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At least 523 records · Page 29Linked to original sources
PubMed2004

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Animals↗
PubMed2000

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog.

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.

Animals↗
PubMed2000

Adaptation of conformation-sensitive gel electrophoresis to an ALFexpress DNA sequencer to screen BRCA1 mutations.

Conformation-sensitive gel electrophoresis (CSGE) has been introduced as the most reliable method for the screening of large and multi-exon genes because of its simplicity, sensitivity and specificity. Based on heteroduplex formation and with the use of mildly denaturing solvents, it allows detection of single-base mutations with accuracy. This is important in genes such as BRCA1 and BRCA2, in which alterations span the entire gene. We have adapted the CSGE assay to a fluorescent platform--a DNA sequencer one-color technology--that reduces the time involved and enhances resolving power for the complete scanning of the BRCA genes. Electrophoresis has high sensitivity and is performed in less than three hours, and the gel does not require staining with ethidium bromide. Eighteen single-base and six frameshift mutations in the BRCA1 gene were analyzed. We compared the manual and fluorescent CSGE methods, and all mutations were detected with accuracy.

BRCA1 Protein↗
PubMed2003

[Screening C282Y mutation with double-stranded probes using synchronous fluorometry].

We described in the paper a new high-throughput screening method for Cys282Tyr mutation in hereditary haemochromatosis with double-stranded probe using synchronous fluorometry. The probe for wild type was labeled with Fam, the probe for mutant type was labeled with Joe. After PCR, reaction tubes were transferred to a spectrofluorometer, where synchronous spectra were scanned in a constant-wavelength mode. The genotype could be obtained through the appearance of the fluorescence peaks corresponding to each probe. The results were totally in agreement with restriction endonuclease analysis. Considering the simplicity,low cost and specificity, this approach could be generally applied to detect varieties of gene mutations.

English Abstract↗
PubMed1993

[Screening for mutations of the precursor of beta-amyloid protein in early forms of Alzheimer disease. French Study Group of Alzheimer Disease].

Genetic linkage studies suggest that Alzheimer's disease (AD) is genetically heterogeneous and that in several early onset families the disease locus is close to the amyloid precursor protein (APP) gene on chromosome 21. Recently, several mutations of APP gene have been described. We screened 100 sporadic and familial cases of early onset AD in order to determine the frequency of two of these mutations, APP 717 Val-->Ile and APP 713 Ala-->Val. The patients included fulfilled NINCDS-ADRDA criteria of probable AD and their age of onset was 60 or lower. Seventy-five cases were sporadic and 25 were familial with at least one first or second degree affected relative. None of our cases presented the mutations. Combined results allow to presume that the APP 717 Val-->Ile mutation accounts for less than 0.8% of all early onset AD, and 3.8 +/- 2.8% of familial cases. While the pathogenic effect of the APP 717 Val-->Ile mutation is established, this is not the case for the APP 713 Ala-->Val mutation. Searching for the APP 717 mutations in early-onset AD is fully justified, taking into account its consequences for genetic counselling.

Aged↗
PubMed1995

Characterization of Japanese families with early-onset type 2 (non-insulin dependent) diabetes mellitus and screening for mutations in the glucokinase and mitochondrial tRNA(Leu(UUR)) genes.

Genetic linkage studies of families with early-onset type 2 diabetes have facilitated the identification of diabetes-susceptibility genes. In order to assess the feasibility of using linkage approaches to identify genes responsible for the development of type 2 diabetes in Japanese subjects, we examined our clinical records for multigenerational families suitable for genetic studies. We identified 16 families in which at least one subject was diagnosed with type 2 diabetes before 25 years of age. Seven of these families had a pattern of inheritance consistent with a diagnosis of maturity-onset diabetes of the young (MODY) and nine families showed a complex pattern of inheritance of type 2 diabetes with transmission of diabetes-susceptibility genes from both parents. The glucokinase and mitochondrial tRNA(Leu(UUR)) genes were screened for mutations in at least one affected subject from each family in order to assess the contribution of mutations in these genes to the development of the diabetes. No mutations were found, which suggests that the diabetes in these families resulted from mutations in other genes.

Adolescent↗
PubMed2006

A fluorescent multiplex-DGGE screening test for mutations in the BRCA1 gene.

Screening for mutations in the BRCA1 gene is challenging because of the wide spectrum of mutations found in this large gene. As the extensive exon 11 is commonly screened by the protein truncation test (PTT), here a fluorescent multiplex denaturing gradient gel electrophoresis (FMD) mutation screening technique was developed to test the remaining numerous small exons and splice sites of the gene. The method is based upon the use of an efficient multiplex polymerase chain reaction (PCR) amplification of the target regions, followed by denaturing gradient gel electrophoresis (DGGE) separation of the amplicon mixture, and the immediate achievement of results by wet gel scanning. The technique was applied to screen 16 samples with different BRCA1 sequence variants distributed over 12 exons. All variants were detected. In addition, 188 DNA samples from ovarian cancer patients were screened, identifying 22 new sequence variants (11.7% of the samples) and 243 common polymorphisms in the BRCA1 locus. Variants included 16 single nucleotide substitutions, 3 deletions of 2 nucleotides, 1 deletion of 4 nucleotides, and 2 insertions of 1 nucleotide. The FMD test provides an accurate, fast, nonradioactive and cost-efficient way to scan the BRCA1 gene with high sensitivity and an ease of result interpretation. This technique may prove to be a useful research tool for the detection of mutations and polymorphisms in the BRCA1 gene and for large-scale epidemiologic studies.

BRCA1 Protein↗
PubMed2006

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31-46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.

Adolescent↗
PubMed2004

Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reverse-hybridization assay in the South African population.

A recently developed strip-assay for hemochromatosis provides a rapid method for simultaneous detection of multiple mutations, which among others includes the HFE gene mutations V53M, V59M, H63D, H63H, S65C, Q127H, E168Q, and C282Y, previously detected in the general South African population using gel-based mutation-screening methods. The objective of the study was to determine the frequency of the relatively rare mutations in samples selected for altered iron parameters or a family history of hereditary hemochromatosis (HH) as part of the validation process of the assay for routine diagnostic purposes. The study population consisted of 451 individuals previously screened for mutations C282Y and H63D by restriction enzyme analysis in order to confirm or possibly exclude a diagnosis of HH. These individuals were subjected to mutation screening using the commercially available hemochromatosis strip-assay. Previous positive results for mutations C282Y and H63D in 233 individuals confirmed the accuracy of the reverse-hybridization assay. Mutation S65C was detected in 13 Caucasians, including three compound heterozygotes. These constituted 2% (13/600) of the chromosomes without mutations C282Y or H63D. The African-specific HFE mutation V53M was detected in one out of 11 (9%) African subjects screened. Mutation E168Q was detected in a single Caucasian individual together with mutation H63D. Our data demonstrate the value of the strip-based technology in providing a rapid and reliable comprehensive test for simultaneous analysis of multiple mutations.

DNA Mutational Analysis↗
PubMed2005

The human secretin gene in children with autistic spectrum disorder: screening for polymorphisms and mutations.

We screened 29 children with autism for mutation in the human secretin gene using single-strand conformation polymorphism. No mutation was detected in exon 2, 3, or 4. Polymerase chain reaction and DNA sequence of 5' variable number of tandem repeats showed two polymorphisms with deletion or duplication of a repeat unit that failed to show any gene expression with transient transfection assay. We did not find evidence of a relationship between human secretin gene mutation and autism.

Adolescent↗
PubMed2001

Assessment of denaturing high-performance liquid chromatography (DHPLC) in screening for mutations in connexin 26 (GJB2).

Mutations in the gene GJB2 encoding connexin 26 (Cx26), a gap junction protein, have been shown to be responsible for a majority of recessive nonsyndromic hereditary hearing impairment in children. Over 60 different mutations in Cx26 have been reported. To obviate the need for direct sequencing of each specimen, a variety of screening techniques have been used to detect mutations in Cx26. However, each of these methods has significant shortcomings including expense, time consumption, and limited sensitivity. Denaturing high-performance liquid chromatography (DHPLC) has been recently introduced as a rapid and highly sensitive method of detecting sequence alterations. We have assessed the efficacy of DHPLC as a screening assay for detecting mutation in Cx26 coding region in 154 patients with hereditary hearing impairment. The GJB2 coding exon was amplified in one or two fragments, analyzed by DHPLC, and sequenced. Sequence analysis identified sequence variations in 34 patients concordant with abnormal DHPLC results. Three novel Cx26 mutations were identified: a single base pair substitution 511G>A, a 4 bp insertion 504insAACG, and a 3 bp deletion 358delAGG in three unrelated patients. In 120 patients with normal Cx26 sequence, DHPLC was normal. These results yield sensitivity and specificity of 100% for DHPLC-based detection of Cx26 mutations, and demonstrate that DHPLC is a highly sensitive and specific method of screening for sequence variations in Cx26 that is time and labor efficient. Further, our experience suggests that DHPLC screening alone followed by DNA sequencing only when DHPLC is abnormal may be adequate for identification of all sequence alterations in Cx26.

Chromatography, High Pressure Liquid↗
PubMed1997

Detection and characterization of a novel splice mutation in the LDL receptor intron 12 resulting in two different mutant mRNA variants.

Using a simple, standardized denaturing gradient gel electrophoresis (DGGE) based mutation screening technique, a novel G-to-A mutation in the last base of the intron 12 splice acceptor site of the LDL receptor gene was found in 2 Danish families with familial hypercholesterolemia (FH). The mutation is shown to result in 2 mRNA splice variants, both leading to truncated LDLR proteins, containing only the first 594 of the normal 839 amino acids. In one of the FH-families harbouring the mutation, a striking difference in the clinical picture amongst biochemically diagnosed FH patients was clarified when genetic analysis showed that 2 hypercholesterolemic family members, who despite advanced age had no atherosclerotic disease, had not inherited the family LDLR mutation. DGGE analyses of the LDLR exons, LDLR promoter, and apolipoprotein B codon 3456-3553 as well as Southern blotting of the LDLR gene were without signs of other mutations in the non-atherosclerotic hypercholesterolemics of the family. Availability of the clinically applicable mutation screening assay for FH may thus aid in defining reasons for phenotypic differences in FH families and potentially supply information allowing a more differentiated therapeutic approach to individual members of FH families.

Adolescent↗
PubMed2006

[Acute intermittent porphyria. A clinical chameleon: case study of a 40-year-old female patient].

Acute porphyrias are rare, but often misdiagnosed and may take a dramatic clinical course. The combination of various internal, psychiatric and neurological symptoms can mimic different other diseases. We report a 40-year-old female patient who was admitted with a subacute tetraparesis. During the last 2 months the patient was treated several times because of abdominal pain and just before admission to our clinic in a psychiatric hospital because of acute mental changes and hallucinations. The typical combination of abdominal pain, motor neuropathy and psychiatric symptoms confirmed by increased amounts of porphyrins and their precursors, led us to promptly diagnose acute intermittent porphyria. Better knowledge about the pathogenesis has clearly improved the prognosis of acute porphyria. In remission, measurement of enzyme activities or mutation screening can be the only diagnostic verification. A mutation screening for family members should be conducted to identify symptom-free carriers, especially in cases of a positive family history.

Abdominal Pain↗
PubMed2000

High-throughput single-strand conformation polymorphism analysis by capillary electrophoresis.

Mutation detection plays a great role in genetic and medical research and clinical diagnosis of inherited diseases and particular cancers. Single-strand conformation polymorphism (SSCP) analysis is one of the most popular methods for detection of mutations. Recently, automated capillary electrophoresis (CE) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis. SSCP analysis in combination with CE is a rapid, simple, sensitive and high-throughput mutation screening tool, and has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes. The new technique has a great potential for mutation screening of large numbers of samples in clinical diagnosis. This review discusses basic issues about the methodology of SSCP analysis based on CE and summarizes several key applications.

Electrophoresis, Capillary↗
PubMed1999

Glycogen storage disease type II: birth prevalence agrees with predicted genotype frequency.

OBJECTIVES: To compare the overall birth prevalence of diagnosed glycogen storage disease type II (GSD II) with the predicted frequency based on mutation screening, in order to determine whether GSD II is an underdiagnosed condition, and to analyze which medical disciplines recognize GSD II. METHODS: Retrospective data on all enzymatic diagnoses of GSD II were collected from diagnostic labs throughout the Netherlands, covering the period from January 1, 1972 to December 31, 1996. Age-specific diagnostic incidence rates were calculated for the entire study period. By adding together the diagnostic incidences for all age groups, we calculated the birth prevalence of diagnosed GSD II and compared these figures with the predicted frequency based on mutation screening in a random sample from the general population. The medical specialization of the referring clinicians was also recorded. RESULTS: GSD II was diagnosed in 154 individuals, including 11 prenatal diagnoses. The birth prevalences of the various phenotypes were 1/101,000 (infantile form), 1/720,000 (juvenile form) and 1/53,000 (adult form). The birth prevalence of the adult and infantile phenotype together was 1/35,000. Eighty-two percent of the patients were diagnosed in university hospitals. Of the patients with infantile GSD II, 71% were diagnosed by a pediatrician, whereas most patients with adult GSD II were diagnosed by a neurologist (80%). CONCLUSIONS: There is no evidence for the underdiagnosis of GSD II in the Netherlands, as the calculated birth prevalences of the disease are consistent with previous predictions based on mutation screening in a random sample of newborns. The worldwide birth prevalence of the disease may well be higher than 1 in 100,000. GSD II is mainly diagnosed in university hospitals.

Journal Article↗
PubMed2002

Mutational analysis of selected genes in the TGFbeta, Wnt, pRb, and p53 pathways in primary uveal melanoma.

PURPOSE: It is known that the pRb pathway cell-cycle inhibitor p16(INK4A) plays a significant role in cutaneous melanoma and that alteration of p16(INK4A), which resides within the 9p21-22 locus that also contains p15(INK4B) and p14(ARF), may occur in up to one third of uveal melanomas. The absence of TGFbeta responsiveness noted in cultured uveal melanoma cells also suggests that the TGFbeta pathway plays a role in the formation of this tumor. Therefore, mutational screening was performed in several key genes in tumor-suppressor pathways that are known to be altered in some uveal melanomas. METHODS: Using denaturing high-performance liquid chromatography (DHPLC) analysis and DNA sequencing, a series of 67 uveal melanomas were screened for inactivating mutations in the TGFbeta pathway members Smad4 and TGFbeta receptor type 2 (TGFbetaR2), the downstream cell-cycle inhibitor p15(INK4B), and the cell-cycle inhibitors p14(ARF) and p16(INK4A). p16(INK4A) was also investigated for promoter hypermethylation. Mutational analysis was also performed on the Wnt pathway gene beta-catenin, known to be mutated in approximately one quarter of cutaneous melanoma cell lines. RESULTS: Polymorphisms in p16(INK4A) were detected in 3 of 50 samples, but no inactivating mutations were detected in any of the genes screened. Promoter hypermethylation of p16(INK4A) was detected in 5 of 55 tumors, and loss of heterozygosity of the p16(INK4A) locus was detected in 5 of 16 tumors. CONCLUSIONS: Most primary uveal melanomas do not appear to contain somatic mutations in Smad4, TGFbetaR2, p14(ARF), p15(INK4B), p16(INK4A), or beta-catenin. However, methylation of the p16(INK4A) promoter and loss of heterozygosity of the p14(ARF)-p16(INK4A) locus occurs in some tumors.

Cell Cycle Proteins↗
PubMed1993

Prions and nucleic acids: search for "residual" nucleic acids and screening for mutations in the PrP-gene.

Studies on prions involve nucleic acid chemistry under two headings: i) do infectious prion particles contain nucleic acids? ii) is it possible by a simple procedure to screen the prion protein (PrP)-gene for mutations? The return refocusing gel electrophoresis technique was developed to detect by sensitive silver staining homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all kinds of nucleic acids from a length of 13 nucleotides up to several thousand could be recovered and detected with a yield over 90%. Despite extensive nuclease digestion some small polynucleotides remained. The results define clear restrictions for a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be smaller than 80 nucleotides in length at a particle-to-infectivity ratio near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To detect mutations in the PrP-gene, either known mutations from human prion diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole PrP-ORF. It will be shown that a combination of PCR and temperature-gradient gel electrophoresis fulfils these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated depending on the position of the mutation. The theoretical results were tested with the mutations known so far.

Animals↗
PubMed1996

Pulmonary function and clinical observations in men with congenital bilateral absence of the vas deferens.

Congenital bilateral absence of the vas deferens (CBAVD) was once thought to be a distinct clinical entity, but genetic similarities in men with cystic fibrosis (CF) and CBAVD are described increasingly. We evaluated the clinical status, growth and nutritional state, and respiratory function of 18 men with CBAVD to determine whether these men with different CF transmembrane regulator (CFTR) genotypes may have clinical evidence of mild CF. Following a thorough history and examination, pulmonary function tests, sweat test, and renal ultrasound were performed. Genetic evaluation for 50 known CF mutations, screening for private mutations (single-strand conformational polymorphism and direct sequencing), and assay of the length of the polypyrimidine tract in the splice site acceptor of intron 8 was performed. A history of pulmonary disease was present in three, and an additional man had some features suggestive of malabsorption. Results of general physical examination and anthropomorphic measurements were unremarkable in all patients, with a mean (SD) body mass index of 26 (3). Pulmonary function tests of large and small airway function as well as lung volumes were normal in all except one whose results were consistent with moderate asthma. Five men were compound heterozygotes for CFTR mutations, four of whom had positive sweat tests (sweat chloride > 60 mEq/L). Twelve men were heterozygotes for CFTR mutations while no mutations were identified in one man. Although putative etiologic factors may suggest that men with CBAVD and CFTR mutations could be considered within the spectrum of clinical CF, the authors suggest that in men with CBAVD without any other clinical features of CF, the diagnosis of CF may not be made.

Adult↗