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PubMed2002

Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: relationship to clinicopathological parameters.

To delineate more precisely the somatic von Hippel-Lindau disease (VHL) gene alteration as well as to elucidate its etiologic role in renal tumorigenesis, we examined a total of 240 sporadic renal cell carcinomas (RCCs) for somatic VHL gene alterations by DNA-SSCP followed by sequencing, methylation-specific PCR assay, microsatellite LOH study, and Southern blot analysis. Intragenic mutation of the VHL gene was found exclusively in clear-cell or variant-type RCCs at a frequency of 51% (104/202). Hypermethylation of the VHL promoter region was detected in an additional 11 clear-cell RCCs. Microsatellite analysis demonstrated that LOH of the VHL locus was found in 140/155 (90%) informative clear-cell RCCs. The VHL gene therefore seems to be inactivated in a two-hit manner by intragenic mutation or hypermethylation plus allelic loss in clear-cell RCC. Genomic rearrangement of the VHL gene detected by Southern analysis was not found (0/216 cases); this is in contrast to germ lines in which Southern aberrations consisted of 7-19% of the mutations. Clinicopathologic data demonstrated that VHL mutation/LOH did not vary according to tumor progression in clear-cell RCC, including tumor diameter, stage, grading, distant metastasis, and lymph node metastasis. Interestingly, VHL mutation was significantly less frequent in RCCs occurring in younger (< or = 55 years) than that in older (> or = 56 years) patients. These data suggested that the inactivation of the VHL tumor-suppressor gene is a specific genetic change in clear-cell RCC, and that it may occur at an early or first step in the clear-cell tumorigenic pathway rather than as a late event.

Adult↗
PubMed2001

MECP2 gene mutation analysis in the British and Italian Rett Syndrome patients: hot spot map of the most recurrent mutations and bioinformatic analysis of a new MECP2 conserved region.

Rett syndrome (RTT) is an X-linked dominant neurological disorder, which appears to be the most common genetic cause of profound combined intellectual and physical disability in Caucasian females. This syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of unknown target genes. We report a detailed mutational analysis of a large cohort of RTT patients from the UK and Italy. This study has permitted us to produce a hot spot map of the mutations identified. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, conserved among brain-specific regulatory factors.

Adolescent↗
PubMed1996

Prognostic value of TP53 and K-ras-2 mutational analysis in stage III carcinoma of the colon.

BACKGROUND: Genetic mutations involving oncogenes and tumor-suppressor genes occur in carcinogenesis, and may affect biologic behavior of neoplasms. In this study, we analyzed the prognostic value of mutational analysis in colon carcinoma. PATIENTS AND METHODS: Archival pathology specimens from 70 consecutive patients, resected for stage III colon carcinoma, were analyzed for point mutations by amplification and direct sequencing of exons from the K-ras-2 and the TP53 genes (topographic genotyping). Mutations were compared with adverse histopathologic features (poor differentiation, vascular and lymphatic invasion, mucin production) as prognostic markers. RESULTS: Five-year survival was 75% in patients with nonmutated lesions, significantly lower (21%) with TP53 mutations (P = 0.01), and intermediate with K-ras-2 only (45%) or both K-ras-2 and TP53 mutations (36%). A TP53 mutation carried the highest relative risk of death (2.39; 95% confidence interval, 1.29 to 4.42; P = 0.006). There was an additive effect on the risk of death between TP53 mutations and adverse histopathologic features. CONCLUSIONS: The information derived from mutational analysis is creating new prognostic variables that may play a role in the choice of therapy for colorectal carcinoma.

Aged↗
PubMed2005

Meningioma arising in Werner syndrome confirmed by mutation analysis.

OBJECTIVE AND IMPORTANCE: Meningioma arising in Werner syndrome has been described previously, but never in association with a mutation analysis. We present the first reported case of meningioma in a patient with Werner syndrome and a confirmed major mutation. In addition, we review 27 previously reported patients with meningioma associated with Werner syndrome. CLINICAL PRESENTATION: We report a 56-year-old Japanese woman with Werner syndrome and a meningioma. She presented with pain and redness of the right eye and a headache. Cranial CT revealed a tumor the in right frontal and temporal lobes. Pathological examination after surgical removal confirmed meningioma. She displayed typical features of Werner syndrome including juvenile cataract, short stature and low weight, a bird-like face, a hoarse voice, and dry, atrophic, pigmented skin. INVESTIGATION: To confirm the clinical diagnosis, a mutation analysis based on the mutant allele-specific amplification (MASA) method was performed. CONCLUSION: Mutation analysis of peripheral blood leukocyte DNA showed amplification of the mutation 4/4. There were 22 patients with Werner syndrome and meningioma reported from Japan and 5 from outside Japan. There was only one malignant meningioma. Meningiomas in Werner syndrome have a higher frequency in males and occur at a younger age than those of the general population.

Adult↗
PubMed2000

Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation.

For many years it has been noted that there is a correlation between acetylation of histones and an increase in transcriptional activity. One prediction, based on this correlation, is that hypomorphic or null mutations in histone deacetylase genes should lead to increased levels of histone acetylation and result in increased levels of transcription. It was therefore surprising when it was reported, in both yeast and fruit flies, that mutations that reduced or eliminated a histone deacetylase resulted in transcriptional silencing of genes subject to telomeric and heterochromatic position effect variegation (PEV). Here we report the first mutational analysis of a histone deacetylase in a multicellular eukaryote by examining six new mutations in HDAC1 of Drosophila melanogaster. We observed a suite of phenotypes accompanying the mutations consistent with the notion that HDAC1 acts as a global transcriptional regulator. However, in contrast to recent findings, here we report that specific missense mutations in the structural gene of HDAC1 suppress the silencing of genes subject to PEV. We propose that the missense mutations reported here are acting as antimorphic mutations that "poison" the deacetylase complex and propose a model that accounts for the various phenotypes associated with lesions in the deacetylase locus.

Alleles↗
PubMed2000

Mutational analysis of GNAS1 in patients with pseudohypoparathyroidism: identification of two novel mutations.

Pseudohypoparathyroidism (PHP) refers to two major variants that generally coexist in the same family, PHP type Ia (PHP Ia), in which both PTH resistance and a constellation of physical features, termed Albright's hereditary osteodystrophy (AHO), are present, and pseudopseudohypoparathyroidism (PPHP), in which AHO occurs without PTH resistance. Most patients with PHP Ia show a partial deficiency (50%) of Gs activity, due to loss of function mutations in Gsalpha gene (GNAS1). The present study reports clinical, biochemical, and molecular data of 8 unrelated families with PHP Ia and PPHP. The 13 exons of GNAS1 were screened for mutations by PCR and direct sequencing of the amplified products. We detected heterozygous mutations in the affected members of the 4 families in which PHP Ia was present. In 2 families 2 previously reported deletions in exons 5 and 7 were found, whereas in the other 2 families, 2 novel frameshift deletions were identified in exons 1 and 11, causing a premature stop codon in the mutant allele. No mutation was detected in the families in which PPHP was the only clinical manifestation. In conclusion, we report the first mutational analysis of Italian patients with PHP Ia and PPHP, and we describe two novel deletions in GNAS1. Furthermore, we confirm that these mutations cannot be detected in families with isolated PPHP, suggesting that these forms of AHO are genetically distinct from PHP Ia.

Adolescent↗
PubMed2000

Mutation analysis of Gaucher disease using dot-blood samples on FTA filter paper.

FTA((R)) filter papers were used as an effective means of blood cell collection, genomic DNA processing, and delivery. Minute blood samples (<1 microL) were collected onto the filters via a simple lateral prick to the patient's finger, circumventing the need for intravenous blood puncture. Collected samples, which are stable at room temperature for several years, were subsequently sent through the postal system to the diagnostic laboratory, bypassing the stringent requirements of courier delivery. Using this method, we performed restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis on prevalent mutations among Canadian and Chinese Gaucher disease patients. Of the 12 alleles (six patients) analyzed, 42% (5/12) have the N370S mutation and 58% (7/12) the L444P mutation, the two most common alleles found among Jewish and non-Jewish Gaucher disease patients. Uniquely, a Chinese Gaucher disease patient was found to have an N370S mutation. Although the presence of the N370S mutation is regarded as common in other ethnic groups, previous to this report it had not been noted in an individual of Asian descent. PvuII polymorphism analysis showed that the N370S mutation found in the Chinese patient was linked to the Pv1.1(-) polymorphism, as has been previously seen in the Jewish population. The use of FTA((R)) filter paper facilitates access of samples to diagnostic centers, and therefore provides an effective means of performing population-based mutational analysis of Gaucher disease internationally.

Adult↗
PubMed1999

Identification of X-linked severe combined immunodeficiency by mutation analysis of blood and hair roots.

Severe combined immunodeficiency is a heterogenous syndrome of varied genetic origins of which the X-linked type is the commonest (XSCID). The most sensitive method for diagnosis of XSCID in the absence of X-linked inheritance pattern is by mutation analysis. In this report we have performed mutation analysis in 13 unrelated boys transplanted (BMT) for SCID without a known cause to determine the frequency of XSCID. Five boys had an affected male relative. We also assessed the utility of hair roots for children without pre-transplant blood stored for mutation analysis since donor genotype was expressed in peripheral blood post BMT. Screening was performed by analysis of single-strand conformational polymorphism (SSCP) followed by sequencing of candidate exons. Mutations were found in 11 cases, of which six were sporadic, and maternal mosaicism was found in one family. Three mothers of the six sporadic cases were identified as carriers. The majority (6/8) of boys with SCID had gammac deficiency despite the absence of X-linked inheritance pattern. The significant frequency of de novo mutations and the occurrence of maternal germline mosaicism highlights the importance of mutation analysis. The strategy of using DNA from hair roots was particularly valuable where no pre-transplant blood was stored. Characterization of the mutations will also enable research into the correction of these genetic defects.

DNA↗
PubMed1995

A fluorescent multiplex ARMS method for rapid mutation analysis.

The amplification refractory mutation system (ARMS) is a powerful technique for the identification of mutations. We present a modification of this technique involving coamplification of normal and mutant alleles with primers that differ in length by three to five bases. Fluorescent labelling allows exact sizing of the amplification products on an automated DNA fragment analyser. We have used the fluorescent multiplex ARMS method for the identification of common phenylketonuria mutations in Northern Ireland (R408W, 165T and F39L) together with the analysis of a polymorphic short tandem repeat site at the human phenylalanine hydroxylase locus. This provides an efficient and inexpensive first step for diagnostic mutation analysis in our population.

Alleles↗
PubMed1999

Mutational analysis of beta-catenin gene in Japanese ovarian carcinomas: frequent mutations in endometrioid carcinomas.

To investigate the contribution of the beta-catenin gene to the development of ovarian carcinomas, mutational analysis of exon 3 of the beta-catenin gene was conducted. We analyzed 61 primary ovarian carcinomas, consisting of 49 non-endometrioid-type and 12 endometrioid-type tumors, for genetic alteration of the beta-catenin gene. Five carcinomas showed beta-catenin mutations (S37C, T41I, T41A), including 4 (33%) of 12 endometrioid-type tumors and 1 (14%) of 7 mucinous-type tumors. All of these mutations altered at the serine/threonine residues that are potential sites of GSK3-beta phosphorylation. We detected no carcinomas with interstitial deletion involving exon 3 of beta-catenin. Furthermore, we immunohistochemically studied 27 of the 61 ovarian carcinomas. Both nuclear and cytoplasmic beta-catenin expressions were demonstrated in 4 of the 27 ovarian carcinomas for which tissue samples were available for examination. All 4 cases exhibited mutations in exon 3 of beta-catenin, including a mucinous carcinoma. Our results suggested that beta-catenin gene mutation at potential GSK3-beta phosphorylation sites results in accumulation of beta-catenin protein within the cells and its translocation to nuclei. Accumulated beta-catenin protein may be involved in the development of endometrioid-type ovarian carcinomas, and some mucinous-type ovarian carcinomas.

Adult↗
PubMed1992

Mutation analysis of phenylketonuria in Spain: prevalence of two Mediterranean mutations.

We have screened seven Spanish phenylketonuric (PKU) families for the most prevalent Mediterranean and Caucasian mutations, and have subsequently found mutations P281L and IVS10. We have analyzed these two mutations in 23 of our patients. The frequencies found correspond to those of Mediterranean countries, such as Italy. This is the first report of a mutation analysis in the Spanish PKU population.

Gene Frequency↗
PubMed1996

Mutational analysis of the catalytic subunit of muscle protein phosphatase-1.

A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli. The selection of the sites to be mutated was based on sequence alignments which showed the existence of a number of invariant residues when eukaroytic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosinetetraphosphatase [Barton et al. (1995) Eur. J. Biochem. 220, 225-237]. In other studies, it had been shown that PP1 is a metalloprotein [Chu et al. (1996) J. Biol. Chem. 271, 2574-2577], and in this study, we have largely focused on invariant histidine and aspartate residues which may be involved in metal binding. The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124, and R96E. The results showed that mutation of H66, H248, D64, and D92 resulted in severe loss of catalytic function. Mutation of D95, N124, and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins. The results of the mutational analysis are consistent with the involvement of conserved His and Asp residues in metal binding, and are discussed in the context of the recently described crystal structure of PP1 [Goldberg et al. (1995) Nature, 376, 745-753], which reveals that PP1 possesses a bimetallic center at the active site. The behavior of the D95, R96, and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group.

Amino Acid Sequence↗
PubMed2003

Toward new strategies to select young endometrial cancer patients for mismatch repair gene mutation analysis.

PURPOSE: To determine the frequency of mismatch repair (MMR) gene germline mutations in endometrial cancer patients who were diagnosed at less than 50 years of age; to relate the presence of mutations to family history, histopathologic data, presence of tumor microsatellite instability (MSI), and immunostaining; and to formulate criteria for genetic testing in these patients. PATIENTS AND METHODS: Endometrial cancer patients (N = 58), who were diagnosed at less than 50 years of age, were included and questioned about their family history. Mutation analysis of the MLH1, MSH2, and MSH6 genes was performed (denaturing gradient gel electrophoresis and sequence analysis to detect small mutations and multiplex ligation-dependent probe amplification to detect large deletions or duplications). For MSI analysis, five consensus markers were used, and immunostaining of the three MMR proteins was performed. RESULTS: In five of 22 patients with a positive first-degree family history for hereditary nonpolyposis colorectal cancer (HNPCC)-related cancers, pathogenic germline mutations were found (one MLH1, three MSH2, and one MSH6). Four mutation carriers belonged to families fulfilling the revised Amsterdam criteria. No mutations were found in the 35 patients without such family history (P =.006). MSI was detected in 20 of 57 cancers, among which four were from mutation carriers. In 23 of 51 cancers, one or more MMR protein was absent; in all five mutation carriers, immunostaining indicated the involved MMR gene. CONCLUSION: In 23% of the young endometrial cancer patients with at least one first-degree relative with an HNPCC-related cancer, an MMR gene mutation was detected. Therefore, presence of an HNPCC-related cancer in a first-degree relative seems to be an important selection criterion for mutation analysis. Subsequent immunostaining of MMR proteins will point to the gene(s) that should be analyzed.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Mutation analysis of the KIF21A gene in an Indian family with CFEOM1: implication of CpG methylation for most frequent mutations.

PURPOSE: To carry out the mutation analysis of the KIF21A gene in a four-generation Indian family affected with CFEOM1 and to find out the molecular basis of the most frequent mutations c.2860C>T and c.2861G>A in exon 21 of the KIF21A gene. METHODS: Mutational analysis was carried out by direct automated sequencing of the PCR products from exons 8, 20, and 21 of the KIF21A gene. Allele specific oligo hybridization analysis was carried out to study the segregation of the mutation within the family. Methylation status of the mutated CpG dinucleotide in exon 21 was detected using bisulfite genomic sequencing technique on genomic DNA isolated from blood and sperms. RESULTS: We found a previously reported missense mutation c.2860C>T (p.954R>W) in exon 21 of the KIF21A gene in our family. This mutation was found in a CpG dinucleotide. Bisulfite genomic sequencing revealed that all the CpG dinucleotides in exon 21 including the one which harbored the two most frequent mutations were methylated both in the genomic DNA from blood and sperms. CONCLUSIONS: CFEOM1 phenotype in our family was caused by a previously reported most frequent missense mutation, c.2860C>T. This mutation occurred at the C residue in a CpG dinucleotide, which was found to be methylated. Previous work has demonstrated that this CpG dinucleotide is a mutational hotspot in the KIF21A gene, and our finding suggests that its high mutability may result, in part, from its methylated state.

CpG Islands↗
PubMed1999

A highly sensitive, fast, and economical technique for mutation analysis in hereditary breast and ovarian cancers.

Mutation analysis of complex genes without hotspots for sequence variations, such as BRCA1, is time-consuming and expensive. Of all currently available methods, direct sequencing has the highest sensitivity, but also the highest costs. Other techniques, such as SSCP, DGGE, and PTT, are more economical but, depending on the experience of the investigator, have at best a sensitivity of 90%. We investigated in a prospective study the feasibility and accuracy of the DHPLC technique. We present the application of the DHPLC protocol for BRCA1 mutation detection on a HPLC device from Bio-Tek Kontron Instruments (Neufahrn, Germany). DNA from 46 women with hereditary breast and ovarian cancer undergoing genetic testing for BRCA1 mutations were tested. Of 1,518 amplicons analyzed by DHPLC, corresponding to 33 fragments spanning the entire BRCA1 gene, 626 were also directly sequenced. The comparison demonstrated that DHPLC detected all alterations found by direct sequencing. No false-positive signals were seen in cases of homozygous sequences. Further, no false-negative results were ever obtained in women with mutations or polymorphisms, or both. In cases of known genetic variations, the nature of the alterations could be predicted by DHPLC. We also compared different separation matrices. Up to about 500 injections, no significant differences in sensitivity could be observed between poly(styrene divinylbenzene) and end-capped silica based columns. However, after more than 500 injections, the resolution of hetero- from homoduplex deteriorated rapidly on silica columns.

BRCA1 Protein↗
PubMed2002

Transferrin receptor 2 (TfR2) and HFE mutational analysis in non-C282Y iron overload: identification of a novel TfR2 mutation.

Hereditary hemochromatosis (HH) is classically associated with a Cys282Tyr (C282Y) mutation of the HFE gene. Non-C282Y HH is a heterogeneous group accounting for 15% of HH in Northern Europe. Pathogenic mutations of the transferrin receptor 2 (TfR2) gene have been identified in 4 Italian pedigrees with the latter syndrome. The goal of this study was to perform a mutational analysis of the TfR2 and HFE genes in a cohort of non-C282Y iron overload patients of mixed ethnic backgrounds. Several sequence variants were identified within the TfR2 gene, including a homozygous missense change in exon 17, c2069 A-->C, which changes a glutamine to a proline residue at position 690. This putative mutation was found in a severely affected Portuguese man and 2 family members with the same genotype. In summary, pathologic TfR2 mutations are present outside of Italy, accounting for a small proportion of non-C282Y HH.

Canada↗
PubMed1993

Genotypic mutation analysis by RFLP/PCR.

In most cases somatic mutations in disease-related genes do not give rise to a functional change of the mutated cell which would allow its isolation or expansion in vitro. Therefore, selection of mutated cells on the basis of an altered phenotype has to be replaced by biochemical separation and detection of the altered sequence of the gene of interest. In the RFLP/PCR (RFLP, restriction fragment length polymorphism) approach of 'genotypic' mutation analysis base pair changes, small deletions and insertions are measured which are located in a restriction enzyme recognition sequence and render this site resistant to cleavage by the corresponding endonuclease. The resistant DNA sequence containing the mutated site is amplified by PCR only after wild-type DNA has been eliminated by restriction digestion. Amplified DNA is directly sequenced or cloned into lambda gt10 and mutants are quantitated by oligonucleotide plaque hybridization. Absolute mutation frequencies are estimated relative to an internal 'mutant standard'. The RFLP/PCR protocol has been developed with mixtures of plasmid constructs containing wild-type inserts of the human c-H-ras1 protooncogene or inserts with base pair changes in an MspI site, a PvuII site and a TaqI site of this gene. As few as 1-5 mutated copies could be rescued from 10(7)-10(9) copies of the corresponding wild-type sequence. The RFLP/PCR protocol was successfully applied to the determination of N-ethyl-N-nitrosourea-induced mutations in codon 12 of c-H-ras1 (MspI site 1695-1698) and codon 248 of the p53 tumor suppressor gene (MspI site 14067-14070) in human skin fibroblasts. The RFLP/PCR approach holds promise for molecular toxicology and epidemiology.

Base Sequence↗