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Results for “mutational screening”

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At least 541 records · Page 30Linked to original sources
PubMed2001

Screening for mutations and polymorphisms in the genes KCNH2 and KCNE2 encoding the cardiac HERG/MiRP1 ion channel: implications for acquired and congenital long Q-T syndrome.

BACKGROUND: The voltage-gated, rapid-delayed rectifier current (I(Kr)) is important for repolarization of the heart, and mutations in the genes coding for the K+-ion channel conducting this current, i.e., KCNH2 for the alpha-subunit HERG and KCNE2 for the beta-subunit MiRP1, cause acquired and congenital long Q-T syndrome (LQTS) and other cardiac arrhythmias. METHODS: We developed a robust single-strand conformation polymorphism-heteroduplex screening analysis, with identical thermocycling conditions for all PCR reactions, covering all of the coding exons in KCNH2 and KCNE2. The method was used to screen 40 unrelated LQTS patients. RESULTS: Eleven mutations, of which six were novel, were found in KCNH2. Interestingly, six mutations were found in the region of the gene coding for the Per-Arnt-Sim (PAS) and PAS-S1 regions of the HERG protein, stressing the need to examine the entire gene when screening for mutations. No mutations were found in KCNE2, suggesting that direct involvement of MiRP1 in LQTS is rare. Furthermore, four novel single-nucleotide polymorphisms (SNPs) and one amino acid polymorphism (R1047L) were identified in KCNH2, and one novel SNP and one previously known amino acid polymorphism (T8A) were found in KCNE2. CONCLUSIONS: The potential role of rare polymorphisms in the HERG/MiRP1 K+-channel should be clarified with respect to drug interactions and susceptibility to arrhythmia and sudden death.

Cation Transport Proteins↗
PubMed1992

Screening for mutations in the muscle promoter region and for exonic deletions in a series of 115 DMD and BMD patients.

Mutations in the muscle promoter region and exonic deletions were screened in a series of 115 unrelated DMD and BMD patients from north-east Italy. No gross mutations of the promoter region were found. In three cases in which dystrophin of normal size was expressed at low levels, the analysis of DNA sequences of the promoter region failed to detect abnormalities. The majority of deletions in coding sequences, detected by cDNA probes, occur in the deletion hot spot identified by the probe P20. Intrafamilial variability in the severity of the disease is reported and discussed.

Adolescent↗
PubMed2004

Denaturing HPLC-based approach for detecting RYR2 mutations involved in malignant arrhythmias.

BACKGROUND: Mutations in the RYR2 gene, which encodes the cardiac ryanodine receptor, have been reported in patients showing either arrhythmogenic right ventricular cardiomyopathy, type 2, or stress-induced polymorphic ventricular tachycardia. Both clinical phenotypes are characterized by a high risk of sudden death. Detection of RYR2 mutations is particularly important because beta-blocker treatment has been shown to be effective in preventing fatal arrhythmias in affected patients. METHODS: We used denaturing HPLC (DHPLC) to identify mutations in the human RYR2 gene. Fifty-three single exons, possibly targeted by mutations, were identified by comparison with the distribution of pathogenic mutations of the RYR1 gene, the skeletal muscle counterpart of RYR2. PCR primers for amplification of the entire coding sequence (116 amplicons, corresponding to 105 exons) were tested, and optimal DHPLC conditions were established. DHPLC analysis of critical exons was performed on 22 unrelated patients with effort-induced polymorphic ventricular arrhythmias but lacking a precise diagnosis. RESULTS: We identified four novel missense mutations among 22 patients. Their pathogenic role was related to present knowledge of the structure and function of RyR2 protein. CONCLUSIONS: Under optimized conditions, DHPLC is a cost-effective, highly sensitive, rapid, and efficient method for mutation screenings. A four-step approach is proposed for mutation screening of the RYR2 gene: (a) DHPLC analysis of 48 critical exons (2-4, 6-15, 17-20, 39-49, 83, 84, 87-97, and 99-105); (b) DNA sequencing of 5 critical exons unsuitable for DHPLC; then, in case of negative results, (c) DHPLC analysis of the remaining 39 exons and (d) DNA sequencing of the last 13 amplicons unsuitable for DHPLC analysis.

Adolescent↗
PubMed1991

Screening for mutations in the phenylalanine hydroxylase gene from Italian patients with phenylketonuria by using the chemical cleavage method: a new splice mutation.

To investigate the molecular basis of phenylketonuria in Italy we applied the chemical cleavage method (CCM) on amplified DNA encompassing exons 7 and 8 of the phenylalanine hydroxylase gene. These exons are in a region likely to be involved in enzyme function. Using this approach, we could simultaneously screen for novel mutations and for seven reported mutations which map in this area. Three mutations were identified. The first was shown to be a not previously described mutation: a G----A substitution at the 5' donor junction splice site of intron 7. The second change was a reported G----A mutation at codon 261. The third change corresponded to a polymorphism at codon 245. Our results indicate that CCM analysis of amplified genomic DNA fragments can be successfully used to search for mutations in large genes whose transcripts are not readily available.

Base Sequence↗
PubMed2003

Expanding the phenotypic spectrum of the CACNA1A gene T666M mutation: a description of 5 families with familial hemiplegic migraine.

BACKGROUND: Familial hemiplegic migraine (FHM) is a rare autosomal dominant subtype of migraine with aura. Missense mutations in the chromosome 19 CACNA1A calcium channel gene have been found in approximately half of the families. The T666M mutation, replacing a threonine by a methionine at residue number 666, is the most frequent mutation, reported in 14 independent FHM families; other mutations have so far been described in only 1 or 2 families each. The clinical features of T666M families have been reported, but the course is unknown. OBJECTIVE: To present a detailed description of the clinical features of new FHM families in which we identified the T666M mutation in our CACNA1A screening program. METHODS: As part of our ongoing genetic screening, mutation analysis of the CACNA1A gene was performed by single-strand conformational polymorphism analysis in 33 probands of families with FHM. RESULTS: We identified the T666M mutation in 5 unrelated FHM families. In 3 of the families, patients displayed cerebellar ataxia. In 1 family, some affected members with the mutation had attacks with confusion but without hemiparesis. In 1 family, patients had progressive cognitive dysfunction. CONCLUSIONS: The T666M mutation is the most frequent CACNA1A mutation in FHM; it was found in 5 of 33 FHM families at our laboratory, and in 19 of 39 families with a known mutation reported in the literature (including the present study). Screening for the T666M mutation should therefore be the first step when screening families with FHM. There is a remarkable clinical heterogeneity among families with the T666M mutation.

Adult↗
PubMed1998

Screening male intracytoplasmic sperm injection candidates for mutations of the follicle stimulating hormone receptor gene.

Follicle stimulating hormone (FSH) is considered to be essential for spermatogenesis. Therefore, genetic abnormalities of FSH signalling on testicular Sertoli cells would be expected to affect sperm production negatively in males. Inactivating FSH receptor mutations have been reported earlier in both males and females. All affected males had elevated FSH serum concentrations and abnormal sperm parameters. We postulated that inactivating FSH receptor mutations might be a cause of oligozoospermia or azoospermia and reviewed the clinical data of 151 male intracytoplasmic sperm injection (ICSI) candidates with special attention to FSH serum concentrations. The exclusion criteria for mutation screening of the FSH receptor gene were: a history of operative sterilization or testicular malignancy, congenital abnormality other than cryptorchidism, and a chromosomal aberration or a Y-chromosome microdeletion. The inclusion criteria were: male (ICSI candidate) with azoospermia or oligoasthenoteratozoospermia (OAT) and elevated FSH serum concentrations. In total, 23 males with OAT and five males with azoospermia were tested for mutations of the coding sequences and the intron-exon boundaries of the FSH receptor gene by polymerase chain reaction (PCR) followed by single strand conformation polymorphism analysis (SSCP). Neutral polymorphisms were readily detected using this technique in both probands and controls. None of the 28 selected patients showed a pathogenic FSH receptor mutation. Mutations in the FSH receptor gene are not a common cause of infertility in ICSI candidates.

Cytoplasm↗
PubMed1995

Detection of ten new mutations by screening the gene encoding factor IX of Danish hemophilia B patients.

Hemophilia B is caused by a wide range of mutations. In order to characterize the mutations among patients in Denmark, we have systematically screened the entire coding region, the promoter region and exon flanking sequences of the gene encoding factor IX using single strand conformation and heteroduplex analyses. Patients from 32 different families were examined, and point mutations (23 different) were found in all of them. Ten of the mutations have not been reported by others; they include a splice site mutation, a single base pair deletion, and missense mutations. Notably, the study contains a female patient and a previously described Leyden mutation. In ten families with sporadic cases of hemophilia B, all 10 mothers were found to be carriers. The origin of two of these mutations was established.

Base Sequence↗
PubMed2007

Genetic screen for mutations affecting development and function of the enteric nervous system.

An intact enteric nervous system is required for normal gastrointestinal tract function. Several human conditions result from decreased innervation by enteric neurons; however, the genetic basis of enteric nervous system development and function is incompletely understood. In an effort to increase our understanding of the mechanisms underlying enteric nervous system development, we screened mutagenized zebrafish for changes in the number or distribution of enteric neurons. We also established a motility assay and rescreened mutants to learn whether enteric neuron number is correlated with gastrointestinal motility in zebrafish. We describe mutations isolated in our screen that affect enteric neurons specifically, as well as mutations that affect other neural crest derivatives or have pleiotropic effects. We show a correlation between the severity of enteric neuron loss and gastrointestinal motility defects. This screen provides biological tools that serve as the basis for future mechanistic studies.

Animals↗
PubMed2000

Functional evaluation of p53 and PTEN gene mutations in gliomas.

We screened mutations of two major tumor suppressor genes, p53 and PTEN, in 66 human brain tumors using a yeast-based functional assay and cDNA-based direct sequencing, respectively. The frequency of p53 mutations was 28.8% (19 of 66) and was higher in anaplastic astrocytoma (9 of 14, 64.3%,) than in glioblastoma multiforme (GBM; 7 of 27, 25.9%,), supporting previous speculation that there are at least two genetic pathways leading to GBM, a de novo pathway without p53 mutation and a "progressive" pathway with p53 mutation. PTEN mutation was observed in 8 of 64 tumors (12.5%), mainly GBMs (7 of 26, 26.9%), both with and without p53 mutation. These results suggest that mutation of the PTEN gene is a later event than that of the p53 gene in glioma progression and is associated with both the genetic pathways. All of the detected PTEN missense mutations and an in-frame small deletion inactivated PTEN phosphoinositide phosphatase activity in vitro. Because the tumors containing PTEN mutations also showed loss of heterozygosity in the chromosome 10q23 region flanking the PTEN gene, our data clearly indicate that inactivation of both PTEN alleles occurs in a subset of high-grade gliomas, therefore confirming the previous idea that PTEN acts as a tumor suppressor gene.

Adolescent↗
PubMedDate not supplied

Ethnic intermarriage and its consequences for cystic fibrosis carrier screening.

Cystic fibrosis gene mutations can vary in frequency between different ethnic populations. However, there is a rising trend of ethnic intermarriage in the United States, a situation suggesting that differences in specific mutation frequencies currently apparent in Europe may not persist for long in this country. Therefore, limited mutation screens targeted at specific ethnic groups and risk calculations based on data from more homogeneous European populations may not be appropriate in the United States. The genetic consequences of ethnic admixture are also likely to extend to other recessive diseases (e.g., Tay-Sachs, thalassemia), which, in the past, have been limited largely to particular ethnic, racial, or religious subgroups, with implications for public health agencies overseeing newborn screening programs for genetic diseases and for clinical genetics programs offering population-based carrier-detection programs, carrier risk assessment, and counseling.

Cystic Fibrosis↗
PubMed1996

Screening for mutations of the human thyroid peroxidase gene in patients with congenital hypothyroidism.

While congenital hypothyroidism in 80-90% of the affected individuals is caused by thyroid dysgenesis (athyrosis, ectopy or hypoplasia), hypothyroidism in patients with a thyroid gland of normal position and size can be due to regulatory or enzymatic defects of thyroid hormone biosynthesis. Beside defects of thyroglobulinsynthesis, defects of the sodium-iodide-transporter or the TSH-receptor, a defect of the thyroidperoxidase, the key-enzyme of thyroid hormone biosynthesis, can cause a total iodide organification defect and thereby congenital hypothyroidism. We screened 14 of 103 patients (13.6%) with non familial congenital hypothyroidism and a normally developed thyroid gland detected by the newborn screening program with the PCR-SSCP (single-stranded-conformational-polymorphism) technique for mutations in the exons 2, 8, 9, 10 and 14 of the human thyroperoxidase gene, and in which mutations had been described previously in Dutch and Brazilian families with total organification defects. Most of the previously reported mutations were found in exons 8, 9 and 10 which code for the caralytic part of the enzyme. In two patients a GGCC-duplication in exon 8 was detected leading to a premature stop codon in exon 9. While one patient without neonatal goiter was homozygous for this mutation, the second patient was only heterozygous thus demanding another mutation on the second TPO-allel to explain the phenotype. Since the GGCC duplication is easily demonstrable by a NaeI digestion, because it creates a restriction site for this enzyme, screening for this mutation is indicated since it is easy to perform. In contrast to the perchlorate discharge test molecular genetic studies are less invasive, but as useful in making a definitive diagnosis in the individual patient. Furthermore it is the first feasible step to study the etiology and epidemiology of the so far only putative defects of thyroid hormone biosynthesis leading to congenital hypothyroidism.

Congenital Hypothyroidism↗
PubMed1999

Screening for mutations in candidate genes for hypospadias.

Hypospadias. a condition with a frontally placed urethral orifice on the penis, is the most common malformation in males. During fetal development several components are necessary for normal male genital development. Testosterone and dihydrotestosterone act via the androgen receptor but a defective receptor function results in different degrees of genital malformations. Testosterone-5alpha-reductase converts testosterone to dihydrotestosterone, which is crucial for normal differentiation, and a total lack of this enzyme results, in syndromes with hypospadias. The Wilms' tumour 1 (WT1) gene is expressed in the fetal gonad and genital malformations can occur due to WT1 gene mutations. These genes are therefore strong candidate genes for hypospadias. We have analysed 35 boys with hypopadias and one girl diagnosed as with complete androgen insensitivity syndrome, using exon by exon polymerase chain reaction (PCR) amplification of the AR, WTI and 5alpha-reductase genes and screened for point mutations and performed subsequent DNA sequencing. No mutations in any of these genes were found in the 26 patients with isolated hypospadias. Two patients with severe hypospadias with cryptorchidism were found to carry mutations in the androgen receptor gene. Also the girl with clinically diagnosed complete androgen insensitivity was found to be homozygous for a splice mutation in the 5alpha-reductase gene. In summary, mutations in the WT1, AR and 5alpha-reductase genes are not common causes of isolated hypospadias.

Base Sequence↗
PubMed1985

A genetic screen for mutations that increase the thermal stability of phage T4 lysozyme.

A method has been developed to screen for mutants of phage T4 lysozyme that are more stable than the wild-type enzyme. Using an assay that detects lysozyme activity on Petri plates [Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, A., Terzaghi, E. & Inouye, M. (1966) Cold Spring Harbor Symp. Quant. Biol. 31, 77-84], protein synthesized during the formation of phage plaques at a permissive temperature (33 degrees C) was tested for its ability to withstand incubation at a temperature that inactivates the wild-type enzyme. In our initial screen of approximately 3 X 10(4) plaques from a T4 phage stock mutagenized with hydroxylamine, greater than 30 mutants that produce lysozyme activity resistant to high temperature incubation were found. Lysozyme produced by two of the mutants was purified and found to denature at a higher temperature than the wild-type enzyme in vitro. We have called such mutants "st" for thermostable. The existence of st mutants indicates that protein stability is not maximized during evolution; instead, it is likely that stability is optimized for the physiology of the organism. Analysis of the structures of these mutants will provide another way to identify and predict interactions that stabilize proteins. The method of finding thermostable variants presented here may be applicable to any protein that can be detected by a plate assay or by a plate screen with antibodies.

Hot Temperature↗
PubMed2005

Comparison of PCR-based mutation detection methods and application for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates.

Reverse genetic approaches to generate mutants of model species are useful tools to assess functions of unknown genes. Recent work has demonstrated the feasibility of such strategies in several organisms, exploiting the power of chemical mutagenesis to disrupt genes randomly throughout the genome. To increase the throughput of gene-driven mutant identification, efficient mutation screening protocols are needed. Given the availability of sequence information for large numbers of unknown genes in many species, mutation detection protocols are preferably based on PCR. Using a set of defined mutations in the Hprt1 gene of mouse embryonic stem (ES) cells, we have systematically compared several PCR-based point mutation and deletion detection methods available for their ability to identify lesions in pooled samples, which is a major criterion for an efficient large-scale mutation screening assay. Results indicate that point mutations are most effectively identified by heteroduplex cleavage using CEL I endonuclease. Small deletions can most effectively be detected employing the recently described "poison" primer PCR technique. Further, we employed the CEL I assay followed by conventional agarose gel electrophoresis analysis for screening a library of chemically mutagenized ES cell clones. This resulted in the isolation of several clones harboring mutations in the mouse Sult1a1 locus, demonstrating the high-throughput compatibility of this approach using simple and inexpensive laboratory equipment.

Animals↗
PubMed1996

Use of the yeast one-hybrid system to screen for mutations in the ligand-binding domain of the estrogen receptor.

This report describes a novel yeast one-hybrid system which easily allows for the detection of mutations in the ligand-binding domain of the estrogen receptor. This screen is based on the observation that a fusion protein consisting of the GAL4 DNA-binding domain and the estrogen receptor can interact with a GAL4 upstream activating sequence and induce the expression of an integrated GAL1-lacZ gene only in the presence of estradiol. Various deletion mutants of the estrogen receptor were tested in this assay and activating function 1 which is present in the N-terminus of the estrogen receptor was found to be responsible for the transactivation produced in the assay. To test if the screen could be used to detect random mutants in the ligand-binding domain of the estrogen receptor the region of the human receptor between amino acids 381 to 403 was mutated by oligonucleotide saturation mutagenesis. Two of the mutants generated by this mutagenesis were characterized to demonstrate that the results obtained from the screen in the yeast screen are relevant to mammalian systems. One of the mutants which has a valine at position number 388 instead of a glycine was able to transactivate in both the yeast and a mammalian system. This mutant was a more potent activator of transcription and also appeared to have a higher affinity for [3H]estradiol in vivo than the wild type receptor. The other mutant which was characterized has five amino acid changes from amino acids 390 through 400. This mutant was nonfunctional in the yeast and mammalian transcription assays and did not bind [3H]estradiol in vivo or in vitro.

Animals↗
PubMed2006

Mutations in the testis-specific NALP14 gene in men suffering from spermatogenic failure.

BACKGROUND: Because of the common use of ICSI and the potential genetic aetiology of spermatogenic failure, concern has been raised about transmitting genetic disorders to ICSI offspring. However, to date, in only approximately 15% of all cases of spermatogenic failure, an underlying genetic cause can be identified. We have previously established an association between spermatogenic failure and chromosomal region 11p15. In this study, we set out to explore whether NALP14, a gene recently mapped to 11p15, has a function in spermatogenesis and whether mutations in NALP14 can cause spermatogenic failure. METHODS: We applied two different multiple tissue northern (MTN) blots to determine tissue specificity of NALP14 and performed immunohistochemistry on human testis with anti-NALP14 antiserum. To determine imprinting status of NALP14, we tested the expression pattern of two single-nucleotide polymorphisms (SNPs) in human testis. Finally, we performed a mutation screen of the NALP14 gene in 157 men with azoospermia or severe oligozoospermia by direct sequencing; 158 normospermic men served as controls. RESULTS: NALP14 was, as are the three other genes in 11p15, exclusively expressed in testis. Within the testis, the NALP14 protein was mainly expressed in A dark spermatogonia, mid and late spermatocytes and spermatids. The mutation screen revealed five mutations that occurred only in the patient group. One of these unique mutations introduced an early stop codon in the NALP14 sequence, predicted to result in a severely truncated protein. CONCLUSION: Our data suggest that NALP14 has a function in spermatogenesis and that mutations in this gene might cause spermatogenic failure.

Amino Acid Sequence↗
PubMed2006

[The mutation R672H in SCN4A gene exists in Chinese patients with hypokalaemic periodic paralysis].

OBJECTIVE: Mutation screening was performed on two Chinese families with HOKPP to locat the corresponding mutations and to specify the clinical features associated with the mutation. METHODS: Target-exon PCR and direct sequencing were used to screen mutation in the CACNA1S and SCN4A gene of all numbers of the two families. The clinical features of patients were summary. RESULTS: A heterozygous point mutation 2015G-->A causing R672H in the SCN4A was found in five patients and five normal relatives of the two families. Features of R672H mutation are incomplete penetrance, especially non-penetrance of phenotype in women and potassium is effective, but acetazolamide is not. CONCLUSION: The SCN4A R672H mutation exists in the Chinese family with HOKPP.

Adolescent↗
PubMed2006

Screening for mutations affecting sexual reproduction after activation tagging in Arabidopsis thaliana.

In this work, a seed-set-based screening was performed on 70 lines of Arabidopsis thaliana after activation tagging mutagenesis to identify mutations in reproductive mechanisms. Five mutants showed significantly lower seed set than the wild type and confirmed the phenotype in the progeny. This phenotype was linked with the marker gene bar carried by T-DNA conferring glufosinate resistance. Genetic analysis revealed that the mutation inheritance was sporophytic in 3 mutants and gametophytic in 2 mutants. In addition, 2 mutants had an extra T-DNA copy. Thus activation tagging can be an effective strategy to identify new mutations affecting sporogenesis or gametogenesis.

Arabidopsis↗