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1.
In the past few years, more than 20 different mutations have been reported in hyperphenylalaninemias. In southwestern Europe and Mediterranean countries, however, the mutant genotypes reported account for only a fraction (27%) of all mutant alleles at the phenylalanine hydroxylase (PAH) locus, and most of the mutations causing the disease remain unknown. In order to develop a strategy for rapid detection of mutation-containing exons, we applied the single-strand conformation-polymorphism (SSCP) technique to exons 3, 5, 7, and 12 of the PAH gene. We observed five abnormal patterns of migration in mutant PAH genes, and we consistently found base substitutions in the corresponding exons, with no false-positive results. By this procedure, two novel putative mutations were detected in the seventh exon of the PAH gene, (A259V and Y277D) and we were able to demonstrate that the delta I94, R158Q, R408W, and E280K mutations were easily detectable by the SSCP technique. This procedure is therefore of particular interest for rapid detection of mutation-containing exons and for determination of further genotype-phenotype correlations in hyperphenylalaninemias.  相似文献   

2.
The phosphomannomutase 2 gene (PMM2; MIM 601785) has been identified as the carbohydrate-deficient glycoprotein syndrome type 1A gene (CDGS type 1A; MIM 212065). The gene spans 8 exons and 741 bp of coding DNA. Previously, we have identified 20 different mutations in the PMM2 gene using mutation screening with single-stranded conformation polymorphism (SSCP) and sequencing of DNA from 61 CDGS type 1A patients. Because eight of these could not be detected by SSCP, we were not satisfied with the sensitivity of the mutation detection technique used. Thus, we wanted to investigate if denaturing high-performance liquid chromatography (DHPLC) was a more suitable mutation screening method for PMM2. DHPLC was set up for PMM2 by optimizing eight different PCR fragments, one for each exon. The mutation detection was optimized empirically with PCR fragments from controls. First, control samples were run at a universal gradient and after modification and shortening of the gradient, also run at 10 different temperatures, 50-70 degrees C with 2-degree intervals, to enable setting of the temperature with the highest resolution. Then, PCR products with known mutations from the previous study were analyzed, and the results were compared to the control chromatograms for aberrations. We detected 19/20 mutations with DHPLC, and several mutations not detected by earlier screening techniques were readily detected by DHPLC. We conclude that DHPLC is a suitable detection technique for a rapid and reliable first scan of CDGS type 1A patients.  相似文献   

3.
We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5′ fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30°C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.  相似文献   

4.
5.
To detect mutations in the glucocerebrosidase gene in Gaucher disease patients, we used the recently described technique of single-strand conformation polymorphism (SSCP) analysis in combination with selective amplification. We analyzed exon 8, 9, 10 and 11 of the glucocerebrosidase gene; these exons were sequentially amplified using the selectively amplified products as templates. We found variant SSCP patterns corresponding to the presence or absence of the 6433C mutation, which was detected by NciI digestion analysis, in exon 10. Furthermore, we detected four variant SSCP patterns in exon 8, 10 and 11. Sequencing analysis consistently revealed four single-base substitutions in the corresponding exons, three novel missense mutations (5409A, 6375G and 6682T) and one silent polymorphism (6594A). These mutations were found only in one patient; therefore, these findings have confirmed the marked genetic heterogeneity of Gaucher disease. SSCP analysis in combination with selective amplification is a rapid and sensitive procedure for the screening of the mutations in the glucocerebrosidase gene of patients with Gaucher disease.  相似文献   

6.
The spectrum and frequencies of RB1 structural defects were studied in tumors and peripheral blood lymphocytes of patients with various forms of retinoblastoma. Single strand conformation polymorphism (SSCP) and heteroduplex (HA) analyses, along with direct sequencing, revealed 47 mutations, including 24 new ones. Of these, 42.5% were nonsense mutations, 15% were missense mutations, 15% affected splicing sites, and 27.5% were frameshifts resulting from microdeletions or microinsertions. Six polymorphisms were found, including three new ones located in the coding region. Microsatellite analysis with markers Rbint2, Rbint20, D13S262, and D13S284 revealed a loss of heterozygosity for at least one marker in 71% tumors.  相似文献   

7.
As a result of intensive studies on hereditary breast and ovarian cancers, two breast cancer susceptibility genes, BRCA1 and BRCA2, have been identified. In each gene, a small number of specific mutations have been found at relatively high frequency in certain ethnic populations. The mutations, 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2, have been identified as common mutations in the Ashkenazi Jewish population, with a combined frequency of 2.0 to 2.5%. Women who have one of the above three common mutations are at a high risk of developing breast or ovarian cancer. Consequently, accurate and cost-effective detection of these three mutations may have important implications for risk assessment in susceptible women and men. In this report, we describe a fast and simple capillary electrophoresis (CE)-based method using a polymer network for screening the three common mutations in BRCA1 and BRCA2. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify three DNA fragments of 258, 296, and 201 bp for detection of the 185delAG, 5382insC, and 6174delT mutations, respectively. After the PCR products were denatured, a single-strand conformation polymorphism (SSCP) profile could be obtained for each mutation in less than 10 min by CE in a polymer network. We demonstrate the potential provided by translating this assay to the microchip format where the SSCP analysis is complete in 120 s, representing only a fraction of the reduction in analysis time that can be achieved with microchip technology. The speed and simplicity of the SSCP methodology for detection of these mutations make it attractive for use in the clinical diagnostic laboratory.  相似文献   

8.
Multiple mutations in several ion channel genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and KCNJ2) have been shown to cause autosomal dominant long QT syndrome (LQTS), a familial cardiac disorder that causes syncope, seizures, and sudden death. Due to their multiple loci and considerable size, mutation detection in these genes represents a challenge that is only partially met by the conventional screening method of single-stranded conformational polymorphism (SSCP). The recently introduced denaturing high-performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of LQTS, we first assessed a cohort of 192 patients from our International LQTS Registry for 14 previously identified mutations (including 10 different missense mutations, 1-bp, 2-bp, 3-bp, and 9-bp deletion mutations), and 2 polymorphisms in the LQTS potassium and sodium channel genes. Applying empirically determined exon-specific melting profiles, all mutations (including four previously undetectable by SSCP) were readily identified by dHPLC. We conclude that the dHPLC technology is a highly sensitive and efficient method for the molecular analysis of LQTS, and the same PCR amplicons developed for SSCP testing can be directly used for dHPLC assay.  相似文献   

9.
Pelizaeus-Merzbacher disease (PMD) is a clinically heterogeneous, slowly progressive leukodystrophy. The recent detection of mutations in the myelin proteolipid protein (PLP) gene in several PMD patients offers the opportunity both to design DNA-based tests that would be useful in diagnosing a proportion of PMD cases and, in particular, to evaluate the diagnostic utility of single-strand conformation polymorphism (SSCP) analysis for this disease. A combination of SSCP analysis and direct sequencing of PCR-amplified DNA was used to screen for PLP mutations in 24 patients affected with leukodystrophies of unknown etiology. Two heretofore undescribed mutations in the PLP gene were identified, Asp202His in exon 4 and Gly73Arg in exon 3. The ease and efficiency of SSCP analysis in detecting new mutations support the utilization of this technique in screening for PLP mutations in patients with unexplained leukodystrophies.  相似文献   

10.
11.
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.  相似文献   

12.
Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.  相似文献   

13.
Hereditary non-polyposis colorectal cancer (HNPCC) is a clinical syndrome characterised by an inherited predisposition to early onset colorectal and uterine cancers and an increased incidence of other cancers. It is caused by germline defects in the human mismatch repair genes. Defects in two of the known mismatch repair genes (namely hMSH2 and hMLH1) account for over 90% of mutations found in HNPCC families. In this study we have identified 14 families that fulfilled the clinical criteria for HNPCC and screened the hMSH2 and hMLH1 genes for germline mutations using single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Seven mutations were identified. Of these, there were five frameshifts, one missense mutation and a further novel mutation that involved separate transition and transversion changes in successive amino acid residues. Three of the mutations were in hMSH2 and four in hMLH1. The identification of germ-line mutations in an HNPCC family enables targeted surveillance and the possibility of early curative intervention. SSCP is a simple and effective method for identifying most mutations in the human mismatch repair genes using DNA from fresh, frozen or archival material. Received: 24 July 1996 / Revised: 26 September 1996  相似文献   

14.
We have analyzed 61 German breast and breast/ovarian cancer families for BRCA1 mutations using single-strand conformation polymorphism analysis (SSCP) followed by sequencing. Forty-seven of the families had at least three cases (at least two under 60 years) and 14 families had only two cases of breast/ovarian cancer (at least one under 50 years). Twenty-eight families were breast/ovarian and 33 were breast cancer-only families. Eighteen mutations in BRCA1 were detected in 11/28 breast/ovarian cancer families and 7/33 breast cancer families and none in the families with only two cases. We identified 17 truncation mutations (8 frameshift, 7 nonsense and 2 splice variants) and one missense mutation. Seven of these are novel and two, the 5382insC and 5622C→T mutations, occurred in two apparently unrelated families. The genotype of the two families with the 5382insC mutation is compatible with the rare haplotype segregating with the 5382insC mutation in different populations, further supporting its European origin. One unclassified missense alteration, R841W, was found in one family but did not segregate with the disease, suggesting that it is more likely a polymorphism. We also report and discuss the sequence of several new unclassified single-nucleotide changes first identified by SSCP. Of the 18 mutations, 13 occurred in the 3′ third of the gene (end of exon 11–24) and ovarian cancers were found in eight of these families. Received: 5 February 1998 / Accepted: 7 April 1998  相似文献   

15.
Previous studies of the molecular basis of 21-hydroxylase deficiency have shown four common gene conversion mutations in exons 7 and 8. Current molecular diagnostic protocols use allele-specific oligonucleotide hybridization (ASOH) to individually detect each of these mutations and the corresponding normal alleles. This method is costly, labor intensive, and may not provide quantitative results. To expedite molecular diagnosis in families with 21-hydroxylase deficiency, we have designed and implemented single-strand conformational polymorphism (SSCP) analysis. We applied SSCP analysis to 12 families in whom mutations in exons 7 or 8 had been previously identified by ASOH. Using a single polymerase chain reaction (PCR) amplification, unique conformers can be assigned to three mutations: V281L, Q318X, and R356W. The fourth mutation, T insertion at nucleotide 1761, was detected by heteroduplex analysis of the same PCR product. Thus, we were able to identify all four mutations using a single PCR product on a single gel.  相似文献   

16.
We describe a new environment of a single-stranded conformational polymorphism (SSCP) analysis using automated capillary array sequencers (e.g., ABI Prism 3100 and 3700). In this environment, electrophoretic conditions, settings for instrument management, and software for data analysis are adjusted for SSCP analysis. Highly reproducible results are obtained with this new system, and fragments with mutations and/or polymorphisms in different capillaries or different runs can be reliably detected. The relative peak heights between alleles are quantitative and reproducible between runs, and so allele frequencies of single nucleotide polymorphisms can be accurately estimated by a pooled DNA strategy. The method allows unattended, low-cost, and quantitative SSCP analysis using instruments that are widely accessible.  相似文献   

17.
To evaluate whether a structural defect in the human glucose transporter gene GLUT1 could be involved in the aetiology of insulin resistance, a key factor of non-insulin-dependent diabetes mellitus (NIDDM) and obesity, we performed single-strand conformation polymorphism (SSCP) analysis in 40 subjects (20 NIDDM patients and 20 subjects with familial obesity). The GLUT1 gene, which is involved in basal glucose transport in most tissues, consists of ten exons and encodes a 492 amino acid protein. Population studies have shown a strong association between the X1 allele of an XbaI restriction fragment length polymorphism of the GLUT1 gene and NIDDM. We therefore performed SSCP analysis in NIDDM subjects known to carry at least one X1 allele. Variant SSCP patterns were detected in exons 2, 4, 5 and 9. Sequence analysis of the SSCP variants revealed the presence, in all exons examined, of silent mutations consisting of single-nucleotide substitutions with no amino acid changes. Both NIDDM and obese patients showed a high frequency of polymorphism in the sequence (50% and 35%, respectively). We conclude that the GLUT1 gene is unlikely to play a role in the aetiology of NIDDM and obesity. However, the strong association between the GLUT1 gene and NIDDM, together with the recent family studies showing linkage between chromosome 1p and NIDDM warrant further studies on this chromosomal region. Received: 18 August 1997 / Accepted: 10 December 1997  相似文献   

18.
Single-strand conformational polymorphism analysis (SSCP) and heteroduplex analysis (HD) were tested as methods for mutation screening with respect to experimental variation, sensitivity, and specificity. Thirty-nine fluorescently labeled PCR products covering the two mismatch repair genes, hMLH1 and hMSH2, were tested in 15 patients for pattern changes, using SSCP and HD at two temperatures, in a total of 2340 runs. SSCP was most efficient in detecting base changes, whereas HD was the method of choice when detecting deletions. SSCP and HD at 20 degrees C were most effective (sensitivity 97%, specificity 49%), and SSCP and HD at 10 degrees C gave no additional information, except in one case where an exon had two base changes. Several mutations only showed a small pattern change in one of the two strands, most explicit at 20 degrees C. No correlation between the type of base change and the size or direction of the pattern changes could be found.  相似文献   

19.
AIMS: To study the frequency of mutations in the Pax8 gene in a cohort of patients with congenital hypothyroidism (CH) in South West Germany. METHODS: A cohort of 95 patients with CH (60 females, 35 males), identified in our newborn screening program, was analyzed for mutations in Pax8 by single-stranded conformational polymorphism (SSCP) and DNA sequencing. RESULTS: SSCP analysis and direct sequencing of exon 3 of a female patient with a hypoplastic thyroid gland revealed two heterozygous mutations in Pax8 resulting in a transition of T to C (codon 34) and G to A (codon 35), replacing isoleucine by threonine and valine by isoleucine. Using allele-specific PCR we could demonstrate that both mutations are located on the same allele. Furthermore, a polymorphism was documented in 24 patients with thyroid hypoplasia in intron 6 at nucleotide +51 (CC, GG, CG). Comparison of the polymorphisms between hypothyroid patients and controls revealed no significant differences suggesting that this polymorphism does not play a role in the pathogenesis of hypothyroidism. No further mutations or polymorphisms were found in the cohort. CONCLUSIONS: These findings confirm the contribution of mutations in the Pax8 gene to the etiology of thyroid dysgenesis with a variable penetrance, but also demonstrate the rare overall incidence in CH.  相似文献   

20.
We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recessive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E203K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E203K mutation created a unique isoform-banding pattern. An asymptomatic sister''s GALT genes carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intraallelic complementation when in cis.  相似文献   

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