Novel point mutations in the dystrophin gene

Duchenne (DMD) and Becker (BMD) type muscular dystrophies are allelic X-linked recessive disorders caused by mutations in the gene encoding dystrophin. About 65% of the cases are caused by deletions, while 5–10% are duplications. The remaining 30% of affected individuals may have smaller mutations (point mutations or small deletions/insertions) which cannot be identified by current diagnostic screening strategies. In order to look for pathogenic small mutations in the dystrophin gene, we have screened the 18 exons located in the hot spot region of this gene through two different single strand conformation polymorphism (SSCP) conditions. Five different pathogenic mutations were identified in 6 out of 192 DMD/BMD patients without detectable deletions: 2 nonsense, 1 bp insertion, 1 bp deletion and 1 intronic. Except for the intronic change, which alters a splice site, all the others cause a premature stop codon. In addition, 8 apparently neutral changes were identified. However, interestingly, one of them was not identified in 195 normal chromosomes, although it was previously described in a DMD patient from a different population. The possibility that this mutation may be pathogenic is discussed. Except for two neutral changes, all the others are apparently here described for the first time. Hum Mutat 10:217–222, 1997. © 1997 Wiley-Liss, Inc.     

Metachromatic leukodystrophy: Identification of the first deletion in exon 1 and nine novel point mutations in the arylsulfatase A gene

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459+1G→A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459+1G→a, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene. Hum Mutat 9:234–242, 1997. © 1997 Wiley-Liss, Inc.     

Dystrophin基因第51号外显子的缺失机制的研究

用DMDcDNA8探针从Dystrophin基因的YAC克隆的Cosmid亚克隆库中筛选到含Dystrophin基因的第51号外显子Cosmid克隆，将该Cosmid亚克隆于pUC118中，获得合Dystrophin基因第51导外星子的3.1kb—HindⅢ片段的亚克隆，该片段的长度为3179bp，含Dystrophin基因第家51号外星子的全部和第50和第51导内含子的部分，对第50和51号内含子的核苷酿顺序分析表明，在这两个内台子中共发现36个短段串联重复顺序．22正向和反向重复顺序和8个同原顺序。提出重复顺序之间的重组可能导致第51号外显子缺失的假设。     

A novel insertional mutation of a single base in exon 12 of the dystrophin gene

A new point mutation in exon 12 of the dystrophin gene was identified in a DMD patient using multiple SSCP analysis, which allows the simultaneous study of several exons. The mutation is an A insertion at position 1580 of the cDNA sequence, leading to a stop codon in the translational reading frame. This mutation was not observed in a sample of 70 DMD patients.     

FH-Sydney 1 and 2: Two novel frameshift mutations in exon 10 of the low-density lipoprotein receptor gene detected by heteroduplex formation

We report two novel frameshift mutations in exon 10 of the low-density lipoprotein receptor gene that lead to familial hypercholesterolemia in separate lineages. The lesions, FH-Sydney 1 and FH-Sydney 2, were detected by a modified heteroduplex analysis of exon-specific polymerase chain reaction (PCR) amplified DNA, and characterized at the molecular level by sequencing. Restriction enzyme digestion of PCR amplified DNA confirmed the presence of the mutant alleles in affected family members and their absence in nonaffected family members in both lineages. FH-Sydney 1 is a 4-bp duplication at position 1373, while FH-Sydney 2 is a 2-bp deletion at position 1478. The predicted result of both mutations is the premature truncation of the receptor at stop codons generated downstream of the mutations. Neither mutation was detected in a survey of 54 unrelated familial hypercholesterolemia patients. © 1994 Wiley-Liss, Inc.     

非缺失/重复型Duchenne肌营养不良症患者的致病点突变分析

目的检测非缺失／重复突变型Duchenne肌营养不良症（Duchenne muscular dystrophy，DMD）的致病点突变。方法对6个家系的6个无关DMD男性患者的DMD基因的79个外显子及5′-3′-非翻译序列进行PER扩增，产物通过变性高效液相色谱（denaturing high performance liquid chromatography，DHPLC）技术进行突变筛查。结果6例非缺失／重复突变型Duchenne肌营养不良症患者，检测出了5例患者的致病点突变，即697-698insGT，C616T，G1255T，C4279T和C2302T。第1个点突变引起移码突变，后4个致病点突变引起翻译的提前终止，最终导致Duchenne肌营养不良症。患者3除致病点突变外，在第39内含子还发现1个T5586＋61A点突变；患者5还检测出了一个位于第8外显子的错义突变；而没有检出致病点突变的患者6，发现了2个外显子突变及2个内含子序列点突变，即C2168＋13T、G5234A、C5280T和5740-13dupG。所有检出的突变有7个点突变未见报道。结论变性高效液相色谱技术结合测序，可用于检测DMD患者的点突变．该方法具有准确，灵敏的特点。     

Rapid detection of exon 1 NRAS gene mutations using universal heteroduplex generator technology

Specific NRAS oncogene missense mutations have been frequently found in some tumors and several hematological diseases, especially in those of myeloid origin. There is a wide range of PCR-based methods for screening and detection of NRAS exon 1 single-base substitutions. However, there are disadvantages and ambiguities associated with these techniques because all of them require either separate probes, separate PCR amplifications, or complicated post-PCR manipulations. This report describes a new approach for detection of NRAS gene mutations at codon 12 and 13 based on the DNA heteroduplex analysis method. The strategy relies upon differential electrophoretic behavior of induced heteroduplex molecules formed by cross-hybridization of two PCR-amplified species, the sample under analysis and the synthetic universal heteroduplex generator (UHG). The screening of a panel of all codon 12 and 13 NRAS mutant DNA variants indicated that this approach discriminates all 12 relevant mutations. The sensitivity of the method was estimated by a competitive assay where mutant alleles could be detected at a dilution level of 1 to 16 wild-type alleles. This UHG technology was tested on some clinical samples previously studied by PCR-ASO. This methodology is highly specific, sensitive, and achieves an appreciable reduction in workload and time because it requires one PCR amplification followed by polyacrylamide gel electrophoresis in standard conditions. We propose that this new approach may be applied as an alternative strategy for codon 12-13 NRAS mutations and it could be easily incorporated into the range of routine assays performed in oncology laboratories.     

Characterization of two nonsense mutations in the human dystrophin gene.

Forty Duchenne muscular dystrophy patients from the province of Moravia in the Czech Republic, who were previously found negative for large deletions in the dystrophin gene, were tested for the presence of point mutations in selected exons. Besides several intron and exon polymorphisms, two cases of nonsense mutations were detected in exon 70, thus causing the loss of the C-terminal domain of dystrophin. One of these, the mutation, S3365X, is newly reported here while the other, R3381X, has been described previously. These mutations, only 16 bp distant from each other, have a very different impact on the mental abilities of the corresponding patients.     

Identification of three novel mutations in the dystrophin gene detected by the heteroduplex/SSCA screening procedure. Mutations in brief no. 222. Online

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked neuromuscular disorders associated with alterations in the dystrophin gene. Analysis of 45 DMD/BMD patients has identified 18 patients with no deletion in the dystrophin gene. Heteroduplex analysis (HD), single strand conformation analysis (SSCA), and subsequent sequencing, identified five mutations and nine polymorphisms. Three out of the 5 mutations (780C>G, 2501-1g-->t, 9812 9813ins9800-9812) are first reported here. Furthermore we compare the relative efficiencies of the two alternatives methods (HD and SSCA) for screening sequence alterations.     

Detection of epidermal growth factor receptor (EGFR) exon 19 E746-A750 deletion and EGFR exon 21 point mutations in lung adenocarcinoma by immunohistochemistry: a comparative study to EGFR exons 19 and 21 mutations analysis using PCR followed by high-resolution melting and pyrosequencing

Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) with adenocarcinoma histology confers susceptibility to treatment with EGFR tyrosine kinase inhibitors (TKI). Analysis of these activating mutations is typically performed by molecular techniques including polymerase chain reaction (PCR) amplification followed by deoxyribonucleic acid (DNA) melting curve analysis and/or direct sequencing. Recently developed mutation-specific immunohistochemical stains for the L858R point mutation in exon 21 and an E746-A750 deletion in exon 19 of the EGFR gene may offer a fast, cost-effective alternative to molecular testing. In this study, EGFR exons 19 and 21 mutations were analyzed using EGFR E746-A750 del and EGFR L585R point mutation-specific antibodies and the results were compared with the exon 19 deletions and exon 21 L588R point mutation as detected by molecular testing. The findings demonstrate a high concordance rate (100% in 12 cases) between the two methodologies for EGFR exon 21 L585R point mutation but a low concordance rate (54% in 22 cases) for exon 19 deletions when using EGFR E746-A750 mutation-specific antibody compared to molecular testing. This low concordance rate between the two methods is due to the fact that EGFR exon 19 mutations include deletions other than E746-A750 as well as insertion/deletions that will not be covered by exon 19 E746-A750 del mutation-specific antibody. Despite this limitation, the current EGFR mutation-specific antibodies can prove useful as an alternative to molecular tests for exon 21 L585R mutations as well as a subset of EGFR exon 19 deletion mutations, particularly in cases with low percentage of tumor concentration and in decalcified tissue specimens that are not suitable for DNA-based molecular assays.     

Screening for mutations in exon 4 of the LDL receptor gene: identification of a new deletion mutation.

DNA from 14 unrelated New Zealand familial hypercholesterolaemia (FH) heterozygotes, originating from the United Kingdom, was screened for mutations in exon 4 of the low density lipoprotein receptor (LDLR) gene. One patient was heterozygous for mutation D206E, which was initially identified in South Africa. The chromosomal background of this mutant allele was compatible with that described previously in Afrikaner and English patients, suggesting that this mutation originated in the United Kingdom. The 2 bp deletion in codon 206 and mutations D154N and D200G, previously reported in English FH patients, were not detected in this sample. In one of the patients, however, a new deletion of 7 bp was identified after nucleotide 581 (or 582) in exon 4 of the LDLR gene.     

Large majority of single-nucleotide mutations along the dystrophin gene can be explained by more than one mechanism of mutagenesis

The present study reports for the first time on the analysis of the possible origin of single-base mutations along the highly mutable human dystrophin gene. Seventy-two mutations were considered and analyzed for consistency with the “slipped-mispairing” and “hypermutable CpG” models of mutagenesis. Moreover, repeated and symmetric elements, which could participate in the formation of secondary structures, were searched in each stretch of sequence including a given mutant base. Unexpectedly, the frequency of CpG mutations was found less than reported for other genes, whereas the frequency of transitions was found to be much higher than expected. Base substitutions in CpG dinucleotides that could be explained by methylation-mediated deamination were all C→T transitions. No G→A transitions in CpG dinucleotides were found. A sequence motif, which has been shown to act as an arrest site for polymerase α, occurred associated with < 50% of single-base mutations. All the mutations but one can be explained by at least two mechanisms of mutagenesis. This would mean that mutation could occur with higher probability at a given position, when it might be produced independently by different mechanisms. According to the present data, direct or inverted repeats seem to play a major role in this context. Therefore, the search for repeats along the dystrophin gene might help in identifying potential sites of mutation. Hum. Mutat. 9:537–547, 1997. © 1997, Wiley-Liss. Inc.     

Protein C deficiency: identification of a novel two-base pair insertion and two point mutations in exon 7 of the protein C gene in Spanish families.

We have applied single-strand conformation polymorphism (SSCP) to the analysis of exon 7 of the anticoagulant protein C (PC) gene, in 13 PC-deficient Spanish families. Abnormal patterns were visualized in three samples from type I or quantitative PC deficient proposita. A previously undescribed mutation due to a TT insertion after nucleotide 6139, between codons Gly-142 and Arg-143 was found in one family. The mutation (6139,ins TT) should result in a frameshift with a stop at codon 156, which agrees with the presence of a type I or quantitative PC deficiency in the affected members of the family. The second mutation identified was a C to T transition at nucleotide 6274, 9 base pairs into intron G. This mutation (6274,C-->T), found for the first time in a Spanish family, is identical to the previously characterized PC Sant Louis. The third mutation was a G to A transition that replaces arginine 178 with glutamine (178,R-->Q). This is the third case of 178,R-->Q mutation in 17 apparently unrelated Spanish families with type I PC deficiency. Furthermore, SSCP analysis allowed the detection of another previously described mutation in a PC-deficient Spanish family (178,R-->W).