Rapid scanning of myotubularin (MTM1) gene by denaturing high-performance liquid chromatography (DHPLC)

Flex, Elisabetta; Luca, Alessandro De; D’Apice, Maria Rosaria; Buccino, Anna; Dallapiccola, Bruno; Novelli, Giuseppe

doi:10.1016/s0960-8966(01)00328-5

Cited by 23 publications

(13 citation statements)

References 12 publications

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“…Thirteen different missense mutations were reported in exon 8, of which nine were associated with a severe phenotype Herman et al 2002;Flex et al 2002;this report). This exon is in frame (type 0 exon), thus the deletion could still allow translation into a protein, although this was not verified experimentally.…”

Section: Resultsmentioning

confidence: 96%

“…DNAs were studied by PCR and single strand conformation polymorphism (SSCP Pharmacia Genephor), DHPLC (Flex et al 2002) or direct sequencing (big dye terminators Applied Biosystem). Mutation screening was performed on an index case or a relative (sister or mother) from each XLMTM family.…”

Section: Methodsmentioning

confidence: 99%

“…To date, mutations have been reported in 251 unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene Herman et al 2002;Tachi et al 2001;Flex et al 2002;Jungbluth et al 2002). While truncating mutations cause, in almost all cases, the severe neonatal form, some missense mutations are associated with milder forms, which may allow survival at adult age.…”

Section: Introductionmentioning

confidence: 99%

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Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype

et al. 2003

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X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts (S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopa-thy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.

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Section: Resultsmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype

et al. 2003

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“…This methodology relies upon the rapid separation of homo-and heteroduplexes DNA molecules (due to mismatches between wild-type and mutant strand) on a stationary phase under partially denaturing conditions [Underhill et al, 1997]. DHPLC was chosen as the preferential mutation scanning method for its rapidity, simplicity of analysis, and for its well-documented detection sensitivity approaching 98 to 100% in several studies [Colosimo et al, 2002;Eng et al, 2001;Flex et al, 2002;Xiao and Oefner, 2001;De Luca et al, 2003].…”

Section: Introductionmentioning

confidence: 99%

Molecular detection of novel WFS1 mutations in patients with Wolfram syndrome by a DHPLC-based assay

et al. 2003

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Communicated by Paolo FortinaWolfram syndrome (WS) is a recessively inherited mendelian form of diabetes and neurodegeneration also known by the acronym DIDMOAD from the major clinical features, including diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Affected individuals may also show renal tract abnormalities as well as multiple neurological and psychiatric symptoms. The causative gene for WS (WFS1) encoding wolframin maps to chromosome 4p16.1 and consists of eight exons, spanning 33.44 Kb of genomic DNA. In this study we report on the mutational analysis of the WFS1 coding region in 19 Italian WS patients and 25 relatives, using a DHPLC-based protocol. A total of 19 different mutations in WFS1 were found in 18 of 19 patients (95%). All these mutations, except one, are novel, preferentially located in WFS1 exon 8, and include deletions, insertions, duplications, and nonsense and missense changes. In particular, a 16 base-pair deletion in WFS1 codon 454 was detected in five different unrelated nuclear families, being the most prevalent alteration in this Italian group. Nine neutral changes and polymorphisms were also identified. Overall, this study represents the molecular characterization of the largest cohort of Italian WS patients and carriers studied so far, and increases the number of identified WFS1 allelic variants worldwide. Hum Mutat 21:622-629,

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“…A relatively new, fast, and cost-effective methodology based on denaturing high-performance liquid chromatography (DHPLC) has been successfully applied for mutational detection of several genes, including MTM1, TSC1, TSC2, CFTR, BRCA1, and BRCA2 [Flex et al, 2001;Gross et al, 2000;Jones et al, 2000;Le Marechal et al, 2001;Wagner et al, 1999;Xiao and Oefner, 2001], approaching 90100% in sensitivity and specificity. Using this technique it is possible to separate heteroduplexes (formed in DNA samples containing mismatches between wild type and mutant strand) from homoduplexes on a stationary phase under partially denaturing conditions.…”

Section: Introductionmentioning

confidence: 99%

Reliability of DHPLC in mutational screening of ?-globin (HBB) alleles

Colosimo

Guida

Luca³

et al. 2002

Hum. Mutat.

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The inherited disorders of hemoglobin represent the most common Mendelian disease worldwide, with a higher prevalence among Mediterraneans, Asians, Africans, and Indians. Altered beta-globin sequences, causing either hemoglobinopathies or beta-thalassemia syndromes, are due to more than 200 different mutations in the beta-globin gene. Prevention programs based on postnatal and prenatal molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. We have developed a rapid and highly specific mutation screening test based on the denaturing high-performance liquid chromatography (DHPLC) system. The sensitivity and specificity of the method were tested on the full genomic region of the beta-globin gene in 30 normal Italian subjects and 40 heterozygous carriers in which 25 different beta-globin mutations had been previously characterized by multiplex-ARMS technique. The results showed DHPLC to be 100% sensitive and specific. All the 25 sequence alterations and two previously undetected polymorphisms were precisely identified with neither false positive nor false negative results. In addition, 12 compound heterozygous and four homozygous patients were successfully subjected to DHPLC. Overall, the method was able to rapidly identify the most common beta-globin mutations, accounting for more than 97% of beta-globin alleles in the Italian population. Compared to classical approaches of mutation screening, this method allows a rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.

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Rapid scanning of myotubularin (MTM1) gene by denaturing high-performance liquid chromatography (DHPLC)

Cited by 23 publications

References 12 publications

Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype

Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype

Molecular detection of novel WFS1 mutations in patients with Wolfram syndrome by a DHPLC-based assay

Reliability of DHPLC in mutational screening of ?-globin (HBB) alleles

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