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PubMed2003

Survey of Cryptosporidium parvum genotypes in humans from the UK by mutation scanning analysis of a heat shock protein gene region.

A mutation scanning-selective sequencing approach was employed for the genotypic identification and differentiation of Cryptosporidium parvum isolates. Genomic DNA samples (n=158) from Cryptosporidium oocysts from humans with clinical cryptosporidiosis (following recent foreign travel or during different outbreaks) in the UK were subjected to PCR-based single-strand conformation polymorphism (SSCP) analysis of a heat shock protein 70 gene region (p-hsp70; 448 bp). Samples representing different SSCP profiles were then subjected to sequencing. The analysis allowed the classification of 149 of the 158 samples as type-1 ( approximately 49%) or type-2 ( approximately 46%); amplicons from the remaining nine samples were consistent in size with p-hsp70 but represented non-specific, faecal contaminants. The percentages reflected those of a previous study in the UK for autochtonous, sporadic cases of cryptosporidiosis ( approximately 49% for type-1 and approximately 47% for type-2; n approximately 4000), but contrasted another survey of sporadic cases where type-2 dominated ( approximately 62%; n approximately 1000). The ability of the present SSCP-sequencing approach to accurately screen for C. parvum genotypes and to reliably discern erroneous amplicons has significant implications for the accurate diagnosis and monitoring of cryptosporidiosis and for population genetic studies.

Animals↗
PubMed2026

Deep mutational scan of the pore of the cold-sensing TRPM8 channel.

Members of the Transient Receptor Potential (TRP) family of ion channels have a nearly ubiquitous role in human physiology, tuning cell signaling to remarkably diverse physical and chemical stimuli. Although there is extensive structural data on TRP channels, a systematic and unbiased interrogation of structure-function relations in these proteins is required to fully elucidate their mechanisms of function. By focusing on a critical pore region of the TRPM8 channel, which is the main detector of cold and cooling agents in sensory neurons, we show how deep mutational scanning can be used in combination with the available structural data to understand how TRP channels respond to stimuli. We define a novel mechanism whereby the extracellular pore loop, which has only been resolved in structures representing desensitized states of the channel, plays an essential role in the response of TRPM8 to menthol or cold by coordinating the movement of the S6 helices that line and gate the pore, and the ion-selectivity filter that binds permeant cations. Moreover, our screen reveals sequence determinants along the S6 helices that explain how their architecture sustains gating and, together, provide strong support for a structural mechanism of TRPM8 pore opening in response to menthol and cold.

Journal Article↗
PubMed2003

Use of GC clamps in DHPLC mutation scanning.

OBJECTIVE: Development of a systematic mutation detection assay strategy for denaturing high performance liquid chromatography (DHPLC). DESIGN: Adaptation of Guanine and Cytosine (GC)-clamping from denaturing gradient gel electrophoresis (DGGE) to DHPLC. METHODS: Three target sequences harboring known allelic variants were studied to develop a general DHPLC assay design strategy. These were exon 10 of the human RET (REarranged during Transfection) gene, exon 52 of the mouse Col1a2 gene, and exon 9 of the human FAS (APO-1, CD-95) gene. Available software was used to analyze melting curves and determine assay conditions. GC clamps of 20 bp or 36 bp were added to polymerase chain reaction (PCR) primers to introduce a high melting temperature (T(m)) domain to each of the target molecules. DHPLC was performed under partially denaturing conditions. RESULTS: DHPLC assays of PCR-amplified sequences can be developed using a personal computer. The following three steps allowed for mutation detection in all three targets. The target sequence should have a uniform T(m)GC clamps of length sufficient to introduce a second melting domain with a T(m) > or = 8 degrees above that of the target sequence should be appended to one of the primers. The DHPLC assay should be performed at the highest temperature at which the target sequence is predicted to be > or = 90% double stranded CONCLUSION: Addition of GC-clamps to primers facilitates mutation detection by DHPLC. The theoretical basis for this observation is identical to that underlying the utility of GC-clamps in DGGE.

Animals↗
PubMed1999

Design and application of 2-D DGGE-based gene mutational scanning tests.

Currently, there is a need for practical, accurate and cost-efficient tests to comprehensively scan human genes for disease-related DNA sequence variation. Two-dimensional gene scanning (TDGS) is a parallel mutation detection system, based on a combination of extensive multiplex PCR amplification ('PCR megaplex') and two-dimensional (2-D) DNA electrophoresis. The latter comprises a size separation step followed by denaturing gradient gel electrophoresis (DGGE), and allows single base pair changes to be distinguished among multiple DNA fragments in parallel. Here, we describe the rapid design of TDGS tests and its application to mutation identification in several large human cancer genes.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Scanning mutations of the 5'UTR regulatory sequence of L-ferritin by denaturing high-performance liquid chromatography: identification of new mutations.

Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l-chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high-performance liquid chromatography (DHPLC) and double-gradient denaturing gradient gel electrophoresis (DG-DGGE). We analysed the sequence encoding the 5' untranslated flanking region of ferritin l-chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C-G transversions. DHPLC and DG-DGGE identified seven abnormal patterns in DNA samples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identification of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del-T156. We conclude that DHPLC, similar to DG-DGGE, detects all the mutations in the l-ferritin 5'UTR sequence in a single run, and that various mutations occur outside the IRE structure.

5' Untranslated Regions↗
PubMed2001

Should newborn mutation scanning for hyperphenylalaninaemia and galactosaemia be implemented? A Polish experience.

OBJECTIVE: To elucidate whether screening for mutations causing hyperphenylalaninaemia (HPA) and classic galactosaemia could provide important, additional information on a clinical phenotype. METHOD: Genotypes that cause disease at the phenylalanine hydroxylase (PAH) gene and galactose-1-phosphate uridyltransferase (GALT) gene in a group of 101 hyperphenylalaninaemic and 77 patients with classic galactosaemia were established. The PAH and GALT mutations were identified in genomic DNA extracted from whole blood leucocytes using single stranded conformational analysis and direct fluorescent sequencing of polymerase chain reaction (PCR) products. RESULTS: Mild HPA and mild phenylketonurea (PKU) were caused by divergent genotypes. In the studied group a total of 26 different mild and intermediate PAH mutations were identified, most of them being rare ones. Classic galactosaemia was caused by two frequent mutations, accounting for 82% of all mutated alleles. CONCLUSIONS: Identification of mild or intermediate mutations causing HPA could provide fast and reliable information about future clinical outcome of a newborn infant. Molecular diagnosis of HPA should be preceded by biochemical analysis and implemented to differentiate mild forms of HPA and cases of ambiguous classification. Because of multiple rare mutations scattered on all exons, scanning of the entire PAH coding sequence could be useful and cost beneficial. Routine genotyping is not proposed in classic phenylketonuria and classic galactosaemia, as it provides limited additional, prospective information on the clinical phenotype.

Adolescent↗
PubMed2000

[Ha-ras and p53 gene mutations scanned by PCR-SSCP in premalignant and malignant lesions of the uterine cervix associated with human papillomavirus].

The aim of this study w trial randomized as to investigate the frequencies of human papillomavirus (HPV) and mutation in Ha-ras oncogene and tumour suppressor p53 gene in cervical cancer and precursor lesions. A total of 30 invasive carcinomas (IC), 36 cervical intraepithelial neoplasia grade III (CIN III) and 12 normal tissues adjacent to the tumor (NT) were included. HPV typification and scanning of possible mutations in Ha-ras and p 53 genes were made by SSCP-PCR. The IC cases showed 93% HPV positivity, 41% having mobility shifts for Ha-ras mutations and 17% for p53 mutations while in CIN III, these percentages were 80%, 18% and 11%, respectively. In normal tissues HPV frequency was 17%. All Ha-ras mutated samples were HPV positive but 33% of p53 mutated cases were HPV negative. All mutations were heterozygous. HPV 16 was more prevalent (44%) than HPV 18 (15%) and the high rate of undetermined HPV types (18%) would indicate the circulation in our country of other types different from the assayed HPV controls (6, 11, 16, 18, 31 and 33), being variants or mixed infections. The low frequency of p53 mutations (17%) strengthens the view that wild type p53 inactivation by HPV probably plays a major role in the pathogenesis of cervical cancer. Because mutated Ha-ras was found in HPV associated premalignant lesions, we speculate that it represents an early marker for progression. Our findings provide additional evidence for an interactive effect between high risk types of HPV and oncogene activation in the development of uterine cervical cancer.

DNA, Viral↗
PubMed1999

Genetic diagnosis of 21-hydroxylase deficiency: DGGE-based mutation scanning of CYP21.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the gene CYP21 encoding the enzyme steroid 21-hydroxylase. In addition to deletions, approximately 20 different point mutations have been reported, and still novel mutations are detected. This makes genetic diagnosis as well as carrier detection of 21-hydroxylase deficiency a complicated matter. We developed a simple nonradioactive assay based on the polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to screen for mutations in the CYP21 gene. DGGE allows a fast scanning of PCR-amplified segments of genes for the presence or absence of any single base pair alterations. We have performed this technique on the coding sequence and intron-exon junctions of CYP21. Our results emphasize that this procedure constitutes a fast and reliable approach when performing diagnosis of 21-hydroxylase deficiency.

Adrenal Hyperplasia, Congenital↗
PubMed2002

Comprehensive mutation scanning of the dystrophin gene in patients with nonsyndromic X-linked dilated cardiomyopathy.

OBJECTIVES: The goal of this study was to perform comprehensive mutation analysis of the dystrophin gene in patients with X-linked dilated cardiomyopathy (XLCM). BACKGROUND: X-linked dilated cardiomyopathy is a familial disease that is characterized by congestive heart failure without clinical signs of skeletal myopathy. Mutations in the dystrophin gene have been associated with the X-linked form of dilated cardiomyopathy. However, the fraction of XLCM with dystrophin mutations and the distribution of those mutations is not clear. Technical difficulties previously limited comprehensive mutation analysis of this very large gene. METHODS: The Detection Of Virtually All Mutations-Single Strand Conformation Polymorphism (SSCP) (DOVAM-S), a robotically enhanced multiplexed scanning method that is a highly sensitive modification of SSCP, has successfully detected all of 240 mutations and polymorphisms in three blinded analyses of the factor VIII, factor IX, and ATM genes. Utilizing this method all 79 coding exons and splice junctions for the muscle dystrophin gene, along with six alternative exon 1 sequences, were scanned in eight patients with XLCM. RESULTS: This is the first comprehensive scanning of the dystrophin gene in XLCM. Three of eight patients have putative mutations, including two splicing mutations and a missense mutation at a highly conserved amino acid. CONCLUSIONS: Mutations within the coding regions and splice junctions in the dystrophin gene only account for some cases of XLCM. Genetic heterogeneity and/or undetected mutations in auxiliary regulatory regions or deep within introns may occur in XLCM.

Adolescent↗
PubMed1998

Mutational scanning of mitochondrial DNA by two-dimensional electrophoresis.

An expedient, accurate, and cost-efficient test was developed to scan critical regions of the mitochondrial genome for all possible mutations by two-dimensional DNA electrophoresis. The test involves a two-step multiplex PCR amplification: a long-distance PCR to amplify almost the entire mitochondrial genome, which then serves as template for the amplification of 25 short PCR fragments in two multiplex groups corresponding to regions implicated in human diseases. The mixture of fragments was subsequently subjected to two-dimensional electrophoretic separation, first by size in a nondenaturant polyacrylamide gel and then on the basis of basepair sequence in a denaturing gradient polyacrylamide gel. This latter process of denaturing gradient gel electrophoresis is a most accurate form of mutation detection on the basis of differences in melting behavior of mutant and wildtype fragments. Evaluation of the method using samples with known homoplasmic and heteroplasmic mutations, as well as CEPH pedigrees to study segregation of polymorphic variants, indicated a very high accuracy; none of the previously identified mutations and polymorphisms escaped detection, and no erroneous segregation patterns of polymorphic variants were observed. In addition, two variants were found to be novel mutations when analyzed by sequence analysis. One of these novel mutations was a heteroplasmic mutation in the COXIII gene that was found to segregate to homoplasmy in the next generation. Heteroplasmic mutations as low as 1% of mtDNA could still be detected.

DNA Mutational Analysis↗
PubMed2004

[The mutation scanning of KCNQ1 gene for 31 long QT syndrome families].

OBJECTIVE: To search for the mutations of potassium voltage-gated channel, KQT-like subfamily member 1(KCNQ1) gene in 31 Chinese long QT syndrome(LQTS) families. METHODS: Due to the genetic heterogeneity, the genotype of patients was first predicted based on the spectrum of ST-T-wave patterns on ECG. Ten of 31 probands were considered as LQT1. Then the mutation of KCNQ1 gene was screened by the polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) technique combined with DNA sequencing in all members of these 10 families. To avoid omitting some LQT1 patients without typical characteristics and also to do methodological comparison, the mutations of KCNQ1 gene on 16 exons were screened by PCR and direct DNA sequencing in the rest 21 non-LQT1 probands only. Co-segregation analysis was carried out after the finding of an abnormal sequence. In case that the abnormality existed in patients only, the test of such exon was performed in 50 irrelevant normal individuals. RESULTS: Two missense mutations and three single nucleotide polymorphisms (SNPs) were found in the LQT1 predicted families. The two mutations were S277L (1 family) and G306V (1 family) in exon 5 and were not reported previously. Three polymorphisms were 435C-->T (7 families), 1632C-->A (1 family), and IVS1+9 C-->G (3 families). Only a splice mutation IVS1+5G-->A (2 families) and a polymorphism IVS10+18C-->T (1 family) were found in the non-LQT1 predicted probands. All three mutations were localized within the functional domain of KCNQ1 and were co-segregated with the disease, and were not found in 50 normal individuals. CONCLUSION: Two novel missense mutations, 1 splice mutation and four SNPs on KCNQ1 gene were found in the 31 LQTS families. Combined with ECG-based genotype prediction, PCR-SSCP could find most mutations on KCNQ1 and be a simple and economic method for screening LQTS.

Adolescent↗
PubMed2005

Mutational scanning of potassium, sodium and chloride ion channels in malignant migrating partial seizures in infancy.

The mutational analysis of potassium (KCNQ2, KCNQ3), sodium (SCN1A, SCN2A), and chloride (CLCN2) ion channels was performed in three children with typical features of the recently described syndrome of migrating partial seizures in infancy. Mutational analysis was performed by PCR and automatic sequencing. The coding regions, including the exon-intron boundaries, were amplified in the patients using appropriate primers sets. No mutations associated to migrating partial seizures have been found. Mutational screening of CLCN2 gene, revealed a homozygous mutation G2003C (exon 17), leading to a Ser/Thr substitution at the codon 668, in two of the three patients. The same variation has been found in 38 out of 100 control alleles. The identification of the genetic basis of this new epileptic encephalopathy requires further studies that might be enforced by familial cases.

Aminopeptidases↗
PubMed2005

Mutation scanning of the p53 tumor suppressor gene in renal and liver transplant patients in Hungary.

INTRODUCTION: The increased incidence of malignancies among transplanted patients is well known. Abnormal function of the p53 tumor suppressor gene has been reported in more than half of all tumors. The aim of our study was to detect point mutations of p53 gene in transplanted patients because the presence of mutations may be a predictive factor for tumor development. An earlier diagnosis can help to develop new strategies for immunosuppressive therapies. METHODS: Three point mutations were chosen based on the literature: exon5-codon175, exon7-codon248, exon8-codon273. Genomic DNA from the plasma of 60 liver, 362 renal transplants, and 45 nontransplanted patients with different tumors and 20 suspected healthy patients were analyzed with a real-time PCR method using the Roche LightCycler. The mutations were evaluated by melting curve analysis. RESULTS: We elaborated a special protocol for scanning the above mentioned p53 point mutations, which were proved by sequencing as well. Among 487 patients, 486 showed a wild-type genotype. The only patient carrying a mutation at codon 273 (heterozygous) was a liver transplant patient, who developed pancreas carcinoma and had already died. CONCLUSION: Our data suggest that mutations of the targeted codons in leukocyte DNA seem to be rare, but a mutation could be lethal. The evaluated three point mutations of p53 gene were not predictive for tumor development.

Base Sequence↗
PubMed2006

Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning.

We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches are detected using direct optical methods in a microplate format. Studies with individual deoxynucleotides demonstrated that the reactivity with potassium permanganate resulted in a specific colour change. Furthermore, studies with synthetic oligonucleotide heteroduplexes demonstrated that this colour change phenomenon could be applied to detect mismatched bases spectrophotometrically. A collection of plasmids carrying single point mutations in the mouse beta-globin promoter region was used as a model system to develop a functional mutation detection assay. Finally, the assay was validated as 100% effective in detecting mismatches in a blinded manner using DNA from patients previously screened for mutations using established techniques, such as sequencing, SSCP and denaturing high-performance liquid chromatography (DHPLC) analysis in DNA fragments up to 300 bp in length.

Animals↗
PubMed1996

Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis.

The large number of possible disease-causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutations in the CFTR gene can be both substantially facilitated and made virtually complete, by two-dimensional DNA electrophoretic separation of polymerase chain reaction (PCR) amplified exons on the basis of size and basepair sequence in denaturing gradient gels. Under a single optimized set of conditions we were able to obtain a pattern of spots representing all 27 exons of the CFTR gene and to readily detect 17 out of 17 identified sequence variations in 9 different exons in DNA from 11 CF patients and carriers. Our results demonstrate the potential of 2-dimensional DNA electrophoresis for comprehensive mutation analysis of the CFTR gene. The approach serves as a model for comprehensive diagnosis of the many other large disease genes for which a variety of mutations have also which been reported.

Cystic Fibrosis↗
PubMed1997

Alanine-scanning mutations in the "primer grip" of p66 HIV-1 reverse transcriptase result in selective loss of RNA priming activity.

Alanine-scanning mutants of the primer grip region of human immunodeficiency virus type 1 reverse transcriptase were tested for their ability to extend RNA and DNA versions of the polypurine tract primer, and an oligonucleotide representing the 18-nucleotide sequence at the 3' end of tRNALys3. A majority of the mutant enzymes were either completely or severely deficient in RNA priming activity, but, with only one exception, were able to efficiently extend DNA versions of the same primers. The mutant enzymes were able to bind to RNA primers, indicating that the defect in RNA priming was not simply a loss of binding activity. Mutations at positions 229, 233, and 235 dramatically reduced the amount of specific RNase H cleavage at the 3' terminus of the polypurine tract, which is required for primer removal. An alanine substitution at position 232 led to loss of cleavage specificity, although total activity was close to the wild-type level. Taken together, these results demonstrate for the first time that there are residues in human immunodeficiency virus type 1 reverse transcriptase which are specifically involved in protein-nucleic acid interactions with RNA primers.

Alanine↗
PubMed1999

Single-step mutation scanning of the 6-pyruvoyltetrahydropterin synthase gene in patients with hyperphenylalaninemia.

BACKGROUND: Deficiency of 6-pyruvoyltetrahydropterin synthase (PTPS) is a recessively inherited disorder that leads to depletion of 5,6,7, 8-tetrahydrobiopterin, the obligatory cofactor for hydroxylation of phenylalanine, tyrosine, and tryptophan. A marker for neonatal detection of PTPS deficiency is hyperphenylalaninemia (HPA). Molecular analysis would provide a simple and reliable means for distinguishing PTPS deficiency from other potential causes of HPA. METHODS: We developed a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) that rapidly scans the six coding sequences and all splice sites of the PTPS gene (PTS) for mutations. This method was used to examine the status of the PTS gene in control samples with known PTS mutations and in five patients with PTPS deficiency. RESULTS: Two features of the PTS gene posed particular problems in relation to DGGE analysis: the very high GC content of exon 1, and a 15-bp poly(dT) stretch in the acceptor splice site of intron 1. Both problems were solved by special design of amplification primers. PCR and DGGE conditions were adjusted to allow simultaneous analysis of all six regions of the PTS gene. Using this one-step approach, all control mutations were readily resolved. Among the five PTPS patients, four mutations were identified, including IVS1-3C-->G, IVS2-7T-->A, V57del, and V97M (289G-->A). The IVS1-3C-->G mutation was shown by reverse transcription-PCR analysis to produce multiple splice variants. CONCLUSIONS: We have established a fast and reliable screening method for detection of mutations and small deletions/insertions in the PTS gene. This method should be useful for rapid diagnosis of PTPS deficiency in newborns with HPA.

Alleles↗
PubMed1999

Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity.

Dideoxy fingerprinting (ddF) was used as a tool to search for a generic set of conditions with sufficient power to detect virtually all mutations. For each condition tested, a very large sample of mutation-containing, single-stranded segments (about 1500) were analyzed with ddF. Correlation coefficients identified pairs of conditions in which single-strand conformation polymorphism (SSCP) mobilities were poorly correlated. The data strongly suggest that tertiary structure (e.g., base-sugar and sugar-sugar interactions) rather than secondary structure is the predominant determinant of mobility shifts by SSCP. Five conditions were selected with sufficient redundancy to detect all the mutations. The sensitivity of detection of virtually all mutations-SSCP (DOVAM-S) was determined by blinded analyses on samples containing additional mutations scattered throughout the eight exons and splice junctions in the factor IX gene. The factor IX gene sequence (2.5 kb) was scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild-type (WT) samples and the second consisting of 50 heterozygous mutant and WT samples. DOVAM-S is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.

Biotechnology↗