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Results for “mutation analysis”

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PubMed2002

Argininosuccinate lyase (ASL) deficiency: mutation analysis in 27 patients and a completed structure of the human ASL gene.

Argininosuccinic aciduria is an urea cycle disorder caused by argininosuccinate lyase (ASL) deficiency and is inherited as an autosomal-recessive trait. To date, mutation analysis has been limited because of incomplete sequence information about the human ASL gene. As a consequence, only 12 different mutations in 12 patients have been reported, so far. This study aimed at the completion of the structure and the sequence of the human ASL gene, the development of a genomic DNA-based system for mutation analysis and, finally, the characterisation of the molecular genetic background of ASL deficiency in 27 unrelated patients. This report provides transcript variants, the complete sequence of the human ASL gene and a complete ASL homologue on chromosome 22. The homologue was formerly thought to be a pseudogene but was found, in this study, to be correlated with an immunoglobulin-lambda-like mRNA. On the basis of the novel sequence data, a polymerase reaction chain system for mutation-screening in all 16 coding exons of the ASL gene was established and applied to the analysis of the ASL-deficient patients. We found mutations in all of the 54 investigated alleles and identified 23 (19 novel) different mutations. Some mutational hot-spots were identified (mainly in exons 4, 5, and 7) as were several predominant mutations: IVS5+1G-->A (15 alleles), c.532G-->A (7), c.346C-->T (6), c.1153C-->T (4). This study introduces a system for mutation analysis in the ASL gene, thereby elucidating the genetic background of ASL deficiency, which was found to be associated with considerable allelic heterogeneity.

Argininosuccinate Lyase↗

PubMed2005

Mutational analysis of the MRP2 gene and long-term follow-up of Dubin-Johnson syndrome in Japan.

BACKGROUND: Recent studies have indicated that dysfunction or loss of the multidrug resistance protein 2 (MRP2) is the molecular basis of Dubin-Johnson syndrome (DJS). To clarify the genetic basis of the disease and the long-term stability of serum bilirubin levels, we conducted a mutational analysis of the MRP2 gene and followed up serum bilirubin levels in Japanese DJS patients 30 years after they were originally diagnosed, based on traditional criteria. METHODS: Patients were interviewed by telephone, and blood tests, including a genetic analysis of MRP2, were performed on the patients and family members who gave informed consent. RESULTS: Over the 30 years, hyperbilirubinemia remained unchanged in four of the five patients studied, while it worsened in 1 patient whose DJS was complicated by chronic hepatitis C. From an MRP2 gene mutational analysis, six mutations, including the novel mutation 1177C>T, were found. Three patients of a consanguineous family were homozygotes for three mutations (298C>T, 1967+2T>C, and 2439+2T>C). Two patients were compound heterozygotes (1177C>T/2302C>T and 1967+2T>C/2026G>C). A familial study showed no difference in serum bilirubin levels between mutant/wild heterozygotes and wild/wild homozygotes. CONCLUSIONS: The hyperbilirubinemia of four Japanese patients with DJS, one of whom had a novel mutation, 1177C>T, of the MRP2 gene, had not worsened with aging.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1997

Persistent hyperinsulinaemic hypoglycaemia of infancy: therapy, clinical outcome and mutational analysis.

UNLABELLED: Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) is an autosomal recessive disorder characterized by irregular insulin secretion leading to hypoglycaemia. Recently, mutations in the sulphonylurea receptor (SUR) have been described in association with PHHI. We studied clinical symptoms, therapy, long-term outcome and mutational analysis in 14 patients with PHHI. In 8 patients subtotal pancreatectomy was performed whereas 6 responded to conservative treatment with diazoxide. Psychomotor retardation was found in 6 patients, most of them after a delayed diagnosis. A G-to-A point mutation in one allele of the SUR gene was detected by loss of a MspI restriction site in only one patient. CONCLUSION: Early diagnosis and therapy in PHHI is essential to prevent brain damage. In one patient mutational analysis suggested compound heterozygosity for a known and an as yet unidentified mutation in the SUR gene.

ATP-Binding Cassette Transporters↗

PubMed2002

14-3-3 protein is a component of Lewy bodies in Parkinson's disease-mutation analysis and association studies of 14-3-3 eta.

Mutations in alpha-synuclein have been identified in some rare families with autosomal dominant Parkinson's disease (PD). The synuclein gene family shares physical and functional homology with 14-3-3 proteins and binds to 14-3-3 proteins and to its ligands. We therefore investigated whether 14-3-3 proteins are also involved in the pathogenesis of PD. Here we demonstrate that 14-3-3 proteins are colocalized with Lewy bodies in PD. We investigated the 14-3-3 eta (YWHAH) gene by mutation analysis and association studies as it maps to human chromosome 22q12.1-q13.1, a region which has been recently implicated in PD and carried out immunohistochemical studies of Lewy bodies with two different 14-3-3 eta antibodies. In 358 sporadic and familial PD patients, disease causing mutations were not identified. Furthermore, association studies with intragenic polymorphisms do not provide evidence for an involvement of 14-3-3 eta in the pathogenesis of PD. In accordance with these findings, there was no staining of substantia nigra Lewy bodies with antibodies specific for the 14-3-3 eta subunit.

14-3-3 Proteins↗

PubMed2003

Mutation analysis of BrCA1, BrCA2, and p53 versus soluble HLA class I and class II in a case of familial endometriosis.

OBJECTIVE: To investigate possible correlation(s) between mutations of BrCA1, BrCA2, and p53 genes versus soluble HLA expression in familial endometriosis. DESIGN: Mutation analysis. SETTING: University teaching departments and hospital. PATIENT(S): A family with seven women in two generations with familial endometriosis. INTERVENTION(S): Mutation analysis of BrCA1, BrCA2, and p53 genes. MAIN OUTCOME MEASURE(S): A point mutation of the BrCA1 gene appears to inhibit soluble HLA secretion. RESULT(S): Among the three genes examined, only the BrCA1 gene showed a T to A mutation at position 3232 that correlates with total abolishment of both class I and class II antigen release. CONCLUSION(S): A possible correlation between a BrCA1 mutation and soluble HLA expression appears to exist. The mutation is not stage dependent and seemingly influences the secretion of both class I and class II antigens that are totally absent from the serum of only one family member.

Aged↗

PubMed2001

Mutational analysis of the Drosophila homothorax gene.

The homothorax (hth) gene is involved in multiple aspects of embryonic and adult fly development. It encodes a homeodomain-containing protein of the MEIS family and was shown to regulate the subcellular localization of the homeotic protein cofactor Extradenticle (EXD). The HTH protein contains a TALE class homeodomain and a conserved MH domain, which is required for its interaction with EXD. In this work, we describe the structure of the hth locus, characterize at the molecular level a collection of mutant alleles of hth, and discuss the correlation between the identified structural defects and their consequent phenotypes. The hth locus spans more than 100 kb and contains 14 exons. Several of the exon-intron boundaries within the homeodomain and the MH domain-coding regions are conserved between Drosophila and Caenorhabditis elegans. The analysis of hth mutations demonstrates that the homeodomain of HTH is not required for nuclear localization of EXD and that the MH domain-containing first 240 residues are sufficient for nuclear localization of both EXD and HTH. Mutations that alter or delete the homeodomain cause only partial homeotic transformations in the PNS, whereas mutations affecting the MH domain cause distinct and more severe PNS phenotypes. These observations may suggest that driving nuclear localization of EXD is the main role of HTH in patterning the embryonic PNS. They may also suggest that homeodomain-defective HTH protein retains some of its transcription-regulating functions by binding DNA via its interaction with EXD.

Alleles↗

PubMedDate not supplied

Alpha-mannosidosis and mutational analysis in a Turkish patient.

We present a case of alpha-mannosidosis with its mutational analysis. She was referred to our hospital with the provisional diagnosis of mucolipidosis. She was the first child of second-degree relative parents. She had a coarse face with flat and wide nasal bridge, hepatosplenomegaly, umbilical hernia, lumbar gibbus, motor and mental retardation and deafness. On peripheral blood smear, lymphocytes revealed vacuoles and neutrophils contained some granules resembling Reilly bodies seen in mucopolysaccharidosis (MPS). Based on these findings, the diagnosis of alpha-mannosidosis was suspected. Her urine oligosaccharide chromatography showed an abnormal pattern with a heavy trisaccharide band. Enzyme studies on white cells confirmed a deficiency of alpha-mannosidase activity, which was 2.6 micromol/g/hr. Her DNA analysis showed a S453Y mutation.

Child, Preschool↗

PubMed2002

Mutation analysis of MLH1 and MSH2 genes performed by denaturing high-performance liquid chromatography.

Mutation analysis of large genes, such as MSH2 and MLH1, is time-consuming and expensive. We investigated the sensitivity and specificity of DHPLC analysis for the detection of mutations within both MSH2 and MLH1. Studies included a series of 46 patients affected by colorectal cancer from HNPCC families. We confirmed 19 changes previously identified by DNA sequencing and, in a blind study, an additional 16 rare alterations including four mutations not previously described. Generally, false negative results were not observed. Elution profiles were highly characteristic for a given change and in 98.5% cases allowed the distinction between novel alterations and previously identified mutations and polymorphisms. For the detection of changes in almost all amplicons, it was sufficient to use just one denaturing temperature. DHPLC was confirmed to be highly sensitive, specific and a cost-effective technique with particularly high potential for the detection of MSH2 and MLH1 gene mutations in the diagnostic setting.

Adaptor Proteins, Signal Transducing↗

PubMed2006

CAV3 gene mutation analysis in patients with idiopathic hyper-CK-emia.

As caveolin-3 deficiencies may explain persistent hyper-CK-emia, we performed CAV3 gene mutation analysis and immunohistochemistry for caveolin-3 in 31 patients with idiopathic hyper-CK-emia. In 2 of 29 patients who donated blood, variants in the CAV3 gene were detected. Although immunohistochemical analysis strongly suggested that caveolin-3 was properly localized in the muscle tissue of the two affected patients, it may not function normally and could thus explain their persistent hyper-CK-emia. Our findings contribute to the clarification of unexplained persistent hyper-CK-emia, but further research is needed before CAV3 gene mutation analysis becomes part of the routine evaluation of these patients.

Adult↗

PubMed2001

Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets.

PHEX is the gene defective in X-linked hypophosphatemic rickets. In this study, analysis of PHEX revealed mutations in 22 hypophosphatemic rickets patients, including 16 of 28 patients in whom all 22 PHEX exons were studied. In 13 patients, in whom no PHEX mutation had been previously detected in 17 exons, the remaining 5 PHEX exons were analyzed and mutations found in 6 patients. Twenty different mutations were identified, including 16 mutations predicted to truncate PHEX and 4 missense mutations. Phenotype analysis was performed on 31 hypophosphatemic rickets patients with PHEX mutations, including the 22 patients identified in this study, 9 patients previously identified, and affected family members. No correlation was found between the severity of disease and the type or location of the mutation. However, among patients with a family history of hypophosphatemic rickets, there was a trend toward more severe skeletal disease in patients with truncating mutations. Family members in more recent generations had a milder phenotype. Postpubertal males had a more severe dental phenotype. In conclusion, although identifying mutations in PHEX may have limited prognostic value, genetic testing may be useful for the early identification and treatment of affected individuals. Furthermore, this study suggests that other genes and environmental factors affect the severity of hypophosphatemic rickets.

Adolescent↗

PubMed1999

Mutation analysis of the BRCA1 and BRCA2 genes in the Belgian patient population and identification of a Belgian founder mutation BRCA1 IVS5 + 3A > G.

Since the identification of the BRCA1 and BRCA2 genes, several hundred different germline mutations in both genes have been reported. Recurrent mutations are rare and mainly due to founder effects. As the mutational spectrum of the BRCA1 and BRCA2 genes in the Belgian patient population is largely unknown, we initiated mutation analysis for the complete coding sequence of both genes in Belgian families with multiple breast and/or ovarian cancer patients and in "sporadic" patients with early onset disease. We completed the analysis in 49 families and in 19 "sporadic" female patients with early onset breast and/or ovarian cancer. In 15 families we identified a mutation (12 mutations in BRCA1 and 3 mutations in BRCA2). In 5 apparently unrelated families the same splice site mutation was identified (BRCA1 IVS5 + 3A > G). Haplotype analysis revealed a common haplotype immediately flanking the mutation in all families suggesting that disease alleles are identical by descent. In none of the 19 sporadic patients was a mutation found.

Adult↗

PubMed2005

Combined small-cell carcinoma of the stomach: p53 and K-ras gene mutational analysis supports a monoclonal origin of three histological components.

Primary small-cell carcinoma (SmCC) is extremely rare in stomach. We reported an autopsy case of combined gastric SmCC with p53 and K-ras mutational analysis. Histologically, the tumour was composed of well-differentiated adenocarcinoma surrounding the central dominant SmCC component with scattered nests of squamous cell carcinoma. Immunohistochemically, all the neoplastic components revealed strong expression for p53 protein. Based on these findings, we hypothesized that these histologically different components originated from a same progenitor cell that possessed p53 mutation. Using Laser-capture microdissection technique and mutational analysis, we identified the same point mutations of p53 gene (A-->G transversion in codon 239) and K-ras gene (G-->A transversion in codon 13) in all the neoplastic components, but not in the adjacent normal gastric epithelium. Our results strongly support a hypothesis that the combined SmCC of stomach in this case was of monoclonal origin.

Adenocarcinoma↗

PubMed2006

SLC7A9 cDNA cloning and mutational analysis of SLC3A1 and SLC7A9 in canine cystinuria.

Cystinuria is a genetic disorder in the domestic dog that leads to recurrent urolith formation. The genetic basis of the disorder is best characterized in humans and is caused by mutations in one of the amino acid transporter genes SLC3A1 or SLC7A9, which results in hyperexcretion of cystine and the dibasic amino acids in the urine and subsequent precipitation of cystine due to its low solubility in urine. In this study we describe the cloning of the canine SLC7A9 cDNA and present a thorough mutation analysis of the coding SLC3A1 and SLC7A9 regions in cystinuric dogs of different breeds. Mutation analysis of the two cystinuria disease genes revealed one SLC7A9 mutation (A217T) and two SLC3A1 mutations (I192V and S698G) in French and English Bulldogs that affect nonconserved amino acid residues, arguing against functional impact on the proteins. The absence of deleterious mutations linked to cystinuria in the remainder of our panel of cystinuric dogs is surprising because SLC3A1 or SLC7A9 mutations explain approximately 70% of all human cystinuria cases studied. The present study, along with previous investigations of canine and human cystinuria, implies that regulatory parts of the SLC3A1 and SLC7A9 genes as well as other unknown genes may harbor mutations causing cystinuria.

Amino Acid Transport Systems, Basic↗

PubMed2003

Mutational analysis of BRCA2 in Spanish breast cancer patients from Castilla-Leon: identification of four novel truncating mutations.

Mutations in BRCA1 and BRCA2 account for approximately 5% of the overall familial risk of breast cancer. We have carried out a mutational analysis of the entire coding sequence of the BRCA2 gene in 150 breast cancer patients from Castilla-Leon by two different methods: Protein Truncation Test (PTT) and Conformation-Sensitive Gel Electrophoresis (CSGE). We have identified 10 distinct truncating mutations of the BRCA2 protein in 17 unrelated patients. Four mutations had not been previously described in any other population: two nonsense, Q2354X and K3083X, and two frameshift deletions, 6126delT and 5374delTATG. Moreover, three further mutations, 1538delAAGA, E1308X and S2219X, had not been reported in Spanish patients until now. Five mutations were recurrent: 3036delACAA, 1538delAAGA, 9538delAA and the novel mutations, 6126delT and 5374delTATG. The most prevalent mutation was 3036delACAA found in four unrelated patients.

Adult↗

PubMed1998

Diagnosis of spinal muscular atrophy in an SMN non-deletion patient using a quantitative PCR screen and mutation analysis.

We report a child with clinical findings consistent with Werdnig-Hoffmann disease (spinal muscular atrophy type I) who was found not to have the homozygous absence of the survival motor neurone (SMN(T)) gene observed in approximately 95% of spinal muscular atrophy patients. A quantitative PCR based dosage assay for SMN(T) copy number showed that this patient possessed a single copy of the SMN(T) gene. Heteroduplex and sequence analysis of the remaining copy of SMN(T) showed a 2 base pair deletion within exon 4 which produces a frameshift and premature termination of the deduced SMN(T) protein. This protocol of initial SMN(T) gene dosage analysis followed by mutation detection allows identification of SMA compound heterozygotes (patients lacking one copy of SMN(T) and having another mutation in their other copy), thereby increasing the sensitivity of SMA molecular diagnosis.

Cyclic AMP Response Element-Binding Protein↗

PubMed1989

Point mutation analysis in a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method.

We have developed a very rapid procedure for DNA sequence analysis of induced mutations in a typically large mammalian gene. We are able to determine the nature of chemical carcinogen-induced point mutations in the 25 kb Chinese hamster ovary (CHO) cell dihydrofolate reductase gene within two days starting with 5 to 10 x 10(6) cells. The approach is based on the use of rapidly prepared total RNA from which a 730 bp dhfr cDNA is synthesized by reverse transcriptase and amplified by the polymerase chain reaction (PCR) procedure. Genomic DNA can simultaneously be prepared from the same cells. The amplified double-stranded cDNA is then sequenced directly by the dideoxy method using Taq polymerase in the Thermal Cycler (Perkin Elmer/Cetus). We have previously shown that nonsense codons in the dhfr coding sequence often result in greatly reduced steady-state levels of dhfr mRNA (2). The methods described are suitable for mutants of this type which contain only 1 to 2 copies of mRNA per cell. This approach is readily adaptable to other selectable genes and other cell types provided the necessary primers can be prepared.

Animals↗

PubMed2000

Mutation analysis of Gsalpha, adrenocorticotropin receptor and p53 genes in Japanese patients with adrenocortical neoplasms: including a case of Gsalpha mutation.

While the mechanisms of tumorigenesis for adrenocortical neoplasms remain unknown, several genes, such as Gsalpha, ACTH receptor (MC2-R), p53, and p16 tumor suppressor genes, are considered to be candidates for adrenocortical neoplasms. Mutation analysis studies have documented these genes in adrenocortical neoplasms, but these studies focused on the mutation of only one of these genes. In the present study we examined the mutations of three of these genes (Gsalpha, MC2-R, and p53) in adrenocortical neoplasms in Japanese patients. We amplified these genes using polymerase chain reaction and directly sequenced them in 30 functioning adrenocortical neoplasms. As for Gsalpha, we identified a heterogeneous substitution of glutamine to histidine at codon 227 and a gain of an Nru I restriction endonuclease site. The mutation was restricted to adenomatous tissue, and did not occur in the adjacent normal adrenal tissue or leukocytes of the patient. We did not find any mutations in MC2-R and p53. In conclusion, although the contribution of these three genes to adrenocortical tumorigenesis remains to be determined, it is suggested that the mutation of Gsalpha might play a role in functional adrenocortical neoplasms.

Adrenal Cortex Neoplasms↗

PubMedDate not supplied

Germline BRCA1 mutation analysis in Indian breast/ovarian cancer families.

Most of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in two tumor suppressor genes BRCA1 and BRCA2. To explore the contribution of BRCA1 mutations to hereditary breast cancer among Indian women, we examined the coding sequence of the BRCA1 gene in 14 breast cancer patients with a positive family history of breast and/or ovarian cancer. Mutation analysis was carried out using conformation sensitive gel electrophoresis (CSGE) followed by sequencing. Three mutations (21%) in the BRCA1 gene were identified. Two of them are novel mutations of which one is a missense mutation in exon 7 near the RING finger domain, while the other is a one base pair deletion in exon 11 which results in protein truncation. The third mutation, 185 delAG, has been previously described in Ashkenazi Jewish families. To our knowledge this is the first report of a study of germline BRCA1 mutation analysis in familial breast cancer in India. Our data from 14 different families suggests a lower prevalence but definite involvement of germline mutations in the BRCA1 gene among Indian women with breast cancer and a family history of breast cancer.

Breast Neoplasms↗