Is CFTR 621+3 A>G a cystic fibrosis causing mutation?

Abstract

The 621+3 A4G variant of the CFTR gene was initially detected in four Greek patients with a severe form of cystic fibrosis, and it is reported to impair CFTR mRNA splicing. We present three lines of evidence that argue against the pathogenicity of this variant. First, its allelic frequency in the Italian population was 0.4%. Even considering the lowest value in the confidence interval we would expect 10% of Italian CF patients to be heterozygotes for this variant, whereas it has been reported only in one patient (0.04% of Italian CF patients). Second, expression of the 621+3 A4G variant in HeLa cells using a hybrid minigene showed that 39.5 ± 1.1% of transcripts were correctly spliced, indicating that its effects on mRNA splicing are similar to those of the CFTR intron 8 5T variant, associated with congenital bilateral absence of vas deferens (CBAVD), but not with CF. Third, we have identified an asymptomatic individual who harbored the 621+3 A4G variant in trans with the Q552X mutation. Because 621+3 A4G is often included in population-screening programs, this information is critical to provide adequate counseling to patients. Further work should be aimed at investigating whether this variant may have a role in CBAVD or atypical CF.

Alport syndrome: impact of digenic inheritance in patients management

“…To obtain the hybrid minigene constructs, we employed as backbone the β‐globin‐pCDNA3.1 vector harboring an artificial multiple cloning site (containing the XhoI, NotI and HindIII sites) as previously reported . We amplified by PCR a portion of each gene containing the variant of interest (c.780+5G>A in COL4A5 , c.931‐2A>G and c.976‐56A>G in COL4A4 ) from genomic DNA of the patients.…”

Parent-of-origin effect of hypomorphic pathogenic variants and somatic mosaicism impact on phenotypic expression of retinoblastoma

“…Concerning the c.1331A>G allele, exon 13 and part of the flanking introns were amplified from human genomic DNA and the specific variant was introduced by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Stratagene). We employed as backbone the β-globin-pCDNA3.1 vector [36] and obtained the hybrid minigene constructs as previously described [37]. For expression analysis, we selected one clone containing the wild-type allele, one clone with each variant and the empty vector.…”

Validation of CFTR intronic variants identified during cystic fibrosis population screening by a minigene splicing assay

“…However, the CFTR gene is not expressed in peripheral blood cells and it is necessary to isolate RNA from other cell types, e.g., cells of the nasal mucosa [13] , a task which can be relatively complex. Moreover, the presence of transcripts originating from the normal allele in trans may cause the misinterpretation of data [12] , especially in the case of alleles which cause only a partial splicing defect. For these reasons hybrid minigenes have been used to study CFTR variants [14] : they do not rely on patient ' s RNA, and the analysis is not affected by the presence of a wild type allele.…”

“…There are several online software programs that predict the consequences of both intronic and exonic variants but their reliability is limited [10,11] , especially when dealing with variants identified in healthy carriers (where no correlation with a specific phenotype can be made). Evaluation of allelic frequencies may be helpful [12] , but since most of these variants are rare (with a frequency < 1:500) the analysis may not yield informative results.…”