) in skin fibroblasts obtained from old donors [Science 286(1999)774]. We have conducted a survey for a specific deletion and for point mutations in several regions of mtDNA from cultured skin fibroblasts derived from eight fetal (12-20 weeks gestational age), ten young (17-33 years of age) and 11 old (78-92 years of age) human donors. Using PCR analysis, detectable levels of the 4977 basepair (bp) 'common deletion' were present in all three age groups, with the highest deletion levels of up to 0.3% of total mtDNA found in several cell lines from old donors, although other old donor cell lines had much lower levels. Single strand conformation polymorphism (SSCP) analysis for point mutations in the non-coding D-loop region and two regions of the cytochrome oxidase 2 gene failed to reveal the presence of any single base mutations. We infer that age-related high level mutational damage in mtDNA from human skin fibroblasts may manifest both sequence and inter-individual specificity.

Adolescent↗
PubMed1996

[Nucleotide sequences at intron 6 and exon 7 junction of fibroblast growth factor receptor 2 and rapid mutational analysis in Apert syndrome].

Apert syndrome, acrocephalosyndactyly Type I, is an autosomal dominant craniosynostosis comprising acrocephaly, facial dysmorphism and severe syndactyly of the hands and feet. Missense mutations at codons 252 and 253 at 5'-end on exon 7 of fibroblast growth factor receptor (FGFR) 2 have been identified in a large number of patients with Apert syndrome. In this study, nucleotide sequences on the intron 6 were determined by vector ligation-PCR and direct sequencing. Five DNA samples from sporadic Apert syndrome were examined by non-RI SSCP and direct sequencing using a primer pair of intron 6 and exon 7. All cases of the syndrome showed abnormal banding pattern in the SSCP and missense mutations from Ser to Trp at codon 252 of the FGFR2 gene. The non-RI SSCP and direct sequencing of the FGFR2 exon 7 from genomic DNAs may be a useful and rapid molecular means for clinical diagnosis of Apert syndrome.

Acrocephalosyndactylia↗
PubMed2003

Mutational analysis of the Myocilin gene in patients with primary open-angle glaucoma in Morocco.

PURPOSE: To investigate the Myocilin (MYOC) gene for mutations and polymorphisms in patients with primary open-angle glaucoma (POAG) in Morocco. METHODS: Fifty-seven patients with severe POAG, who suffered from complete or almost complete visual field loss, were included in the study. The MYOC coding region, including exon I, exon II, and the coding part of exon III, were screened for sequence alteration using denaturing high-performance liquid chromatography (DHPLC). Variant amplicons were sequenced bidirectionally. The control group consisted of 60 subjects from the general population. RESULTS: One disease-causing mutation, T377M, was observed in one POAG patient. In addition, 10 polymorphisms, namely P13P, R76K, R82H, G122G, T135I, L159L (often associated with P13P), T285T, T325T, Y347Y, and E396E, were detected in patients or in controls. The Q368X mutation that has been documented in Caucasian POAG patients was absent. CONCLUSIONS: MYOC is an infrequent genetic cause of severe POAG in Morocco. The absence of the POAG-associated Q368X mutation and the presence of particular polymorphisms, including P13P + L159L and T325T, could be specific features of the MYOC sequence in African populations.

Adult↗
PubMed2006

Thiamine-responsive megaloblastic anaemia syndrome: long-term follow-up and mutation analysis of seven families.

AIM: Thiamine-responsive megaloblastic anaemia syndrome (TRMA) is the association of diabetes mellitus, anaemia and deafness, due to mutations in SLC19A2, encoding a thiamine transporter protein. This is a unique monogenic form of vitamin-dependent diabetes for which there is limited long-term data. We aimed to study genotype-phenotype relationships and long-term follow-up in our cohort. METHODS: We have studied 13 patients from seven families and have follow-up data for a median of 9 y (2-30 y). RESULTS: All patients originated from Kashmir or Punjab, and presented with non-immune, insulin-deficient diabetes mellitus, sensorineural deafness and a variable anaemia in the first 5 y of life, the anaemia progressing to megaloblastic and sideroblastic changes in the bone marrow. The anaemia and diabetes mellitus responded to oral thiamine hydrochloride 25 mg/d, but during puberty thiamine supplements became ineffective, and almost all patients require insulin therapy and regular blood transfusions in adulthood. All patients are homozygous for mutations in the SLC19A2 gene. We have identified a novel missense mutation (T158R) that was excluded in 100 control alleles. CONCLUSION: Diabetes in this syndrome is due to an insulin insufficiency that initially responds to thiamine supplements; however, most patients become fully insulin dependent after puberty. A mutation screening strategy is feasible and likely to identify mutations in almost all cases.

Adolescent↗
PubMed2006

Accuracy of BRCA1 and BRCA2 founder mutation analysis in formalin-fixed and paraffin-embedded (FFPE) tissue.

BACKGROUND: A major limitation in counseling unaffected women from families with inherited breast and ovarian cancer is that a "true-negative" interpretation of wild type BRCA analysis of the proband cannot be inferred in the absence of demonstration of a BRCA mutation segregating in the kindred. Documentation of familial BRCA mutations from paraffin-derived DNA of deceased patients has been limited due to reports of technical complications leading to lack of reproducibility of BRCA testing of archival material. METHODS: DNA was extracted from formalin-fixed paraffin-embedded (FFPE) morphologically normal tissue of 161 blinded, coded samples from women previously genotyped for the three Ashkenazi Jewish BRCA founder mutations from lymphocyte-derived DNA. Multiplex PCR followed by denaturing polyacrylamide gel electrophoresis was performed for the three founder mutations to determine if analysis on FFPE tissue could produce results concordant with those of the lymphocyte-derived DNA. RESULTS: After disclosure of the sample codes, the results were compared with the original lymphocyte-derived DNA genotypes. Excluding one sample unevaluable due to PCR failure, there was 100% concordance of 160 genotypes (120 mutation samples) derived from DNA from archival FFPE tissue compared to peripheral lymphocytes. CONCLUSIONS: The method described reliably detected BRCA founder mutations in archival DNA derived from FFPE tissue. These results suggests that this technique may be useful in clinical settings to inform wild type BRCA results of unaffected probands, leading to avoidance of unnecessary intensified surveillance or risk-reducing surgery. With further validation this approach can also be applied to other populations where founder mutations are observed.

DNA↗
PubMed2005

Mutation analysis of the HFE gene associated with hereditary hemochromatosis in a Venezuelan sample.

The frequency of the C282Y, H63D and S65C alleles of the HFE gene was determined in a sample of the Venezuelan population. Two new sets of primers were tested for amplifying the regions mapping these mutations, and genotyping was performed by restriction fragment length polymorphism (RFLP). DNA sequencing was used to validate the RFLP analysis. Serum ferritin levels were also determined. Two hundred and fourteen individuals were tested, extracting DNA from whole blood cells (n=177) or from serum (n=37). The frequency of heterozygous subjects was 3.7, 18.2 and 1.7% for the C282Y, H63D and S65C mutations, respectively, and the allele frequencies were 0.019+/-0.01 for C282Y, 0.119+/-0.016 for H63D and 0.009+/-0.005 for S65C. The results suggest that the admixture of native populations with subjects of South European origin might have had an important role in the diffusion of HFE alleles in Venezuela. C282Y homozygous subjects were not found in this study. No HFE genotype studied here was associated with a significant elevation of serum ferritin concentrations, except for C282Y/H63D compound heterozygote found in one asymptomatic male. This finding supports the theory that the H63D mutation could be involved in alterations of iron parameters when inherited together with C282Y. Our results indicate that C282Y homozygotes will be rarely detected. Performance of HFE mutation analysis in individuals with high iron determinations would be recommended.

Adolescent↗
PubMed1992

Mutational analysis of a plant heat shock element.

A total of 32 mutations were generated within the TATA-proximal site 1 (-72 to -47) of soybean heat shock gene Gmhsp17.5E in order to functionally define the optimal configuration of sequences within the heat shock element (HSE). Mutants were tested in vivo utilizing sunflower tumors transformed by a T-DNA based vector. Promoter activity was determined by S1 nuclease hybrid protection analysis of tumor transcripts. A total of five repeats (5'-nGAAn-3' or 5'-nTTCn-3') which comprise the HSE at site 1 were required for full transcription induction by heat stress. Analysis of non-conserved bases flanking the central trinucleotide block indicated that 5'-aGAAg'-3' is the optimum sequence for the 5 bp repeat.

Base Sequence↗
PubMed2003

Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography.

Mutations in the DPYD gene, which encodes dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in the catabolism of pyrimidines, are responsible for an inborn error of metabolism associated with thymine-uraciluria and neurological symptoms. Because the antimetabolite 5-fluorouracil (5-FU) is metabolized by the same enzyme, deficient DPYD alleles may also constitute a risk factor for severe toxicity following treatment with this anticancer drug. The aim of this study was to develop a comprehensive and rapid method to detect sequence variations within the DPYD gene. Using polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC), we established a protocol that makes it possible to screen all 23 exons of the DPYD gene and their exon-intron boundaries for both known and unknown mutations under identical conditions. A novel one-step PCR mutagenesis procedure was developed to generate heterozygous mutant amplicons as positive controls to optimize DHPLC detection of any sequence variation. DHPLC analysis was shown to result in mutation-specific elution profiles and to be able to distinguish different base changes within the same exon or different heterozygous combinations of mutations within the same exon. By analyzing the DPYD gene in 16 affected individuals, a total of 47 base changes were detected, representing eight known mutations and three novel intronic base changes. Sequence analysis confirmed all base changes detected. This method will be useful in identifying patients at risk for toxicity prior to 5-FU treatment, as well as in the analysis of individual patients with thymine-uraciluria.

Alleles↗
PubMed2003

Patterns of beta-catenin expression in gastric carcinoma: clinicopathological relevance and mutation analysis.

Studies on the expression of beta-catenin (beta-ct) in gastric carcinoma have provided conflicting results, and the role played by beta-ct mutations in gastric carcinogenesis remains unclear. In an attempt to clarify the aforementioned issues we undertook the retrospective study of 157 gastric carcinomas by using immunohistochemistry and molecular genetics. Reduced/absent membranous beta-ct expression was significantly associated with isolated-cell/diffuse histotype both in "pure" diffuse gastric carcinomas and in the isolated-cell/diffuse component of mixed carcinomas. Cytoplasmic and/or nuclear beta-ct expression was particularly prevalent in mixed carcinomas and was significantly associated with lymphatic vessel invasion and lymph node metastases. beta-ct mutations were not detected in any case. We conclude that the pattern of beta-ct expression is closely related to gastric carcinoma histotype. The activation of Wnt/beta-ct pathway is associated with mixed gastric carcinoma and with features of clinical aggressiveness; the mechanism(s) underlying this pathway in gastric carcinoma are not due to beta-ct mutations and remain to be elucidated.

Cell Nucleus↗
PubMed1996

Mutational analysis of RAG-1 in lymphoid malignancies.

Illegitimate recombinase activity promoted by the recombination activating RAG-1 and RAG-2 is assumed to be involved in the pathogenesis of the chromosomal translocations observed in lymphoid neoplasias. We analyzed the complete coding region of the RAG-1 gene in patients with lymphoid neoplasis using a multiple PCR-SSCP (single strand conformation polymorphism) strategy. Nine multiple myelomas, 17 non-Hodgkin's lymphomas, 18 acute lymphocytic leukemias, 37 chronic lymphocytic leukemias and 33 non-neoplastic controls were studied. To screen the entire RAG-1 gene we used primers overlapping genomic segments of the RAG-1 coding sequence (nucleotides 87 to 3311). Samples with an abnormal band pattern in the SSCP were cloned and sequenced. Successful amplification was achieved with our protocol. The multiple PCR-SSCP analysis proved to be a feasible and sensitive strategy for studying variations in the sequence of the RAG-1 gene. No mutations other than the three previously reported sequence variations were detected. Although mutations in this gene do not appear to be common in lymphoid neoplasias, it would be interesting to ascertain whether the different variant forms of RAG-1 protein have an abnormal recombinase activity.

DNA Mutational Analysis↗
PubMed1998

Mutation analysis of CYP2D6 locus in the Korean population: identification of rare poor metabolizer alleles at the nucleotide level.

The CYP2D6 loci of one hundred and eight genetically unrelated Koreans were analyzed. The G1934A mutation which causes aberrant splicing, thereby producing nonfunctional enzymes and is responsible for 70% of poor metabolizer (PM) phenotype among Caucasians, was found in three heterozygous individuals. The mutation frequency of Koreans at 1934 (1.5%) was one thirteenth of that of Caucasians (20.7%). Such a low mutation frequency could be the major genetic explanation for rare PMs in Koreans. A substitution allele of Gly169-Arg(G1846A) which also produces a nonfunctional enzyme was detected in one heterozygous individual. This PM allele has been reported only in Chinese but not in Caucasians. This is the first report which demonstrates the presence of CYP2D6 PM alleles at the nucleotide level in Koreans. No base changes were detected at 1795, 2637, and 3023 whose mutations would also make nonfunctional enzymes. Individuals with two nonfunctional alleles are thereby expected to be PMs phenotypically and were not detected in this study. Mutant alleles with Arg296-Cys(C2938T) and Ser476-Thr(G4268C) substitutions known to reduce enzyme activity and with silent mutations at 1749 were studied; mutation frequencies were 17.9%, 64.7%, and 66%, respectively. The mutation frequency at 2938 was especially reduced by half when compared to that of Caucasians (32.4%).

Alleles↗
PubMed2005

Mutation analysis of p53, K-ras, and BRAF genes in colorectal cancer progression.

Gene mutations in APC, K-ras, and p53 are thought to be essential events for colorectal cancer development. Recent data seem to indicate that K-ras and p53 mutations rarely co-exist in the same tumor, indicating that these alterations do not represent a synergistic evolutionary pathway. Moreover, an inverse relation between K-ras gene activation and BRAF mutations has been demonstrated, suggesting alternative pathways for colorectal cancer transformation. To reconstruct the chronological modulation of these gene mutations during cell transformation and colorectal cancer progression, mutations of p53, K-ras, and BRAF genes were analyzed by Single Strand Conformation Polymorphism (SSCP) or sequencing analysis in 100 colorectal cancer samples, evenly distributed among different Dukes' stages. We found mutations in p53, K-ras, and BRAF genes in 35%, 30%, and 4% of tumors, respectively, and observed a minimal or no co-presence of these gene alterations. Moreover, the frequency of molecular p53 mutations increased as tumor stage increased, suggesting an important role for this gene in the progression of colorectal cancer. Conversely, K-ras or BRAF genes were not related to tumor stage or location. These data seem to indicate the absence of a co-presence of the genes, highlighting the possibility of multiple pathways for colorectal tumor progression. Moreover, mutations in p53, K-ras, and BRAF are not present in about one-third of colorectal cancers and therefore other gene mutations need to be investigated to better understand molecular mechanisms at the basis of cell transformation and the progression of colorectal cancer.

Adult↗