PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “DNA Mutational Analysis”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1997

Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders.

AIM: To facilitate DNA mutation analysis by use of capillary electrophoresis. METHODS: The usefulness and applications of capillary electrophoresis in DNA fragment sizing and sequencing were evaluated. RESULTS: DNA mutation testing in disorders such as cystic fibrosis, Huntington disease, alpha thalassaemia, and hereditary fructose intolerance were undertaken effectively. However, sizing the (CAG)n repeat in the case of Huntington disease was a potential problem when using capillary electrophoresis. Separation polymers used in capillary electrophoresis are still in the developmental phase, with improved ones being released regularly. CONCLUSIONS: In the DNA diagnostic setting, capillary electrophoresis is a valuable development because it expands the scope for automation and has useful analytical properties. The potential to perform complex multiplexing within one electrophoresis run facilitates DNA diagnosis. The different mobility of DNA fragments in capillary electrophoresis compared with conventional gel electrophoresis will require, in some circumstances, additional care when results are being interpreted or reported. Capillary electrophoresis is a cheap alternative for combined automated sequencing and fragment analysis that utilises multicolour fluorescence capability. However, in its present form, it is not useful for large scale sequencing.

Costs and Cost Analysis↗
PubMed2004

Diagnostic value of mitochondrial DNA mutation analysis in juvenile unilateral ptosis.

PURPOSE: To highlight the diagnostic relevance of mitochondrial DNA (mtDNA) mutation analysis in acquired juvenile unilateral upper eyelid ptosis. METHODS: A 13-year-old boy presented with acquired, slowly progressive unilateral ptosis. We performed ophthalmological and neurological examinations, laboratory testing, skeletal muscle biopsy including histological and histochemical investigations, biochemical analysis of respiratory chain enzymes in skeletal muscle homogenate and molecular genetic testing of skeletal muscle DNA. RESULTS: Though clinical, laboratory, histological and biochemical analyses did not reveal any hints suggesting a mitochondrial cytopathy, molecular genetic testing by Southern blot analysis of total DNA from skeletal muscle tissue showed a 5.8 kb mtDNA deletion thus proving the diagnosis of mitochondrial chronic progressive external ophthalmoplegia (CPEO). CONCLUSIONS: In patients with unexplained acquired juvenile unilateral ptosis, an underlying mitochondrial cytopathy should be considered even in cases of inconspicuous ancillary examinations comprising skeletal muscle histology and biochemistry. To establish the diagnosis, molecular genetic testing of DNA derived from skeletal muscle tissue is essential in those patients.

Adolescent↗
PubMed2005

[Mitochondrial DNA mutation analysis in patients with mitochondrial myopathy].

OBJECTIVE: To examine mitochondrial DNA mutations in mitochondrial myopathy. METHODS: Three suspected cases of mitochondrial myopathy were examined by HE staining, histochemical staining methods and electron microscopy. The mutations in all 22 tRNA genes of mitochondrial genome were screened by polymerase chain reaction-single strand conformation polymorphism and DNA sequencing. RESULTS: The three cases were diagnosed as mitochondrial myopathy. The examinations revealed that patient 1 had a homoplasmic A1627G mutation in tRNA-Val gene, and patient 2 had a heteroplasmic A1627G/A mutation in tRNA-Val gene, and patient 3 had two mutationsuone was homoplasmic T5554C mutation in tRNA-Trp gene, the other was heteroplasmic A10412C/A mutation in tRNA-Arg gene. CONCLUSION: tRNA genes mutations of mtDNA might be one of the etiologies of mitochondrial myopathy.

Adult↗
PubMed2000

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection.

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.

Automation↗
PubMed2002

Mitochondrial DNA mutation analysis in human skin fibroblasts from fetal, young, and old donors.

Multibase deletions in mitochondrial DNA (mtDNA) have been shown to accumulate with age in several tissues, including skin, whereas point mutations have only recently been demonstrated to increase during aging, with several specific mutations occurring at high levels (up to 50%) in skin fibroblasts obtained from old donors [Science 286(1999)774]. We have conducted a survey for a specific deletion and for point mutations in several regions of mtDNA from cultured skin fibroblasts derived from eight fetal (12-20 weeks gestational age), ten young (17-33 years of age) and 11 old (78-92 years of age) human donors. Using PCR analysis, detectable levels of the 4977 basepair (bp) 'common deletion' were present in all three age groups, with the highest deletion levels of up to 0.3% of total mtDNA found in several cell lines from old donors, although other old donor cell lines had much lower levels. Single strand conformation polymorphism (SSCP) analysis for point mutations in the non-coding D-loop region and two regions of the cytochrome oxidase 2 gene failed to reveal the presence of any single base mutations. We infer that age-related high level mutational damage in mtDNA from human skin fibroblasts may manifest both sequence and inter-individual specificity.

Adolescent↗
PubMed1994

DNA mutational analysis of type 1 and type 3 Gaucher patients: how well do mutations predict phenotype?

The wide spectrum of clinical manifestations resulting from glucocerebrosidase deficiency complicates genetic counseling for Gaucher disease. The identification of mutations in the glucocerebrosidase gene has enabled studies of genotype-phenotype correlation. However, a genotypic analysis of 60 type 1 and type 3 Gaucher patients reveals that the 5 most common Gaucher mutations, N370S, L444P, R463C, 84insG, and IVS2 + 1 G-->A, can be found both in patients with and without neurologic manifestations. Moreover, although some generalizations can be made about mutations that are more frequently encountered in particular patient populations, Gaucher patients sharing identical genotypes can exhibit considerable clinical heterogeneity. Thus in considering rationale for population screening one cannot rely solely on PCR determined DNA mutation analysis to reliably predict prognosis in Gaucher disease.

Adolescent↗
PubMed1999

Prenatal diagnosis of glycogen storage disease type IV using PCR-based DNA mutation analysis.

Deficiency of glycogen branching enzyme activity causes glycogen storage disease type IV (GSD-IV). Clinically, GSD-IV has variable clinical presentations ranging from a fatal neonatal neuromuscular disease, to a progressive liver cirrhosis form, and to a milder liver disease without progression. Current methods for prenatal and postnatal diagnosis are based on an indirect method of measuring the enzyme activity, which has a limited sensitivity and cannot be used to distinguish patients with these variable clinical phenotypes. In this study, a GSD-IV family with a non-progressive hepatic form of the disease requested prenatal diagnosis. Determination of the branching enzyme activity in cultivated amniocytes showed 20 per cent residual activity overlapping with the level detected in the heterozygotes. Mutation analysis revealed that the fetus carried two mutant alleles, L224P and Y329S, the same as the proband of this family. The fetus was predicted to be affected and postnatally his clinical presentation is consistent with the diagnosis. We conclude that DNA mutation analysis should be used in the prenatal diagnosis of GSD-IV, especially in the situation of high residual enzyme activity.

Child, Preschool↗
PubMed1998

Buccal cell DNA mutation analysis for diagnosis of cystic fibrosis in newborns and infants inaccessible to sweat chloride measurement.

OBJECTIVES: To assess the application of DNA-based cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis as a primary cystic fibrosis (CF) diagnostic test in preterm and term newborns and infants for whom the quantitative pilocarpine iontophoresis test (QPIT) cannot be used. DESIGN: Retrospective survey. SETTING: DNA Diagnostic Laboratory, Children's Hospital, Boston, Massachusetts. Buccal cell DNA samples were received from inpatients, outpatients, and three neonatal intensive care units. OUTCOME MEASURE: Detection of at least 1 of 12 CFTR mutations. PATIENTS: Between November 1, 1992, and April 30, 1994, 28 newborns and infants under 12 months of age at risk for CF had CFTR DNA mutation analysis performed because a sweat chloride (SC) value could not be obtained. QPIT was either not performed (infant weight <2 kg, QPIT not available at site of hospitalization, or infant not accessible to QPIT laboratory) or was inconclusive (sweat volume <75 mg or indeterminate SC [>/=40, <60 mEq/L]). The postnatal age at time of testing ranged from 1 day to 11 months, and gestational age at birth from 25 to 40 weeks. RESULTS: Six (21%) of 28 infants with unobtainable or indeterminate QPIT had 1 or 2 CFTR mutations detected. Immediate CF diagnosis by direct detection of 2 CFTR mutations was made in 5 of these 6 patients. Definitive CF diagnosis in the infant with 1 CFTR mutation was delayed until an elevation in SC could be documented. The patients with no CFTR mutations detected had a low likelihood of CF. CONCLUSIONS: For infants in whom CF is suspected but QPIT cannot be obtained, buccal cell DNA-based CFTR mutation analysis can be used as a rapid, noninvasive primary diagnostic test. This simple mode of DNA collection may aid in the diagnosis of other inherited disorders in newborns.

Cystic Fibrosis↗
PubMed2001

Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons.

BACKGROUND: Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses. METHODS: We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction. To evaluate this procedure, we examined the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with an increased risk for cardiovascular disease and neural tube defects. DNA was isolated from 10 microL of fresh EDTA-blood samples by use of a robotic workstation. The DNA samples were analyzed using molecular beacons as well as conventional methods. RESULTS: Both methods were compared, and no differences were found between outcomes of genotyping. CONCLUSIONS: The described assay enables robust and automated extraction of DNA and analysis of up to 96 samples (10 microL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high-throughput analyses.

Autoanalysis↗
PubMed2006

DNA mutation analysis in heterotaxy.

Heterotaxy refers to the abnormal arrangement of internal organs in relation to each other. It is characterized by complex cardiac malformations that are thought to result from abnormal left-right patterning in early embryonic development. Mutations in four genes have been identified in human heterotaxy. ZIC3, a zinc finger transcription factor, causes X-linked heterotaxy. EGF-CFC, ACVR2B, and LEFTYA are all members of a transforming growth factor-beta signal transduction pathway that is critical for proper left-right development. Point mutations have been identified in each of these genes using polymerase chain reaction-based mutation analysis strategies. ZIC3 mutation screening will be used to illustrate the methods for molecular sequence data acquisition and examination. These techniques are applicable to any gene of interest and will be useful for further evaluation of candidate genes for heterotaxy.

Abnormalities, Multiple↗
PubMed1992

DNA mutation analysis of Gaucher patients.

We evaluated 62 Gaucher patients to determine whether patients with similar phenotypes had the same DNA point mutations. Genomic DNA from these Gaucher patients was screened for the 3 most frequent single-point mutations, occurring in 69% of the 124 patient alleles, and resulting in changes in amino acids 370, 444, and 463. Many different genotypes were observed, at least one of which is present in all 3 types of Gaucher disease. No specific symptom complex could be correlated with a unique genotype. Even the more clinically homogeneous subgroups of Gaucher patients contained several genotypes. This study further emphasizes the need for caution in making clinical predictions on the basis of current genotype analysis, especially since one might not discern a fetus affected with type 2 disease by current DNA studies. The severity of involvement in type 1 disease could also not be predicted. Thus, even limiting our focus to 3 isolated common point mutations, a given genotype cannot be uniquely correlated with a specific prognosis.

Adolescent↗
PubMed1996

Prenatal diagnosis in congenital erythropoietic porphyria by metabolic measurement and DNA mutation analysis.

Identification of uroporphyrinogen III synthase (UROIIIS) gene mutations in patients with congenital erythropoietic porphyria (CEP) allows fast and reliable carrier detection and prenatal diagnosis. We describe here the first case of prenatal diagnosis by concomitant measurement of uroporphyrin I in amniotic fluid and direct detection of the gene mutation. A French couple, whose first child was diagnosed with CEP, requested prenatal diagnosis at 16 weeks of gestation. Uroporphyrin I was dramatically increased in amniotic fluid and the fetus was homozygous for the C73R mutation, the most common mutation in this disease. The pregnancy was then terminated.

Amniotic Fluid↗
PubMed2003

Efficiency and cost effectiveness: PAGE-SSCP versus MDE and Phast gels for the identification of unknown beta thalassaemia mutations.

BACKGROUND: Prenatal diagnosis for beta thalassaemia has proved to be very effective in preventing the birth of an affected child and hence in controlling the disease. The success of prenatal diagnosis depends on the delineation of the underlying mutations in the population at risk. Each population carries a limited number of frequent defects (89-91%) and a variable number of rare alleles (4-5%), whereas 2-3% of alleles remain uncharacterised. To offer prenatal diagnosis when the parental mutation is unknown, the application of a non-specific detection method (such as single stranded conformational polymorphism (SSCP)) to localise the mutation, followed by direct sequencing of the amplified gene sequence, is required. With this objective in mind, this study was designed to devise the best protocol and system of SSCP for the rapid screening of unknown mutations in the beta globin gene. METHODS: To detect mutations in this disease, three different systems-Phast gels, MDE gels, and polyacrylamide gels-were used under varying conditions. RESULTS: Polyacrylamide gels were found to be the most efficient, both in terms of resolution and cost. CONCLUSION: Polyacrylamide gels are the most rapid, efficient, reliable, and cost effective means for DNA mutation analysis of the beta globin gene.

Autoradiography↗
PubMed1995

DNA-based mutation analysis of Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinaemia.

The identification of the BTK (Bruton's tyrosine kinase) gene defective in human immunoglobulin deficiency X-linked agammaglobulinaemia (XLA) and characterisation of BTK exon-intron boundaries has now allowed the analysis of mutations and polymorphisms at the level of genomic DNA. Using Southern blot analysis and the polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) assay, amplifying all 19 exons and the putative promoter region with a single annealling temperature, mutations have been identified in 19 out of 24 unrelated patients diagnosed as having XLA. Apart from a large deletion involving exon 19, nine missense (F25S, R288W, 1370M, M509V, R525P, N526K, R562W, A582V and G594R), two nonsense (E277X and R525X), five frameshift and two splice site mutations have been found affecting most coding exons and all major enzyme domains. No mutations or polymorphisms were detected in the putative promoter region. A single nucleotide deletion located in the last exon, resulting in a truncation of the eight C-terminal residues of Btk and a typical XLA phenotype, indicates structural and/or functional importance of Btk helix I in the catalytic domain. Although allelic heterogeneity at the BTK locus may partly explain clinical variability in families with XLA, compensatory and redundant mechanisms involved in B-cell development must play a role in the phenotypic diversity of the disease.

Adolescent↗
PubMed1999

A mutation within exon 14 of the TGFBI (BIGH3) gene on chromosome 5q31 causes an asymmetric, late-onset form of lattice corneal dystrophy.

PURPOSE: Two forms of autosomal-dominant lattice corneal dystrophy (LCD), types I and IIIA, have previously been shown to be caused by different mutations within the transforming growth factor, beta-induced (TGFBI) gene. A clinical and molecular analysis of three unrelated kindreds with a clinically distinct late-onset LCD was undertaken to determine whether this phenotype is also caused by mutations within the TGFBI gene. DESIGN: Experimental study. PARTICIPANTS: Thirty-two members of three kindreds with corneal dystrophy. DNA from 100 normal control subjects was used as a control population. METHODS: Members of three kindreds with LCD were examined clinically, and blood samples were taken for DNA analysis. Mutation analysis was undertaken on all individuals for the coding region of the TGFBI gene by means of polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism/heteroduplex analysis, subcloning, and sequencing. MAIN OUTCOME MEASURES: Detection of mutations within the TGFBI gene. RESULTS: Clinical examination revealed a form of LCD that was bilateral in all but one case, with onset around the fourth to fifth decade. The majority of cases showed significant asymmetry, and in one case there was evidence of onset directly after minor superficial corneal trauma. Molecular analysis in all families demonstrated sequence changes within exon 14 of the TGFBI gene on chromosome 5q31, at codon 622 in family 3, and at codon 626 in families 1 and 2, which are presumed to be responsible for the disease. CONCLUSIONS: Previously, a late-onset form of LCD, termed IIIA, was shown to be caused by a P501T mutation in exon 11 of TGFBI. The authors present the first description of mutations in exon 14 of TGFBI causing an LCD, also of late onset. Although the condition presented is morphologically and histopathologically typical of an isolated lattice dystrophy, the age of onset and clinical course is not typical of type I, III, or IIIA lattice dystrophy. This, in conjunction with recent developments in our understanding of the molecular genetics of these disorders, calls into question the usefulness and validity of the current classification of the isolated lattice dystrophies.

Adult↗
PubMed1993

Minisatellite variant repeat mapping: application to DNA typing and mutation analysis.

Most DNA typing systems assay allele length variation at tandemly repeated loci such as minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at minisatellite loci and of the mutational mechanisms that generate ultravariability.

Alleles↗
PubMed2002

Molecular analysis of mutations in DNA polymerase eta in xeroderma pigmentosum-variant patients.

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase eta, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation.

Cell Line↗