Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability

Bateman, John F.; Freddi, Susanna; Lamandé, Shireen R.; Byers, Peter H.; Nasioulas, Steven; Douglas, Jenny; Otway, Robyn; Kohonen‐Corish, Maija; Edkins, Edward; Forrest, Susan M.

doi:10.1002/(sici)1098-1004(1999)13:4<311::aid-humu8>3.3.co;2-g

Cited by 20 publications

(28 citation statements)

References 17 publications

(22 reference statements)

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“…21,22 Thus, we performed cycloheximide (CHX) treatment of chondrocytes isolated from the tumor cartilage to "stabilize" the mutant mRNA. 23 RT-PCR of mRNA extracted from CHX treatment chondrocytes showed amplification of an addition 206-bp fragment ( Fig. 2A).…”

Section: Molecular Consequence Of the C1173 + 1g > T Mutationmentioning

confidence: 99%

A splice‐site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas

Liu

Hui

Chan

et al. 2010

Journal Orthopaedic Research

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Multiple osteochondromas (MO) is an autosomal-dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice-site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173 + 1G > T (EXT2) mutation was identified in a three-generation 34-member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon-reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation.

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Section: Molecular Consequence Of the C1173 + 1g > T Mutationmentioning

confidence: 99%

A splice‐site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas

Liu

Hui

Chan

et al. 2010

Journal Orthopaedic Research

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“…To assess aberrant splicing, fibroblasts were grown in 100 lg/ml cycloheximide (Sigma) overnight prior to total RNA isolation (Bateman et al 1999). For immunoprecipitation, fibroblasts were labeled overnight with […”

Section: Immunohistochemical Molecular and Biochemical Analysesmentioning

confidence: 99%

A homozygous COL6A2 intron mutation causes in-frame triple-helical deletion and nonsense-mediated mRNA decay in a patient with Ullrich congenital muscular dystrophy

et al. 2005

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Ullrich congenital muscular dystrophy (UCMD) is a severe disorder caused, in most cases, by a deficiency in collagen VI microfibrils. Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have been identified in eight of the nine UCMD patients reported thus far. A heterozygous COL6A1 gene deletion, resulting in a mutant protein that exerts a dominant negative effect, has recently been described in a severely affected UCMD patient. Here we describe a patient in whom reverse transcription-PCR analysis of fibroblast RNA suggested a heterozygous in-frame deletion of exon 13 in the triple-helical domain of COL6A2, which is predicted to be dominantly acting. However, a homozygous A fi G mutation at À10 of intron 12 was found in the genomic DNA. The intron mutation activated numerous cryptic splice acceptor sites, generating normal and exon 13-deleted COL6A2 mRNA, and multiple aberrant transcripts containing frameshifts that were degraded through a nonsense-mediated decay mechanism. Northern analysis indicated diminished COL6A2 mRNA expression as the primary pathogenic mechanism in this UCMD patient. Our results underscore the importance of multifaceted analyses in the accurate molecular diagnosis and interpretation of genotype-phenotype correlations of UCMD.

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“…The principle of IVTT is based on targeting mutations that generate truncated proteins induced by premature translation termination. 21 IVTT enables to pinpoint the site of a mutation, offers good sensitivity, and a low false-positive rate. 22 Further tight junction proteins (occludin and ZO-1) were co-investigated in this study.…”

mentioning

confidence: 99%

Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

Zheng

Sieuwerts

Luider

et al. 2004

The American Journal of Pathology

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Caldesmon is a cytoskeleton-associated protein whichhas not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal brain microvasculature. To exclude sources of splice variant expression from non-vascular components all possible cellular components present in control and glioma samples were prescreened by laser-capture microdissection followed by RT-PCR before the cohort study. We discovered differential expression of the splicing variants of CALD1 in the tumor microvessels in contrast to normal brain microvasculature. Missplicing of exons 1, 1 ؉ 4, and 1 ؉ 4 of the gene is exclusively found in glioma microvessels. To exclude the possibility that this missplicing results from splice-site mutations, Genome-wide analyses have revealed that 40 to 60% of human genes undergo alternative splicing. 1 Alternative splicing, therefore, seems to contribute considerably to enable the highly complex and diverse functions encoded by the human genome. Alternative splicing permits vertebrate pre-mRNA to be processed into multiple mRNAs differing in their precise combination of exon sequences, resulting in the encoding of different protein isoforms. 2 Multiple modes of alternative splicing exist, such as alternative 5Ј or 3Јsplice-site usage, differential inclusion or skipping of particular exons, mutually exclusion of exons, and more. 3 Importantly, alternative splicing is often tightly regulated in a cell type-or developmental stage-specific mode. 3 The essential nature of this process is underscored by the fact that misregulation (missplicing events) is often related to human disease. 4 -6

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Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability

Cited by 20 publications

References 17 publications

A splice‐site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas

A splice‐site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas

A homozygous COL6A2 intron mutation causes in-frame triple-helical deletion and nonsense-mediated mRNA decay in a patient with Ullrich congenital muscular dystrophy

Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

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