A T3 allele in theCFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with Congenital Bilateral Absence of Vas Deferens (CBAVD)

Abstract

The different alleles at the (TG)m(T)n polymorphic loci at the 3' end of the human CFTR intron 8 determine the efficiency by which exon 9 is spliced. We identified a novel TG12T3 allele in a congenital bilateral absence of vas deferens (CBAVD) patient who carries a [TG11T7; p.Phe508Cys; p.Met470Val] haplotype on the other chromosome. To better understand the complex regulation of exon 9 splicing, we analyzed the levels of correctly spliced CFTR transcripts in six CFTR-expressing epithelial cell lines derived from lung, colon, testis, vas deferens, and epididymis transiently transfected with four CFTR minigenes (pTG11T7, pTG12T7, pTG12T5, and pTG12T3). In this work, we show that a decrease in the Ts at the polymorphic locus in a TG12 background determines a cell-type dependent reduction in exon 9+ transcripts that is not related to the basal splicing efficiency in the cell line. These data emphasize the role of the T5 allele in CBAVD and identify the T3 allele as a severe cystic fibrosis (CF) disease-causing mutation. Finally, UV cross-linking experiments demonstrated that tissue-specific trans-acting splicing factors do not contribute to the different patterns of exon 9 splicing found between the cell lines. However, we observed that lower numbers of Ts can alter the binding of TDP-43 (TDP43 or TARDBP) to its specific target ug12 in a tissue-specific manner. Our results support the idea that the ratio of general splicing factors plays a role in the tissue variability of exon 9 alternative splicing.

Functional analysis of a promoter variant identified in the CFTR gene in cis of a frameshift mutation

“…Hybrid minigene assay showed no difference between WT and mutated contexts ( Figure 1e). As alternative splicing events have been proposed to differ between cell lines, 10 we verified that the same results were obtained in the other tested cell line A549 (data not shown). These results indicate that degradation of the transcript bearing the PTC in exon 18 is the result of NMD rather than NAS.…”

Comprehensive and Rapid Genotyping of Mutations and Haplotypes in Congenital Bilateral Absence of the Vas Deferens and Other Cystic Fibrosis Transmembrane Conductance Regulator-Related Disorders

“…However, the splicing efficiency of the IVS8-T5 allele shows interand intraindividual variability, and its incomplete penetrance in CBAVD is largely influenced by the IVS8-(TG)m tract. 14,15 The determination of (TG)m repeat number is predictive of pathogenic T5 alleles. 16 Longer TG repeats increase exon 9 skipping and raise the proportion of nonfunctional CFTR protein, emphasizing the importance of assessing the length not only of the Tn but also of the (TG)m tract for diagnostic purposes.…”

Multi-Functional Consequences of a Single Mutation of CFTR of the Importance to Analyze Mutations by Functional Tests

“…The low number of T repeats could affect the splicing by reducing the distance separating the AG 3' intronic site from the UG repeats, here the TDP-43 protein binds [16]. In fact, this protein has been demonstrated to affect exon 10 splicing [17] and its affinity to mRNA is higher with a high number of UG [16].…”

“…The low number of T repeats could affect the splicing by reducing the distance separating the AG 3' intronic site from the UG repeats, here the TDP-43 protein binds [16]. In fact, this protein has been demonstrated to affect exon 10 splicing [17] and its affinity to mRNA is higher with a high number of UG [16]. Furthermore, the TDP-43 protein has a higher level of expression in a testicular cell line [16], which could explain the occurrence of CBVAD in TG(13)T(5) patients carrying in trans a CF mutation.…”