An optimized DHPLC protocol for molecular testing of the <i>EXT1</i> and <i>EXT2</i> genes in hereditary multiple osteochondromas

Wuyts, Wim; Radersma, Reinder; Storm, Katrien; Vits, Lieve

doi:10.1111/j.1399-0004.2005.00538.x

Cited by 37 publications

(39 citation statements)

References 24 publications

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“…To facilitate and reduce the cost of this mutation screening, optimization of a DHPLC-based protocol for all EXT1 and EXT2 coding exons has been described. 17,20 After all, most MO patients have point mutations or small deletions or insertions of a few bp in one of both genes and the EXT1 and EXT2 coding regions contain very little polymorphisms, making them very suitable for DHPLC analysis. However, because mutation screening was performed almost exclusively at the sequence level, quantitative (deletions and duplications) and positional (inversions and translocations) changes were not detected by this technique.…”

Section: Discussionmentioning

confidence: 99%

“…17 If a fragment showed an aberrant chromatograph in DH-PLC analysis it was PCR reamplified and the sequence was determined using ABI v1.1 chemistry with sequencing analysis on an ABI3130xl genetic analyzer (Applied Biosystems, Foster City, CA). DNA mutation and nucleotide numbering for EXT1 and EXT2 were based on the cDNA reference sequences (GenBank accession numbers NT_023811.12 and NT_009237.13, respectively) with base one corresponding to the first base of the initiation codon.…”

Section: Dhplc Sequencing Analysis and Fluorescence In Situ Hybridimentioning

confidence: 99%

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Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set in Patients with Multiple Osteochondromas

Jennes

Entius

Hul

et al. 2008

The Journal of Molecular Diagnostics

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Multiple osteochondromas (MO) is an autosomaldominant skeletal disorder characterized by the formation of multiple cartilage-capped protuberances. MO is genetically heterogeneous and is associated with mutations in the EXT1 and EXT2 genes. In this study we describe extensive mutation screening in a set of 63 patients with clinical and radiographical diagnosis of MO. Denaturing high-performance liquid chromatography analysis revealed mutations in 43 patients. Additional deletion analysis by fluorescence in situ hybridization and a newly developed multiplex ligation-dependent probe amplification probe set identified one patient with an intragenic EXT1 translocation , three patients with a partial EXT1 deletion , and one patient with a partial EXT2 deletion. Thirty-six patients harbored an EXT1 mutation (57%) , and 12 had an EXT2 mutation (19%). We show that our optimized denaturing high-performance liquid chromatography/sequencing/multiplex ligation-dependent probe amplification protocol represents a reliable and highly sensitive diagnostic strategy for mutation screening in MO patients. Clinical analysis showed no clear genotype-phenotype correlation in our cohort of MO patients.

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Section: Discussionmentioning

confidence: 99%

Section: Dhplc Sequencing Analysis and Fluorescence In Situ Hybridimentioning

confidence: 99%

Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set in Patients with Multiple Osteochondromas

Jennes

Entius

Hul

et al. 2008

The Journal of Molecular Diagnostics

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“…Además, en el gen EXT2 de este caso, se consiguió una mutación ya conocida, pero de patogenicidad dudosa. 8 Las mutaciones encontradas en el gen EXT1 fueron Se presentó un caso de EMH con diagnóstico clínico, radiológico y molecular, y se encontraron dos mutaciones patogénicas no informadas previamente en el gen EXT1 y otra considerada dudosa en el gen EXT2. Este hallazgo molecular muestra mutaciones que hacen importante el seguimiento clínico, ante la posibilidad de asociar estas mutaciones a una eventual característica de presentación de la entidad.…”

Section: Discussionunclassified

“…7 posición 710 del exón 6, que produce un cambio de aminoácido serina por leucina en la posición 237 de la exostosina 2. Esta mutación ha sido documentada por Wuyts, et al 8 que la consideran de patogenecidad dudosa, pues la presenta un paciente y también su padre no afecto.…”

Section: Caso Clínicounclassified

Alelo doble mutante en el gen EXT1 no informado previamente en una adolescente con exostosis múltiple hereditaria

et al. 2015

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Presentación de casos clínicos RESUMENLas formas hereditarias de exostosis múltiple, actualmente denominada EXT1/EXT2-CDG dentro de los desórdenes congénitos de la glicosilación, son los tumores óseos benignos más comunes y se caracterizan por la formación de lesiones óseas cubiertas de cartílago, localizadas en yuxtaposición a epífisis de huesos largos, aunque, en los casos graves, pueden presentar una amplia distribución. El inicio es variable desde los 2-3 años hasta los 13-15 y presenta una incidencia estimada que va de 1/18 000 a 1/50 000 casos en los países europeos. Se presenta el caso de un doble alelo mutante en el gen EXT1 no informado previamente en una adolescente y su familia con exostosis múltiple hereditaria. Palabras clave: exostosis múltiple hereditaria, EXT1/EXT2-CDG. ABSTRACTHereditary forms of multiple exostoses, now called EXT1/ EXT2-CDG within Congenital Disorders of Glycosylation, are the most common benign bone tumors in humans and clinical description consists of the formation of several cartilagecapped bone tumors, usually benign and localized in the juxta-epiphyseal region of long bones, although wide body dissemination in severe cases is not uncommon. Onset of the Alelo doble mutante en el gen EXT1 no informado previamente en una adolescente con exostosis múltiple hereditaria Double mutant alleles in the EXT1 gene not previously reported in a teenager with hereditary multiple exostoses disease is variable ranging from 2-3 years up to 13-15 years with an estimated incidence ranging from 1/18 000 to 1/50 000 cases in European countries. We present a double mutant alleles in the EXT1 gene not previously reported in a teenager and her family with hereditary multiple exostoses.

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“…With the currently used methods it is possible to detect point mutations or gross deletions in almost 90% of MO patients [21][22][23][61][62][63].…”

Section: Diagnostic Methodsmentioning

confidence: 99%

Multiple Osteochondromas

Encyclopedia of Cancer

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Multiple osteochondromas (MO) is characterised by development of two or more cartilage capped bony outgrowths (osteochondromas) of the long bones. The prevalence is estimated at 1:50,000, and it seems to be higher in males (male-to-female ratio 1.5:1). Osteochondromas develop and increase in size in the first decade of life, ceasing to grow when the growth plates close at puberty. They are pedunculated or sessile (broad base) and can vary widely in size. The number of osteochondromas may vary significantly within and between families, the mean number of locations is 15-18. The majority are asymptomatic and located in bones that develop from cartilage, especially the long bones of the extremities, predominantly around the knee. The facial bones are not affected. Osteochondromas may cause pain, functional problems and deformities, especially of the forearm, that may be reason for surgical removal. The most important complication is malignant transformation of osteochondroma towards secondary peripheral chondrosarcoma, which is estimated to occur in 0.5-5%. MO is an autosomal dominant disorder and is genetically heterogeneous. In almost 90% of MO patients germline mutations in the tumour suppressor genes EXT1 or EXT2 are found. The EXT genes encode glycosyltransferases, catalyzing heparan sulphate polymerization. The diagnosis is based on radiological and clinical documentation, supplemented with, if available, histological evaluation of osteochondromas. If the exact mutation is known antenatal diagnosis is technically possible. MO should be distinguished from metachondromatosis, dysplasia epiphysealis hemimelica and Ollier disease. Osteochondromas are benign lesions and do not affect life expectancy. Management includes removal of osteochondromas when they give complaints. Removed osteochondromas should be examined for malignant transformation towards secondary peripheral chondrosarcoma. Patients should be well instructed and regular follow-up for early detection of malignancy seems justified. For secondary peripheral chondrosarcoma, en-bloc resection of the lesion and its pseudocapsule with tumour-free margins, preferably in a bone tumour referral centre, should be performed.

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An optimized DHPLC protocol for molecular testing of the EXT1 and EXT2 genes in hereditary multiple osteochondromas

Cited by 37 publications

References 24 publications

Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set in Patients with Multiple Osteochondromas

Mutation Screening of EXT1 and EXT2 by Denaturing High-Performance Liquid Chromatography, Direct Sequencing Analysis, Fluorescence in Situ Hybridization, and a New Multiplex Ligation-Dependent Probe Amplification Probe Set in Patients with Multiple Osteochondromas

Alelo doble mutante en el gen EXT1 no informado previamente en una adolescente con exostosis múltiple hereditaria

Multiple Osteochondromas

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