Similar levels of mRNA from the W1282X and the delta F508 cystic fibrosis alleles, in nasal epithelial cells.

Abstract

The effect of nonsense mutations on mRNA levels is variable. The levels of some mRNAs are not affected and truncated proteins are produced, while the levels of others are severely decreased and null phenotypes are observed. The effect on mRNA levels is important for the understanding of phenotype-genotype association. Cystic fibrosis (CF) is a lethal autosomal recessive disease with variable clinical presentation. Recently, two CF patients with mild pulmonary disease carrying nonsense mutations (R553X, W1316X) were found to have severe deficiency of mRNA. In the Jewish Ashkenazi CF patient population, 60% of the chromosomes carry a nonsense mutation, W1282X. Patients homozygous for this mutation have severe disease presentation with variable pulmonary disease. The presence of CF transcripts in a group of patients homozygous and heterozygous for this mutation was studied by reverse transcriptase PCR of various regions of the gene. Subsequent hybridization to specific CF PCR probes and densitometry analysis indicated that the CF mRNA levels in patients homozygous for the W1282X mutation are not significantly decreased by the mutation. mRNA levels were compared for patients heterozygous for the W1282X mutation. The relative levels of mRNA with the W1282X, and the AF508 or the normal alleles, were similar in each patient. These results indicate that the severe clinical phenotype of patients carrying the W1282X mutation is not due to a severe deficiency of mRNA. In addition, the severity, progression, and variability of the pulmonary disease are affected by other, as yet unknown factors. (J. Clin. Invest.

Nasal potential difference measurements in patients with atypical cystic fibrosis

“…Deoxyribonucleic acid (DNA) sequences spanning individual exons of the CF gene were amplified by polymerase chain reaction (PCR) with oligonucleotide primers located in the respective flanking introns of the CF gene [23,24]. The amplified genomic DNA fragments eluted from 5% polyacrylamide gels were extracted with chloroform and subjected to the dideoxy-chain termination sequencing method as described, using the US Biochemicals Sequenase kit (Cleveland, OH, USA) with either one of the PCR primers or internal oligonucleotides as sequencing primers.…”

Applicability of Different Antibodies for Immunohistochemical Localization of CFTR in Sweat Glands from Healthy Controls and from Patients with Cystic Fibrosis

“…With respect to the W1282X mutation, conflicting data are reported in the literature. mRNA levels were found to be severely decreased by Will et al (1995) and Hamosh et al (1992) but within normal range by Shoshani et al (1994). However, expression of full-length protein has never been demonstrated under normal conditions in this stop mutation (Bedwell et al 1997).…”

Restoration of W1282X CFTR Activity by Enhanced Expression

“…Over 1,400 disease-causing mutations in CFTR have been identified, and can be divided into six major classes (1). Nonsense mutations (a class I mutation type) are due to the presence of a premature termination codon (PTC or stop mutation), resulting in absent functional protein and typically a severe CF phenotype (2,3). PTCs are relatively common in humans, accounting for approximately 5 to 10% of diseasecausing CF mutations in white populations, and are the proximate cause of approximately 30% of human genetic diseases (4).…”