Artículos de Revista en Ciencias Médicas · 2007

New polymorphism in the ncf-2 gene leading to alternative splicing without altering gene expression or the respiratory burst activity

Resumen

The p67-phox protein is an essential component for activating the NADPH oxidase of phagocytes. Mutations affecting this gene lead to an autosomal recessive form of chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent severe infections. We have previously characterized the genetic basis of CGD in patients lacking p67-phox, one of the less frequent forms of CGD. In addition to the mutations responsible for the CGD phenotype, we have also identified other nucleotide changes in different regions of this gene in CGD patients, and in healthy individuals. One of these nucleotide substitutions is an A ? G transition at position 21 of intron 10 (IVS10-21A ? G), which is located in the branch acceptor sequence, important for intron removal during pre- mRNA splicing. The aim of this work was to investigate the frequency of this single nucleotide substitution, as well as its consequences on NCF-2 gene expression. From 114 investigated subjects, 41 were homozygous for A, 49 were heterozygous and 24 were homozygous for G, leading to allelic frequencies of 57.5% for A and 42.5% for G, confirming a polymorphism of the NCF2 gene. This group was found to be in Hardy Weinberg equilibrium (0.50 > P > 0.30). In the current work, the SSCP assay detected the 21 A to G substitution in intron 10. This nucleotide substitution was confirmed by DNA sequencing, and has been submitted to the GenBank data bank with accession number DQ517931. There was a complete correlation between sequencing data and SSCP migration pattern. We further investigated the cDNA structure of NCF2 gene between exons 9 and 14. The RT-PCR analysis followed by cDNA sequencing revealed complete (507 bp) and alternative (284 bp) splicing products, without exon 11 fragment in all genotypes. Sequencing of this p67-phox cDNA region corroborated these data (Fig. 1). The DHR test as assayed by flow cytometry was 99.6% positive cells for G/G, 99.45% positive cells for A/G and 99% positive cells for A/A individuals ( P ¼ 0.81, Kruskal-Wallis test), showing normal respiratory burst activity. The NCF2 gene expression as assayed by real-time PCR was similar among all genotypes ( P ¼ 0.937, Kruskal-Wallis test). In our work, with A or G in position 21, we still observed two cDNA species, one normal and one alternative without exon 11. In most of the cases, normal splicing took place because the 5 0 end of the intron 10 recognizes the branch acceptor site of the same intron; however, in some circumstances the lariat formation occurred between the 5 0 end of intron 10 and the branch acceptor sequence of intron 11, which caused exon 11 to be skipped during the mRNA splicing. However we found that the presence of this intronic transition did not alter the expression of NCF2 gene. We concluded that the IVS10-21A ? G transition is a polymorphism of the NCF2 gene, and its location gives rise to alternative splicing without altering gene expression or the respiratory burst activity of normal subjects under the current experimental conditions.