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At least 109 records · Page 6Linked to original sources
PubMed1996

A sensitive mutation screening strategy for Fabry disease: detection of nine mutations in the alpha-galactosidase A gene.

Fabry disease is an X-linked recessive lysosomal storage disorder caused by a deficiency of alpha-galactosidase A (alpha-gal; EC 3.2.1.22). In the past, it has been difficult to give an unequivocal diagnosis of carrier status in Fabry disease because of the overlap between normal and heterozygote enzyme levels. To facilitate rapid and accurate carrier and hemizygote detection, a mutation detection strategy was devised to determine the lesion in our Fabry disease patients. The seven alpha-gal exons and adjacent intron boundaries from a representative member of each kindred were PCR amplified and analysed for the presence of sequence alterations by single-stranded conformation polymorphism (SSCP) analysis followed by PCR sequencing. Here we report the use of this strategy in the detection and analysis of the causative mutations in 9 patients with classic severe Fabry disease. Three deletions of 1-, 2-, and 3-bp (987delC, 717delAA, and delta E358), five amino acid substitutions (C52R, G128E, P205T, M284T, and N298K) and a mutation that affects the initiating methionine (M1I) were found in these patients. Counting a previously reported mutation, this strategy has now successfully detected all the Fabry disease mutations present in the 10 kindreds that have been analysed.

Adult↗
PubMed2006

Mutation screening of the PTPN11 gene in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disease and a major cause of sudden death. It is an autosomal dominant disorder predominantly caused by mutations in genes encoding for sarcomeric proteins. Only 50-60% of HCM probands have mutations in known genes suggesting the presence of additional disease genes. Noonan and LEOPARD syndromes are characterised by multiple dysmorphia and cardiac defects with HCM present in approximately 20% of cases. Both syndromes are caused by mutations in the PTPN11 gene which codes for the protein tyrosine phosphatase SHP-2. It is suspected but unproven that the cardiac phenotype may predominate or even be present in isolation. In order to determine possible involvement of this gene in the pathogenesis of HCM, we performed mutation screening of the PTPN11 coding region in 250 selected HCM probands (200 patients without mutations in sarcomeric genes and 50 with identified mutations). No mutations in PTPN11 were identified. Our data suggests that mutations in the PTPN11 gene are not a cause of HCM in the absence of Noonan/LEOPARD syndromes.

Adult↗
PubMedDate not supplied

The genetic background of familial adenomatous polyposis. Linkage analysis, the APC gene identification and mutation screening.

Familial adenomatous polyposis (FAP) is an autosomal, dominantly inherited disease that predisposes to colorectal cancer and is characterized by the presence of hundreds to thousands of adenomas covering the colon and rectum. Mapping of the FAP locus to 5q21-q22 by linkage studies in families ultimately allowed the identification of the APC (Adenomatous Polyposis Coli) gene itself. The APC gene comprises 15 exons with a 9 kilobase RNA transcript and a 312 kilodalton final protein product. This discovery transformed the diagnosis of FAP and offered direct identification of defective gene carriers by mutation screening. Currently used techniques have been successful in detecting mutations in 15 to 67 percent of patients. To date, at least 136 different mutations have been described in 301 unrelated FAP patients, most of which (98%) are translation terminating mutations leading to a truncated final protein product. Promising applications or development of novel procedures, like the protein truncation test (PTT), are under way for the remaining FAP patients. With the exception of the description of a critical boundary in exon 9 for the presence or absence of CHRPE, there are no clear genotype-phenotype relationships, but mutations located in the 5' half of exon 15 seem to lead to a more severe phenotype. Very little is know about the APC protein product function. The APC protein could be involved in cell-to-cell signalling and/or cell adhesion functions. The APC gene is a tumour suppressor gene involved in early stages of sporadic colorectal carcinogenesis. Further understanding of the APC gene function may define a rational approach for early detection, prevention strategies, assessment of prognosis and treatment of colorectal cancer. In this regard, animal models of FAP, like the MIN (Multiple Intestinal Neoplasia) mouse or the APC 1638 mouse, are promising and powerful tools.

Adenomatous Polyposis Coli↗
PubMed1999

Mutation screening of the RYR1 gene and identification of two novel mutations in Italian malignant hyperthermia families.

Point mutations in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia, an autosomal dominant disorder triggered in susceptible people (MHS) by volatile anaesthetics and depolarising skeletal muscle relaxants. To date, 17 missense point mutations have been identified in the human RYR1 gene by screening of the cDNA obtained from muscle biopsies. Here we report single strand conformation polymorphism (SSCP) screening for nine of the most frequent RYR1 mutations using genomic DNA isolated from MHS patients. In addition, the Argl63Cys mutation was analysed by restriction enzyme digestion. We analysed 57 unrelated patients and detected seven of the known RYR1 point mutations. Furthermore, we found a new mutation, Arg2454His, segregating with the MHS phenotype in a large pedigree and a novel amino acid substitution at position 2436 in another patient, indicating a 15.8% frequency of these mutations in Italian patients. A new polymorphic site in intron 16 that causes the substitution of a G at position -7 with a C residue was identified.

Caffeine↗
PubMedDate not supplied

Molecular analysis of acute intermittent porphyria: mutation screening in 20 patients in Germany reveals 11 novel mutations.

Acute intermittent porphyria (AIP) is a very rare autosomal dominant disorder with low penetrance. Mutations in the gene of the porphobilinogen deaminase (PBG-D), also called hydroxymethylbilane synthase (HMBS), cause a partial deficiency of this enzyme of the heme biosynthetic pathway. Overstimulation of heme biosynthesis causes clinical symptoms. Because of the variability of the symptoms, diagnosis is often delayed. Using two approaches for genetic analysis, first in a stepwise manner, then sequencing extensive parts of the gene, the screening of the DNA of 20 unrelated individuals revealed 20 different mutations, 11 of which had not been reported previously. The novel mutations affected intron 1 (33 + 2 T-->C), exon 5 (181 G-->C), intron 6 (267-61 del 8 bp), intron 7 (345-1 G-->C), intron 9 (498 + 15 G-->T and 499-13 Delta-14 bp indel TGA), intron 13 (825 + 1 G-->C and 825 + 2 T-->C), exon 15 (962 G-A, 1067 del A and 1067-1068 ins 5 bp). The other nine mutations detected affected intron 14, exons 6, 7, 8, 9, 10 (3x) and 12. In the majority of AIP patients, the genotype does not predict phenotypic expression. Since the sudden manifestation of the disease maybe prevented by early diagnosis, identification of AIP gene carriers is the best preventive measure. This was performed in five families, revealing 10 additional AIP gene carriers.

Adult↗
PubMed1999

Mutation screening and isoform prevalence of the follicle stimulating hormone receptor gene in women with premature ovarian failure, resistant ovary syndrome and polycystic ovary syndrome.

OBJECTIVE: To determine whether mutations in the FSH receptor gene are associated with premature ovarian failure (POF) or resistant ovary syndrome (ROS) in women in the UK. To determine whether an allelic variant of the FSH receptor gene affects fertility parameters in women with polycystic ovary syndrome (PCOS). DESIGN: A mutation screen using DNA from women with POF and ROS. Restriction digest of amplified DNA from women with POF, ROS, PCOS and controls to determine allelic variant status. Fertility parameters were compared between allelic variant subgroups of women with PCOS. PATIENTS: The study population comprised 49 women with POF, 5 with ROS, 93 with PCOS and 51 controls. MEASUREMENTS: In women with PCOS, fertility and menstrual status was recorded and serum FSH and ovarian volume were measured. RESULTS: No mutation of the FSH receptor gene was found in women with POF or ROS. The allelic variant Thr307/Ser680 was found to be similarly prevalent in all study groups. The Thr307/Ser680 variant was found to have no phenotype in terms of fertility parameters in women with PCOS. CONCLUSIONS: Mutations of the FSH receptor gene are rare in women with premature ovarian failure or resistant ovary syndrome in the UK. Polymorphisms of the FSH receptor gene do not appear to have pathophysiological significance with regard to ovarian function.

Adult↗
PubMed2003

Mutation screening in patients with isolated cytochrome c oxidase deficiency.

Cytochrome c oxidase (COX) deficiency has been associated with a variety of clinical conditions and can be due to mutations in nuclear or mitochondrial genes. Despite recent progress in our understanding of the molecular bases of COX deficiency, the genetic defect remains elusive in many cases. We performed mutation screening in 30 patients with biochemical evidence of isolated COX deficiency and heterogeneous clinical phenotypes. Sixteen patients had various forms of encephalomyopathy, and six of these had the neuroradiological features of Leigh syndrome. Four patients had encephalohepatopathy, six had hypertrophic cardiomyopathy, and four had other phenotypes. We studied the three mtDNA genes encoding COX subunits, the 22 mtDNA tRNA genes, and seven COX assembly genes: SCO1, SCO2, SURF1, COX10, COX11, COX15, and COX17. We report two novel pathogenic SURF1 mutations in a patient with Leigh syndrome and one novel SCO2 mutation in a patient with hypertrophic cardiomyopathy. These data show that heterogeneous clinical phenotypes are associated with COX deficiency, that mutations in mtDNA COX genes are rare, and that mutations in additional genes remain to be identified.

Age of Onset↗
PubMed2001

DHPLC-based germline mutation screening in the analysis of the VHL tumor suppressor gene: usefulness and limitations.

In order to evaluate the sensitivity and specificity of the recently introduced high-throughput method DHPLC (denaturing high performance liquid chromatography) for mutation screening in the VHL tumor suppressor gene, we subjected DNA from 43 unrelated VHL patients with previously sequenced VHL germline mutations to this method. In addition, 36 genomic DNAs of unrelated individuals suspected of being VHL carriers but with unknown germline status were analyzed by DHPLC and sequencing. The aims of the present study were to compare mutation results obtained by direct sequencing and DHPLC, and a comparison of two different DHPLC systems. The sensitivity of DHPLC was tested with two commercial devices and protocols, i.e., the Varian-Helix system and the Wave Nucleic Acid Fragment Analysis system. Both resolved all but one mutation in exons 2 and 3 of the VHL gene. In contrast, the GC-rich exon 1 showed discrepancies in the rate of mutation detection. Whereas the Varian-Helix system detected 10/15 (67%) of the known mutations, the Wave Nucleic Acid Fragment Analysis system detected 13/14 (93%). All three mutations in samples with unknown mutation status were revealed by both systems raising the mutation detection rate to 72% and 94%, respectively. Cases with different substitutions at the same nucleotide showed different elution profiles, but similar elution profiles could be obtained from different mutations. The Wave Nucleic Acid Fragment Analysis system detected most VHL mutations; however, when a 100% detection rate is needed, sequencing is still required and must therefore be the standard VHL mutation detection procedure. Once a family-specific mutation has been established, DHPLC may be suitable for the rapid and cost-effective determination of VHL carrier status in family members.

Chromatography, High Pressure Liquid↗
PubMed2000

Rapid mutation screening of phenylketonuria by polymerase chain reaction-linked restriction enzyme assay and direct sequence of the phenylalanine hydroxylase gene: clinical application in northern Japan and northern China.

We describe a simple and technically feasible method for mutation screening of the phenylalanine hydroxylase (PAH) gene and its application to Japanese and Chinese patients with hyperphenylalaninemia. The strategy is based on the identification of a nucleotide substitution by restriction enzyme analysis, coupled with PCR and direct sequencing of exon 7 of the PAH gene. Because the detection of various mutations can proceed simultaneously using the same technique, it is quite rapid and reproducible, making it possible to perform effective molecular diagnosis and carrier screening in most laboratories. Using this procedure, we found that the most common molecular defects were R413P in Hokkaido, Japan (35 %) and R243Q in Heilongjiang, China (50%). R111X, IVS4nt-1, and five mutations in exon 7 (R241C, R243Q, R252W, A259T, and S273P) accounted for 55% of phenylketonuria (PKU) alleles in Hokkaido. In Heilongjiang, the R111X, Y356X, and R408W mutations accounted for 35% of PKU alleles. Clinically, homozygotes or compound heterozygotes of null alleles, which express nonfunctional enzyme activity, were all associated with classic PKU. On the other hand, patients heterozygous for the R241C allele had a benign phenotype of mild hyperphenylalaninemia. The DNA diagnosis in early infancy can predict various PKU phenotypes, and can prove useful in decision-making concerning dietary therapy.

Adolescent↗
PubMed2006

Mutation screen of the TUB gene in patients with retinitis pigmentosa and Leber congenital amaurosis.

TUB is the first identified member of the TULP family of four proteins with unknown function. A spontaneous mutation in murine tub causes retinal degeneration, obesity, and deafness. Mutations in another member of the TULP family, TULP1, are a cause of autosomal recessive retinitis pigmentosa (RP). These findings prompted us to investigate TUB as a candidate gene for RP and Leber congenital amaurosis (LCA). A mutation screen of the entire coding region of the TUB gene in 159 unrelated patients with autosomal recessive RP, 114 unrelated patients with simplex RP, and 21 unrelated patients with LCA uncovered 18 sequence variations. Of these, seven were missense mutations, six were isocoding changes, and five were intronic polymorphisms. All seven missense mutations were identified as heterozygous changes and no defect could be found in the other allele. None of the isocoding variants or intronic polymorphisms are predicted to create or destroy splice donor or acceptor sites based on splice-site prediction software. Although variant alleles of the TUB gene were found, none could be definitively associated with a specific retinal disease.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Mutational screening of thrombopoietin receptor gene (c-mpl) in patients with congenital thrombocytopenia and absent radii (TAR).

Thrombocytopenia with absent radii (TAR) is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. We performed mutational screening of coding and promoter regions of the c-mpl gene, encoding thrombopoietin (TPO) receptor, by sequence analysis in four unrelated patients affected by TAR syndrome. Our results indicate that c-mpl gene mutations are not a common cause of thrombocytopenia in TAR syndrome.

Adolescent↗
PubMed2004

Characterization of the human nasal embryonic LHRH factor gene, NELF, and a mutation screening among 65 patients with idiopathic hypogonadotropic hypogonadism (IHH).

As the mouse nasal embryonic LHRH factor gene (Nelf) encodes a guidance molecule for the migration of the olfactory axon and gonadotropin-releasing hormone neurons, its human homolog, NELF, is a candidate gene for Kallmann syndrome, a disease of idiopathic hypogonadotropic hypogonadism (IHH) with anosmia or hyposmia. We report here characterization of NELF and results of mutation analysis in 65 IHH patients. Assembling EST clones, RACE, and sequencing showed that NELF mapped to 9q34.3 is composed of 16 exons and 15 introns with a 1,590-bp ORF encoding 530 amino acids. RT-PCR on a fetal brain cDNA library revealed five alternatively spliced variants. Among them, NELF-v1 has 93-94% identity at the amino acid level to mouse/rat Nelf, and four other transcripts are also highly conserved among the three species. A 3.0-kb transcript is expressed most highly in the adult and fetal brain, testis, and kidney, indicating that NELF plays a role in the function of these tissues. Mutation screening detected in a patient with IHH one novel heterozygous missense mutation (1438A>G, T480A) at the donor-splice site in exon 15 of NELF. As this mutation was not found in 100 normal control individuals, T480A may be associated with IHH. Four other novel SNPs (102C > T and 1029C > T within the coding region, and two IVS14+47C > T and IVS15+41G > A) were also identified in NELF.

Adult↗
PubMed2005

Mutation screening for the prothrombin variant G20210A by melting point analysis with the Light Cycler system: atypical results, detection of the variant C20209T and possible clinical implications.

In the differential diagnosis of thrombophilic disorders genotyping of prothrombin and factor V are nowadays performed as a routine analysis. In the following we describe the unusual results of the mutation screening using melting point analysis for two patients and the consecutive detection of the mutation C20209T by sequencing the corresponding gene fragments. The molecular result is discussed with special respect to the medical history, ethnic background and clinical findings of both patients.

Adult↗
PubMed2005

Mutation screen and association study of EZH2 as a susceptibility gene for aggressive prostate cancer.

BACKGROUND: Several linkage studies have provided evidence for a prostate cancer aggressiveness gene on chromosome 7q. This report details the results of the first mutation screen and association study of EZH2 (located at 7q35) as a potential candidate gene for the development of aggressive prostate cancer. METHODS: In 10 families with linkage of chromosome 7q31-33 to aggressive prostate cancer, we sequenced the promoter region and all 20 exons of EZH2. We genotyped 11 variants in 287 prostate cancer probands and 96 controls. Association between the disease and the variants/haplotypes was evaluated taking into account clinical data and disease recurrence. RESULTS: The individual variation sites did not show significant differences in the allele frequencies between cases and controls. In contrast, one haplotype had a higher frequency in controls, and another haplotype was significantly more frequent in cases with low grade tumors (GI/II) and progression free survival (NED). CONCLUSION: We have possibly identified haplotypes which mark alleles that have a beneficial effect on the development of prostate cancer. Moreover, our results suggest that genetic variations of the EZH2 gene are not responsible for the linkage of 7q to aggressive prostate cancer.

Adult↗
PubMed2000

Recurrent astrocytoma in a child: a report of cytogenetics and TP53 gene mutation screening.

An 8-year-old girl presented with a cerebral tumor and 3 recurrences within 15 months. The primary tumor was a low-grade astrocytoma, but the recurrences showed progressively malignant phenotypes with increasing mitotic activity and MIB-1 labeling indices. Radiotherapy was given between the first and the second recurrences. Cytogenetic analysis of the first and the second recurrences showed abnormal karyotypes. There seemed to be 2 common breakpoints in these 2 recurrences. TP53 gene mutation screening, using comprehensive denaturing gradient gel electrophoresis, revealed among others a possibly causative mutation of exon 5 in 3 of 4 tumor samples. The meaning of TP53 mutations in low-grade astrocytomas is still unclear, but the highly abnormal karyotypes, which are unusual in these tumors, probably provide genetic evidence for the unexpected aggressive behavior of the tumor in this patient.

Antigens, Nuclear↗
PubMed2006

Mutation screening for JAK2V617F: when to order the test and how to interpret the results.

With the application of adequately sensitive tests, it is now becoming evident that more than 90% of patients with conventionally-defined polycythemia vera (PV) carry the somatic JAK2V617F mutation in their granulocytes. However, the specific mutation is also found in other classic and atypical myeloproliferative disorders (MPD), albeit at a lesser frequency. In contrast, JAK2V617F has not been reported in patients with either reactive myeloproliferation or lymphoid disorders. Therefore, mutation screening for JAK2V617F can be considered as a myeloid-specific clonality assay and it is diagnostically most useful in the evaluation of "polycythemia".

Amino Acid Substitution↗
PubMed2006

A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting.

Rapid and reliable identification of deleterious changes in the breast cancer genes BRCA1 and BRCA2 has become one of the major issues in most DNA services laboratories. To rapidly detect all possible changes within the coding and splice site determining sequences of the breast cancer genes, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. All exons of both genes are covered by the DGGE scan, comprising 120 amplicons. We use a semiautomated approach, amplifying all individual amplicons with the same PCR program, after which the amplicons are pooled. DGGE is performed using three slightly different gel conditions. Validation was performed using DNA samples with known sequence variants in 107 of the 120 amplicons; all variants were detected. This DGGE mutation scanning, in combination with a PCR test for two Dutch founder deletions in BRCA1 was then applied in 431 families in which 52 deleterious changes and 70 unclassified variants were found. Fifteen unclassified variants were not reported before. The system was easily adopted by five other laboratories, where in another 3,593 families both exons 11 were analyzed by the protein truncation test (PTT) and the remaining exons by DGGE. In total, a deleterious change (nonsense, frameshift, splice-site mutation, or large deletion) was found in 661 families (16.4%), 462 in BRCA1 (11.5%), 197 in BRCA2 (4.9%), and in two index cases a deleterious change in both BRCA1 and BRCA2 was identified. Eleven deleterious changes in BRCA1 and 36 in BRCA2 had not been reported before. In conclusion, this DGGE mutation screening method for BRCA1 and BRCA2 is proven to be highly sensitive and is easy to adopt, which makes screening of large numbers of patients feasible. The results of screening of BRCA1 and BRCA2 in more than 4,000 families present a valuable overview of mutations in the Dutch population.

Ambulatory Care Facilities↗
PubMedDate not supplied

Genomic organisation of the human chordin gene and mutation screening of candidate Cornelia de Lange syndrome genes.

We have determined the genomic organisation of the human chordin gene, CHRD, and have shown that it maps within a gene cluster at 3q27 containing THPO (thrombopoietin), CLCN2 (a voltage-gated chloride-channel gene) and EIF4G1 (a eukaryotic translation-initiation-factor-gamma gene). The CHRD and THPO genes are very close neighbours and are transcribed from opposing DNA strands from promoters that are spaced less than 2 kb apart. We considered that the CHRD gene and the chordin-regulating GSC (goosecoid) gene could be candidate genes for Cornelia de Lange syndrome (CDLS), a developmental malformation syndrome which is primarily characterised by mental handicap, growth retardation, distinctive facial features and limb-reduction defects. CDLS patients typically occur as sporadic cases, but several reports have suggested dominant inheritance. The candidacy of the CHRD and GSC genes was supported by several lines of evidence: prior evidence for a CDLS gene at 3q26.3-q27; a report suggesting a significant association between CDLS and thrombocytopenia; suspected genetic heterogeneity in CDLS; location of the GSC gene in close proximity to a 14q32 breakpoint detected in a CDLS patient with a balanced de novo translocation; known regulation of chordin expression by goosecoid; and the pattern of embryonic expression of the mouse GSC gene. Another candidate gene at 3q27, SOX2, was also considered because of its suspected role as a transcription factor in early development and because of known examples of SOX genes that are loci for dominantly inherited developmental disorders. However, mutation screening failed to identify CDLS patient-specific mutations in CHRD, GSC or SOX2.

Amino Acid Sequence↗