Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR

Summary About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.     

Polarity Effects in the Hisg Gene of Salmonella Require a Site within the Coding Sequence

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.     

A high-throughput denaturing high-performance liquid chromatography method for the identification of variant alleles associated with dihydropyrimidine dehydrogenase deficiency

Dihydropyrimidine dehydrogenase (DPD) is the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). A pharmacogenetic syndrome has been described in which DPD-deficient patients are at risk for toxicity following administration of 5-FU. To date, there are at least 21 previously described mutations and/or polymorphisms that have been associated with DPD deficiency. In this study we describe the development of a highly specific, sensitive, inexpensive, and robust denaturing HPLC (DHPLC) method for rapidly identifying sequence variations (mutations and/or polymorphisms) in the gene (DPYD) that codes for the DPD enzyme. DHPLC conditions were optimized at three temperatures for analysis of the 23 exons of the DPYD gene using 25 amplicons representing the entire coding sequence, including all intron/exon boundaries (splice sites). Resolution of all 25 amplicons at the optimized temperature can be performed in 4.2 h. All 21 previously described sequence variations (mutations and/or polymorphisms) were prepared using site-directed mutagenesis from the wild-type DPYD gene, confirmed by sequence analysis, and subsequently resolved by DHPLC using the optimized conditions. These analyses generated reference chromatogram patterns for all known sequence variations previously encountered in DPD-deficient patients. In order to examine the utility and sensitivity of this approach, samples from patients with known sequence variations in the DPYD gene were analyzed. This DHPLC technique resolved 100% of the known DPYD sequence variations and differentiated between homozygous and heterozygous genotypes. We conclude that this DHPLC method is a highly specific and sensitive technique for rapidly detecting known sequence variations in the DPYD gene. In addition, this approach can be used to identify currently unrecognized unknown sequence variations in the DPYD gene and should be useful in future pharmacogenetic studies examining DPD deficiency.     

Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle

We have determined the entire myostatin coding sequence for 32 double-muscled cattle sampled from ten European cattle breeds. Seven DNA sequence polymorphisms were identified, of which five would be predicted to disrupt the function of the protein, one is a conservative amino acid substitution, and one a silent DNA sequence variant. Four additional DNA sequence polymorphisms were identified in myostatin intronic sequences. In all but two breeds, all double-muscled animals were either homozygous or compound heterozygotes for one of the five loss-of-function mutations. The absence of obvious loss-of-function mutations in the coding sequence of the two remaining breeds points either towards additional mutations in unexplored segments of the gene, or towards locus heterogeneity of double-muscling. Received: 20 September 1997 / Accepted: 11 October 1997     

Spectrum of small mutations in the dystrophin coding region.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.     

Protein conformation and disease: pathological consequences of analogous mutations in homologous proteins

The antibody light chain variable domain (V(L))(1) and myelin protein zero (MPZ) are representatives of the functionally diverse immunoglobulin superfamily. The V(L) is a subunit of the antigen-binding component of antibodies, while MPZ is the major membrane-linked constituent of the myelin sheaths that coat peripheral nerves. Despite limited amino acid sequence homology, the conformations of the core structures of the two proteins are largely superimposable. Amino acid variations in V(L) account for various conformational disease outcomes, including amyloidosis. However, the specific amino acid changes in V(L) that are responsible for disease have been obscured by multiple concurrent primary structure alterations. Recently, certain demyelination disorders have been linked to point mutations and single amino acid polymorphisms in MPZ. We demonstrate here that some pathogenic variations in MPZ correspond to changes suspected of determining amyloidosis in V(L). This unanticipated observation suggests that studies of the biophysical origin of conformational disease in one member of a superfamily of homologous proteins may have implications throughout the superfamily. In some cases, findings may account for overt disease; in other cases, due to the natural repertoire of inherited polymorphisms, variations in a representative protein may predict subclinical impairment of homologous proteins.     

Analysis of sequence variations in 59 microRNAs in hepatocellular carcinomas

It is well demonstrated that mutations in protein-coding genes play a key role during carcinogenesis. Whether sequence variations in microRNA genes are also associated with tumorigenesis is still unknown and thus require extensive investigations. In the present study, genomic sequences coding for the precursors of 59 microRNA genes were analyzed in both hepatocellular carcinoma (HCC) tissues and liver cancer derived cell lines. In total, four variations in three microRNAs, including miR-106b, miR-192 and let-7a-2, were found in four out of 96 HCC tissues. Further investigation in the corresponding adjacent non-cancerous tissues identified the same sequence variations, suggesting the possibility of germline mutations or natural polymorphisms. Moreover, no variation was found in eight liver cancer derived cell lines. Among four sequence alterations observed in this study, two were located in miR-106b and identified as known single nucleotide polymorphisms, while the other two found in miR-192 and let-7a-2 had not been reported before. In conclusion, our data suggest that mutation of microRNA is a rare event in HCC and thus may not represent a main mechanism underlying hepatocarcinogenesis.     

A large MSH2 Alu insertion mutation causes HNPCC in a German kindred

Hereditary non-polyposis colorectal cancer (HNPCC) syndrome is an autosomal, dominantly inherited disease accounting for about 1%–5% of all colorectal cancer cases. HNPCC predisposition is caused by germline mutations in at least five genes coding for DNA mismatch repair (MMR) proteins. More than 400 MMR gene mutations have been identified in HNPCC patients. About 90% of mutations affect the MLH1 and MSH2 genes. The mutational spectrum mainly includes point mutations and small deletions or insertions. Here, we report a large 184 base-pair Alu insertion mutation in exon 6 of the MSH2 gene in a German HNPCC family. The inserted sequence contains repetitive Alu sequence elements that present the highest homology with the old Alu J subfamily. The Alu J insertion was most likely derived from Alu-mediated recombination, since Alu J elements have been found close to the insertion site in adjacent introns, and since elements pivotal for Alu retrotransposition are missing. Our results suggest that the recombination event occurred at least one generation ago. This is the first report of an Alu insertion in the coding sequence of a MMR gene as the cause of HNPCC. Our data thus further extend the spectrum of MMR gene mutations causative for HNPCC.M. Kloor and C. Sutter contributed equally to this work     

Evaluation of dHPLC for CX26 mutation screening in patients from southern France with sensorineural deafness

The GJB2 gene (or CX26 for connexin 26) is one of the major genes causing nonsyndromic sensorineural hearing loss (NSSNHL). More than 50 sequence variations have been identified as polymorphisms or associated with autosomal or recessive forms of deafness. Though a major mutation, 35delG, is easily detectable by PCR digest; it is often present in the compound heterozygous state in our population in trans with recurrent, but less frequent, mutations. The CX26 gene is composed of a single coding exon that facilitates sequencing strategies. However, for mutation screening purposes, it is necessary to use high-throughput and cost-effective genotyping methods. Therefore, we have assessed denaturing high-performance liquid chromatography (dHPLC) in patients with known mutations in the CX26 gene. We conclude that dHPLC analysis is suitable for rapid and reliable scanning of the gene in deaf patients.     

Wide Distribution of Mitochondrial Genome Rearrangements in Wild Strains of the Cultivated Basidiomycete Agrocybe aegerita

We used restriction fragment length polymorphisms to examine mitochondrial genome rearrangements in 36 wild strains of the cultivated basidiomycete Agrocybe aegerita, collected from widely distributed locations in Europe. We identified two polymorphic regions within the mitochondrial DNA which varied independently: one carrying the Cox II coding sequence and the other carrying the Cox I, ATP6, and ATP8 coding sequences. Two types of mutations were responsible for the restriction fragment length polymorphisms that we observed and, accordingly, were involved in the A. aegerita mitochondrial genome evolution: (i) point mutations, which resulted in strain-specific mitochondrial markers, and (ii) length mutations due to genome rearrangements, such as deletions, insertions, or duplications. Within each polymorphic region, the length differences defined only two mitochondrial types, suggesting that these length mutations were not randomly generated but resulted from a precise rearrangement mechanism. For each of the two polymorphic regions, the two molecular types were distributed among the 36 strains without obvious correlation with their geographic origin. On the basis of these two polymorphisms, it is possible to define four mitochondrial haplotypes. The four mitochondrial haplotypes could be the result of intermolecular recombination between allelic forms present in the population long enough to reach linkage equilibrium. All of the 36 dikaryotic strains contained only a single mitochondrial type, confirming the previously described mitochondrial sorting out after cytoplasmic mixing in basidiomycetes.     

Predicting the insurgence of human genetic diseases associated to single point protein mutations with support vector machines and evolutionary information

MOTIVATION: Human single nucleotide polymorphisms (SNPs) are the most frequent type of genetic variation in human population. One of the most important goals of SNP projects is to understand which human genotype variations are related to Mendelian and complex diseases. Great interest is focused on non-synonymous coding SNPs (nsSNPs) that are responsible of protein single point mutation. nsSNPs can be neutral or disease associated. It is known that the mutation of only one residue in a protein sequence can be related to a number of pathological conditions of dramatic social impact such as Alzheimer's, Parkinson's and Creutzfeldt-Jakob's diseases. The quality and completeness of presently available SNPs databases allows the application of machine learning techniques to predict the insurgence of human diseases due to single point protein mutation starting from the protein sequence. RESULTS: In this paper, we develop a method based on support vector machines (SVMs) that starting from the protein sequence information can predict whether a new phenotype derived from a nsSNP can be related to a genetic disease in humans. Using a dataset of 21 185 single point mutations, 61% of which are disease-related, out of 3587 proteins, we show that our predictor can reach more than 74% accuracy in the specific task of predicting whether a single point mutation can be disease related or not. Our method, although based on less information, outperforms other web-available predictors implementing different approaches. AVAILABILITY: A beta version of the web tool is available at http://gpcr.biocomp.unibo.it/cgi/predictors/PhD-SNP/PhD-SNP.cgi     

Mutations in three subdomains of the carboxy-terminal region of collagen type X account for most of the Schmid metaphyseal dysplasias

We have used the polymerase chain reaction and single strand conformation polymorphism (SSCP) methods to analyse the COL10A1 gene, which encodes collagen type X, in DNA samples from patients with metaphyseal dysplasia type Schmid (SMCD) and other related forms of metaphyseal dysplasia. Five cases of SMCD were sporadic and three others were familial. Abnormal SSCP profiles were observed in six instances. In two families, the altered pattern segregated with the phenotype. The heterozygous mutations corresponded to a glycine substitution by glutamic acid at position 595 and to an asparagine substitution by lysine at position 617. In one sporadic case, the sequence studies demonstrated that the individual was heterozygous for a single base deletion (del T 1908) that produced a premature stop codon. Three additional mutations were single base substitutions that affected highly conserved residues at positions 597, 644 and 648. In two additional individuals with SMCD, in two patients with unclassifiable forms of metaphyseal dysplasia, and in one family with epiphyso-metaphyseal dysplasia, SSCP analysis detected neutral polymorphisms in the entire coding sequence of the gene but no mutations. Our results demonstrate that mutations in the carboxy-terminal region of collagen X are specific for the SMCD phenotype. Mutations appear to be clustered into three small subdomains: one of them is rich an aromatic residues, the second includes the putative N-linked oligosaccharide attachment site and the third contains mostly hydrophilic residues. The absence of clinical variability between patients carrying heterozygous single base substitutions or small deletions suggests that, in both instances, the mutant collagen chains either fail to be incorporated into stable trimers or disturb type X collagen assembly.     

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.     

PRNP and SPRN genes polymorphism in atypical bovine spongiform encephalopathy cases diagnosed in Polish cattle

Polymorphisms in the coding region of the prion protein gene (PRNP) have been associated with the susceptibility and incubation period of prion diseases in humans and sheep. However, polymorphisms in this part of the bovine PRNP gene do not affect the classical bovine spongiform encephalopathy (BSE) susceptibility in cattle. Studies carried out in Germany have shown that insertion/deletion-type polymorphisms located in the promoter region of the bovine prion gene are possible genetic factors modulating BSE susceptibility by changing the level of PRNP expression. No such association was observed for atypical BSE cases; however, due to the rare nature of the disease, these results should be confirmed. Additionally, a single nonsynonymous mutation in PRNP codon 211 (E211K) was described in one H-type BSE case in the USA; however, it was not found in any other cases. Here, we performed genetic characterization of PRNP promoter indel variations and determined the polymorphism of open reading frames (ORFs) of PRNP and bovine prion-like Shadoo (SPRN) genes in six Polish atypical BSE cases and compared these results to the population of clinically healthy Polish Holstein cattle. No potentially pathogenic mutations were found in the PRNP ORF in atypical BSE-affected cattle, but our study showed a high frequency of deletions at the indel loci of PRNP promoter in these animals. Additionally, a rare sequence variation in the SPRN protein-coding sequence was found in one L-type atypical BSE-affected animal.     

Cloning of the human phospholipase A2 activating protein (hPLAP) gene on the chromosome 9p21 melanoma deleted region

Cutaneous malignant melanoma (CMM) is a common skin cancer. About 50% of CMM sporadic tumours have lost one copy of the chromosome 9p21 region. To identify genes involved in the initiation and/or progression of CMM we have characterised the 9p21 melanoma deleted region and screened the human expressed sequence tag (EST) databases (dbEST) to search for expressed genes. We have identified the gene that encodes the human orthologue of the rat phospholipase A2 activating protein (PLAP). PLAP was considered a potential candidate to be involved in malignant melanoma because it maps to the critical region for CMM and because the PLA2 gene has been identified as a modifier of the APC gene, responsible for the adenomatous polyposis phenotype in the mouse. PLAP encodes a protein of 738 amino acids and has a high DNA (90%) and protein (97%) sequence similarity with the rat and mouse PLAP protein. PLAP has a region of WD40 repeats in the amino-terminus, which allows us to include this protein in the superfamily of beta-transducin proteins. Northern blot hybridisation gave a fragment of 4.5 kb, with higher expression in heart compared to other tissues. PLAP was localised at chromosome 9p21, between marker AFM218xg11 and TEK. SSCP analysis of the coding region of PLAP revealed no variants in the studied samples, but one of six CMM samples analysed by RT-PCR showed specific inactivation of PLAP. Despite PLAP's important role in mediating several cellular responses and its localisation to the chromosome 9p21 region deleted in CMM, it is unlikely that point mutations or deletions in the coding region of PLAP are responsible for the initiation or progression of CMM. Further studies on PLAP inactivation should be performed to clarify its potential involvement in CMM.     

Sequence variations of latent membrane protein 2A in Epstein-Barr virus-associated gastric carcinomas from Guangzhou, southern China

Latent membrane protein 2A (LMP2A), expressed in most Epstein-Barr virus (EBV)-associated malignancies, has been demonstrated to be responsible for the maintenance of latent infection and epithelial cell transformation. Besides, it could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence variations of LMP2A in EBV-associated gastric carcinoma (EBVaGC) and healthy EBV carriers from Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were investigated. Widespread sequence variations in the LMP2A gene were found, with no sequence identical to the B95.8 prototype. No consistent mutation was detected in all isolates. The immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs in the amino terminus of LMP2A were strictly conserved, suggesting their important roles in virus infection; while 8 of the 17 identified CTL epitopes in the transmembrane region of LMP2A were affected by at least one point mutation, which may implicate that the effect of LMP2A polymorphisms should be considered when LMP2A-targeted immunotherapy is conducted. The polymorphisms of LMP2A in EBVaGC in gastric remnant carcinoma (GRC) were for the first time investigated in the world. The LMP2A sequence variations in EBVaGC in GRC were somewhat different from those in EBVaGC in conventional gastric carcinoma. The sequence variations of LMP2A in EBVaGC were similar to those in throat washing of healthy EBV carriers, indicating that these variations are due to geographic-associated polymorphisms rather than EBVaGC-associated mutations. This, to our best knowledge, is the first detailed investigation of LMP2A polymorphisms in EBVaGC in Guangzhou, southern China, where NPC is endemic.     

Structural analyses of the polymorphic area in type II collagen gene

The structure of type II collagen gene is extremely well conserved but contains a cluster of high frequency polymorphisms in a 2.2 kb area. Here we report the nucleotide sequence of this DNA area, essential for the PCR-facilitated RFLP-analyses of this gene. In the structural analyses we found four differences in the deduced human amino acid sequence when compared to the published bovine amino acid sequence. The donor and acceptor signals and branch point signals required for the splicing events were in agreement with mammalian consensus sequences. The frequency of inverted repeats which could provoke the DNA strand to loop formation and consequently to deletion mutations did not differ from that found in other sequenced genes coding for fibrillar collagens. mutations did not differ from that found in other sequenced genes coding for fibrillar collagens.     

Frequency of alterations in the MEFV gene and clinical signs in familial Mediterranean fever in Central Anatolia, Turkey

Familial Mediterranean fever is a recessive autoinflammatory disease that is frequent in Armenians, Jews, Arabs, and Turks. The MEFV gene is responsible for this disease. We looked for MEFV gene variations (polymorphism and mutations) in a population that resides in Central Anatolia, Turkey. DNA was extracted from peripheral blood leukocytes of 802 familial Mediterranean fever patients. The DNA sequence data were examined for approximately 150 different mutations and polymorphisms, including single nucleotide polymorphisms in different exons of the MEFV gene. The male:female ratio of these patients was 1.44:1. Mutations were detected in 48.1% of the patients; 7.5% were homozygous, 11.1% were compound heterozygous and 31.5% had only one identifiable mutant allele. No mutations were detected in 51.9% of the patients. The main clinical characteristics of the patients were: abdominal pain in 20.6%, arthritis in 22.9% and amyloidosis in 4.6%. Sixty-six percent of patients had a family history of familial Mediterranean fever; 19.4% of the patients were found to have parental consanguinity. We conclude that the genetics of familial Mediterranean fever is more complex than has previously been reported; heterozygous patients presenting a severe phenotype should be further analyzed for less common secondary MEFV mutations, using gene sequencing.     

An unusual mutation in RECQ4 gene leading to Rothmund-Thomson syndrome

Rothmund-Thomson syndrome (OMIM #268400) is a severe autosomal recessive genodermatosis: characterised by growth retardation, hyperpigmentation and frequently accompanied by congenital bone defects, brittle hair and hypogonadism. Mutations in helicase RECQ4 gene are responsible for a subset of cases of RTS. Only six mutations have been reported, thus, far and each affecting the coding sequence or the splice junctions. We report the first homozygous mutation in RECQ4 helicase: 2746-2756-delTGGGCTGAGGC in IVS8 responsible for the severe phenotype associated with RTS in a Malaysian pedigree. We report also a 5321 G-->A transition in exon 17 and the updated list of the RECQ4 gene mutations.