Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene.
Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.
Medical Genetics Laboratory, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy.
Lucy
L
Costantino
Damiana
D
Rusconi
Giulia
G
Soldà
Manuela
M
Seia
Valentina
V
Paracchini
Luigi
L
Porcaro
Rosanna
R
Asselta
Carla
C
Colombo
Stefano
S
Duga
D000328:Adult; D017398:Alternative Splicing; D001483:Base Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D005260:Female; D006239:Haplotypes; D006367:HeLa Cells; D006801:Humans; D007223:Infant; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009297:Nasal Mucosa / Q000378:metabolism; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion
C505032:CFTR protein, human; D022821:RNA Splice Sites; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator