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Results for “mutational screening”

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At least 271 records · Page 15Linked to original sources
PubMed2004

Mutation screening and association study of the beta-adrenergic receptor kinase 2 gene in schizophrenia families.

Chromosome 22q12 is one of the most promising regions for harboring a risk gene for schizophrenia. We have reported significant linkage of intermediate phenotypes for schizophrenia with markers within or near the beta-adrenergic receptor kinase 2 (ADRBK2, or GRK3) gene, which is highly expressed in dopaminergic pathways in the central nervous system, and mediates homologous desensitization for a variety of neurotransmitters and hormones through phosphorylation of G protein-coupled receptors (GPCRs). A polymorphism in the promoter region of the ADRBK2 was reported to be associated with bipolar disorder. We screened the putative promoter region, and all 21 exonic and flanking intronic regions of the ADRBK2 gene for mutations in 48 schizophrenia probands (including 16 Japanese and 32 Chinese patients), and evaluated the detected polymorphisms and those reported in the JSNP database for associations with schizophrenia in 113 family trios of schizophrenia probands. Four single nucleotide variants in the 5'-UTR/promoter region, and 16 rare variants in exonic and flanking regions, were identified. Among them, the Cys208Ser variant was the only non-synonymous mutation. Cys208Ser was found in one family without cosegregation between the variant and schizophrenia. Moreover, allelic, genotypic and haplotypic analyses provided no evidence for association between alleles at these polymorphisms and schizophrenia. The present study indicates that the ADRBK2 gene is unlikely to contribute strongly to schizophrenia susceptibility in this set of families.

Alleles↗
PubMed1999

Mutation screening of the CHRNA4 and CHRNB2 nicotinic cholinergic receptor genes in Alzheimer's disease.

Potential genomic changes leading to decreased nicotine binding, crucial for cognitive dysfunction in Alzheimer's disease (AD), have not yet been studied. A search for mutations of the genes coding for the most widely distributed nicotinic receptor subtype alpha4beta2 (CHRNA4/CHRNB2) has been performed in AD patients by screening the coding regions of both genes by single strand conformation analysis and heteroduplex analysis of fibroblast-derived genomic DNA. Polymorphisms in CHRNA4, none of which led to amino acid changes in the predicted sequence, were found in three patients. Although the other receptor subunits have yet to be screened, it appears likely that the reduction of nicotine binding sites in AD is not due to genomic changes.

Aged↗
PubMed1996

A biotinylated MutS fusion protein and its use in a rapid mutation screening technique.

The Escherichia coli DNA mismatch repair protein, MutS, binds single base pair mismatches and short deletions in vivo and in vitro. To adapt this protein for mutation detection, a fusion protein of E. coli MutS with a biotinylated peptide domain has been constructed (MutSb). The biotinylation tag facilitates MutS detection and binding by avidin without significantly altering the DNA mismatch binding properties of MutS in vitro. We describe a novel and rapid mutation detection method with MutSb using streptavidin-coated magnetic beads and demonstrate that MutSb can also be used to remove mismatch containing DNA fragments from a mixture of DNA fragments in solution.

Adenosine Triphosphatases↗
PubMed2001

Mutation screening of the metabotropic glutamate receptor mGluR4 (GRM4) gene in patients with schizophrenia.

Disturbances in glutamate function have been implicated in the pathophysiology of schizophrenia. We searched for mutations in the exons of the metabotropic glutamate receptor mGluR4 (GRM4) gene on human chromosome 6p21.3 and evaluated associations between these polymorphisms with schizophrenia in Japanese patients. Nine nuclear variants of 450G > T, 1455T > C, 2202A > G, 2389G > A (Val797 > Ile797), 2890A > G, 3601C > T, 3639C > T, IVS4-36G > A, and IVS5 + 29(CCGGG)1-2, were found. The Val797Ile variant, although found in both the patient and control groups, was rare and the only variant that causes a non-synonymous amino acid change. There was no statistically significant association between any mGluR4 gene polymorphism and schizophrenia. Thus, this study did not provide evidence for the contribution of the mGluR4 gene to schizophrenia in the Japanese.

Adult↗
PubMed2003

Mutation screening of patients with Leber Congenital Amaurosis or the enhanced S-Cone Syndrome reveals a lack of sequence variations in the NRL gene.

PURPOSE: To determine if mutations in the retinal transcription factor gene NRL are associated with retinopathies other than autosomal dominant retinitis pigmentosa (adRP). METHODS: Genomic DNA was isolated from blood samples obtained from 50 patients with Leber Congenital Amaurosis (LCA), 17 patients with the Enhanced S-Cone Syndrome (ESCS), and a patient with an atypical retinal degeneration that causes photoreceptor rosettes with blue cone opsin. The 5' upstream region (putative promoter), untranslated exon 1, coding exons 2 and 3, and exon-intron boundaries of the NRL gene were analyzed by direct sequencing of the PCR-amplified products. RESULTS: Complete sequencing of the NRL gene in DNA samples from this cohort of patients revealed only one nucleotide change. The C->G transversion at nucleotide 711 of NRL exon 3 was detected in one LCA patient; however, this change did not alter the amino acid (L237L). CONCLUSIONS: No potential disease causing mutation was identified in the NRL gene in patients with LCA, ESCS, or the atypical retinal degeneration. Together with previous studies, our results demonstrate that mutations in the NRL gene are not a major cause of retinopathy. To date, only missense changes have been reported in adRP patients, and sequence variations are rare. It is possible that the loss of NRL function in humans is associated with a more complex clinical phenotype due to its expression in pineal gland in addition to rod photoreceptors.

Basic-Leucine Zipper Transcription Factors↗
PubMed2001

C-kit mutation screening in patients with acute myeloid leukaemia: adaptation of a Giemsa-stained bone-marrow smear DNA extraction technique.

The scarcity of viable tissue samples for leukaemia research is widely recognised and currently restrictive. Archival bone-marrow smears present a valuable resource that can be exploited easily for mutational analysis. Here, a modified technique to extract DNA is described, and used subsequently for mutation/polymorphism screening of the stem-cell factor receptor proto-oncogene c-kit in 23 patients with acute myeloid leukaemia (AML). The selected method was straightforward and used bone-marrow material scraped from periodic acid-Schiff, sudan black B and May-Grünwald/Giemsa-stained preparations, and treated initially with proteinase K prepared in digestion buffer to digest all proteinaceous matter. Following incubation, saturated sodium chloride was added and DNA extracted from the supernatant by phenol/chloroform/isoamyl alcohol treatment. Retrieved DNA was precipitated with ethanol at -20 degrees C overnight, washed with 95% ethanol, air-dried, resuspended using purite water and stored at -20 degrees C prior to use in mutational analysis. The extraction method described was compared with a commercial reagent for combined DNA, RNA and protein isolation using cryopreserved cells from 20 patients with AML. The quality of extracted DNA isolated by the two methods was comparable by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) techniques. Bone-marrow biopsies are performed regularly on each AML patient to monitor the disease; therefore, an extraction method using this resource could liberate a valuable source of DNA for study (e.g. molecular investigations, including mutation/polymorphism screening etc.). This would allow fresh and programme-frozen cells to be reserved for those investigations requiring intact, viable cells. The use of archived bone-marrow smears would permit vast increase in the scope for retrospective testing and large-scale analyses.

Acute Disease↗
PubMed1998

Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease with a wide range of clinical manifestations. It is most often caused by deficiency of steroid 21-hydroxylase, reflecting any of a wide range of mutations in the 21-hydroxylase (CYP21) gene. A major challenge in molecular diagnostics of CAH is the high homology between the CYP21 gene and the CYP21P pseudogene and the phenomenon of apparent gene conversion, which inactivates the functional gene. In this study we devised an improved stepwise diagnostic procedure involving nonradioactive Southern blotting and direct DNA sequencing. This strategy led to a successful elucidation of the molecular cause of the disease in 181 out of 182 unrelated alleles in a total of 91 clinically and biochemically characterized patients. We were able to identify all classical known disease-causing mutations of the 21-hydroxylase gene and a novel nonsense mutation (bp 670, A-->C, Y97X). Our method also allows the reliable, secure diagnosis of the heterozygous configuration and may therefore be used for pre-, peri-, and postnatal diagnosis of CAH, even when informative data of the index patient are lacking. Furthermore, it can be used to confirm the diagnosis of CAH in newborns detected in 17-hydroxyprogesterone screening programs.

Adrenal Hyperplasia, Congenital↗
PubMed1996

Human adenosine A2a receptor (A2aAR) gene: systematic mutation screening in patients with schizophrenia.

Several lines of evidence suggest an involvement of adenosine A2a receptor (A2aAR) mediated adenosinergic neuromodulation in the etiopathogenesis of schizophrenia. We therefore performed a systematic mutation scan of the complete coding region of the human A2aAR gene in a sample of 42 schizophrenic patients. We detected one rare naturally occurring receptor variant (Gly-340-Ser) and two silent mutations (405C/T and 1083C/T). To our knowledge the Gly-340-Ser substitution is the first naturally occurring molecular variant of the A2aAR identified. Determining the frequency of the three variants in 42 unrelated healthy controls, we observed a significant trend towards an overrepresentation of the 1083T variant in patients when compared to controls (p = 0.041). This trend was followed up in a large independent replication sample. However, we were not able to confirm the original trend in the second sample (p = 0.367). The Ser-340 variant was found in a single schizophrenic individual. Investigation of the patient's family revealed independent segregation between the Ser-340 variant and psychiatric illness. Our data suggest that genetically determined structural variation of the A2aAR does not play a major role in the development of schizophrenia.

Alleles↗
PubMed2005

Mutation screen of GABRA1, GABRB2 and GABRG2 genes in Japanese patients with absence seizures.

Absence seizures are classified into typical and atypical absences according to clinical and EEG characteristics. Although missense mutations in the GABA(A) receptor gamma2 subunits (GABRG2) gene have recently been detected in two families with typical absence seizures, no study has been carried out to clarify the relationship between atypical absence and GABA(A) receptors. We performed mutation analysis of all the coding exons of GABA(A) receptor alpha1, beta2 and gamma2 subunit (GABRA1, GABRB2 and GABRG2) genes by direct sequencing to clarify whether there was common molecular biological mechanism underlying both typical and atypical absences. We recruited 52 unrelated Japanese patients, thirty-eight with typical absences and 14 with atypical absences. They consisted of 38 with childhood absence epilepsy, three with Lennox-Gastaut syndrome, two with epilepsy with myoclonic-astatic seizures and nine with epilepsy with continuous spike-waves during slow wave sleep. All of the subjects were idiopathic or cryptogenic cases without any organic brain lesions or underlying diseases. We detected five polymorphisms (T156C in GABRA1, C1194T in GABRB2, and C315T, T588C and C1230T in GABRG2), and they are silent mutations. In conclusion, mutations in GABRA1, GABRB2 and GABRG2 do not seem to be a major genetic cause of epilepsy with typical and atypical absences in Japanese subjects.

DNA Mutational Analysis↗
PubMed1991

Mutation screening of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerase chain reaction.

We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses.

Animals↗
PubMed2003

Molecular characterization and mutational screening of the PRKAG3 gene in the horse.

The PRKAG3 gene encodes a muscle-specific isoform of the regulatory gamma subunit of AMP-activated protein kinase (AMPK). A major part of the coding PRKAG3 sequence was isolated from horse muscle cDNA using reverse-transcriptase (RT)-PCR analysis. Horse-specific primers were used to amplify genomic fragments containing 12 exons. Comparative sequence analysis of horse, pig, mouse, human, Fugu, and zebrafish was performed to establish the exon/intron organization of horse PRKAG3 and to study the homology among different isoforms of AMPK gamma genes in vertebrates. The results showed conclusively that the three different isoforms (gamma1, gamma2, and gamma3) were established already in bony fishes. Seven single nucleotide polymorphisms (SNPs), five causing amino acid substitutions, were identified in a screening across horse breeds with widely different phenotypes as regards muscle development and intended performance. The screening of a major part of the PRKAG3 coding sequence in a small case/control material of horses affected with polysaccharide storage myopathy did not reveal any mutation that was exclusively associated with this muscle storage disease. The breed comparison revealed several potentially interesting SNPs. One of these (Pro258Leu) occurs at a residue that is highly conserved among AMPK gamma genes. In an SNP screening, the variant allele was only found in horse breeds that can be classified as heavy (Belgian) or moderately heavy (North Swedish Trotter, Fjord, and Swedish Warmblood) but not in light horse breeds selected for speed or racing performance (Standardbred, Thoroughbred, and Quarter horse) or in ponies (Icelandic horses and Shetland pony). The results will facilitate future studies of the possible functional significance of PRKAG3 polymorphisms in horses.

AMP-Activated Protein Kinases↗
PubMed1998

PCR based mutation screening of the laminin alpha2 chain gene (LAMA2): application to prenatal diagnosis and search for founder effects in congenital muscular dystrophy.

Classical congenital muscular dystrophy with merosin deficiency is caused by mutations in the laminin alpha2 chain gene (LAMA2). Extended sequencing of the introns flanking the 64 LAMA2 exons was carried out and, based on these sequences, oligonucleotide primers were designed to amplify the coding region of each exon separately. By PCR-SSCP analysis, we identified eight new mutations in nine families originating from various countries. All induced a premature truncation of the protein, either in the short arm or in the globular C-terminal domain. A 2 bp deletion in exon 13, 2098delAG, was found in three French non-consanguineous families and a nonsense mutation of exon 20, Cys967stop, in two other non-consanguineous families originating from Italy. Determination of rare intragenic polymorphisms permitted us to show evidence of founder effects for these two mutations suggesting a remote degree of consanguinity between the families. Other, more frequent polymorphisms, G to A 1905 (exon 12), A to G 2848 (exon 19), A to G 5551 (exon 37), and G to A 6286 (exon 42), were used as intragenic markers for prenatal diagnosis. This study provides valuable methods for determining the molecular defects in LAMA2 causing merosin deficient congenital muscular dystrophy.

Child↗