ABCMdb

PMID: 12719375

Pagani F, Stuani C, Tzetis M, Kanavakis E, Efthymiadou A, Doudounakis S, Casals T, Baralle FE
New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12.
Hum Mol Genet. 2003 May 15;12(10):1111-20., 2003-05-15 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly This is the case in both natural mutations D565G and G576A (the latter having previously considered a neutral polymorphism) and several site-directed silent substitutions. Login to comment 29 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly This is the case for two interesting and enigmatic missense mutations, D565G and G576A. Login to comment 30 ABCC7 p.Asp565Gly The D565G mutation was previously reported in a young subject during a screening program and suspected of inducing exon skipping (19). Login to comment 31 ABCC7 p.Gly576Ala The conservative G576A missense substitution was originally listed as a neutral polymorphism in the Cystic Fibrosis Genetic Analysis Consortium (http://genet.sickkids.on.ca). Login to comment 38 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly Mutations inducing exon skipping include both missense D565G and G576A mutants and several neutral substitutions. Login to comment 40 ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly RESULTS D565G and G576A missense mutations cause CFTR exon 12 skipping in vivo We evaluated, in nasal epithelial cells, the pattern of CFTR exon 12 splicing in both normal subjects and heterozygous individuals with D565G and G576A alleles. Login to comment 41 ABCC7 p.Arg668Cys ABCC7 p.Asp565Gly The missense D565G mutation was detected in seven Greek subjects, always in cis with the common polymorphism R668C (2134C/T) in exon 13 (Table 1). Login to comment 43 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala The patient carrying the G576A missense mutation was affected by testicular azoospermia and in this case the G576A allele was also in cis with the R668C polymorphism. Login to comment 44 ABCC7 p.Arg668Cys To distinguish between the transcripts produced from the normal and mutant alleles we took advantage of the presence of the R668C polymorphism in exon 13 in cis with both mutations, and designed allele-specific primers. Login to comment 46 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly Two PCR`s were set up for the nasal epithelial cell cDNA derived from each of the D565G and G576A heterozygotes and from heterozygous controls for R668C, using the common F3 forward primer in exon 11 and each of the two allele specific primers of exon 13 (Fig. 1A). Login to comment 48 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly In heterozygous individuals the 668C allele carrying the mutations D565G or G576A clearly showed a significantly lower proportion of normal transcripts containing exon 12 than the 668R allele (Fig. 1B, lanes 7-20). Login to comment 53 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly This analysis showed that the mutant D565G and G576A alleles produced about 40 and 22% of exon inclusion, respectively (Table 1). Login to comment 54 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly These results indicate that, in nasal epithelial cells, the D565G and G576A missense mutations cause a splicing defect affecting the recognition of CFTR exon 12. Login to comment 55 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly Defective CFTR exon 12 recognition in hybrid minigenes containing D565G and G576A missense mutations In order to study in more detail the splicing regulation of CFTR exon 12 we have developed a faithful splicing assay that mimics the in vivo situation. Login to comment 61 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly Allele-specific PCR transcript analysis F3/668R F3/668C Subjects with D565G mutation 89.7Æ 5.9 40Æ 8.3a Subject with G576A mutation 88Æ 3 22Æ 4b Normal controls (heterozygotes for polymorphism R668C) 91.7Æ 5.1 89.3Æ 8.1 Data from six subjects with the D565G mutation, one patient with the G576A mutation and four controls are calculated from the experimental proportions of CFTR exon 12 inclusion adjusted according to the graph shown in Figure 1C. Login to comment 63 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly a Transcripts containing exon 12 derived from the D565G allele. b Transcripts containing exon 12 derived from the G576A allele. Login to comment 65 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly RT-PCR allele-specific amplification experiments in D565G and G576A carriers. Login to comment 67 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly The position of the missense substitutions (D565G and G576A) in exon 12 and of the C668R polymorphic variant in exon 13 is indicated. Login to comment 70 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly The D565G and G576A carriers presented the missense mutation in cis with the 668C variant. Login to comment 72 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly RNA extracted from nasal epithelial cells from R688C heterozygous controls (lanes 1-6), from D565G carriers (lanes 7-18) and from the G576A carrier (lanes 19-20), was reverse transcribed and amplified with F3/668C primers (even-numbered lanes) and with F3/668R primers (odd-numbered lanes). Login to comment 77 ABCC7 p.Arg668Cys The specificity of the 668R and 668C primers was evaluated by amplification experiments in homozygous R668C individuals. Login to comment 89 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly ABCC7 p.Tyr577Phe We then studied the pattern of splicing of a minigene with the missense mutations D565G, G576A and Y577F, the latter associated to classical CF. Login to comment 90 ABCC7 p.Asp565Gly The D565G showed about 35% of normal transcripts containing exon 12 (Fig. 2C). Login to comment 91 ABCC7 p.Gly576Ala The G576A mutation resulted in a severe splicing defect, with only 7% of normal exon 12þ mRNA transcripts. Login to comment 92 ABCC7 p.Tyr577Phe Instead, the nearby Y577F mutation found in classical CF did not produce aberrant skipping but surprisingly increased exon inclusion in comparison with WTB (Fig. 2C). Login to comment 93 ABCC7 p.Asp565Gly ABCC7 p.Tyr577Phe This indicates that, unlike G565A and D565G, the disease-causing effect of Y577F cannot be attributed to a splicing abnormality but rather to a protein defect. Login to comment 94 ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly These results indicate that the two missense D565G and G576A mutations associated with non-classical CF induce variable proportion of exon 12 skipping, with G576A most severely affected. Login to comment 97 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly ABCC7 p.Tyr577Phe To evaluate their role in CFTR exon 12 we transfected normal and the three D565G, G576A and Y577F minigenes in different cell lines (Fig. 3A). Login to comment 98 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly ABCC7 p.Tyr577Phe For each cell line tested, the three variants cause comparable changes in splicing efficiency, with D565G and G576A inducing exon skipping and Y577F exon inclusion. Login to comment 100 ABCC7 p.Gly576Ala For example, G576A showed 15% of exon inclusion in NT2, 7-9% in HeLa, COS1 and T84, and complete skipping in CFPAC. Login to comment 118 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly In WTB, D565G and G576A, overexpression of any of the two splicing factors caused an increase in CFTR exon 12 skipping. Login to comment 119 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly The amount of normal transcript containing the exon 12 were reduced in WTB (40-51%), very low in D565G (7-12%) and virtually absent in the G576A mutant (Fig. 3B). Login to comment 121 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly These results indicate that, in the presence of high concentration of inhibitory splicing factors, the D565G and G576A missense mutations produce very low levels of normal CFTR transcripts. Login to comment 124 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly ABCC7 p.Tyr577Phe A total number of 26 hybrid minigenes were analysed containing site-directed mutations at two target sequences of the exon: the AAGATGC sequence at the 50 end from position 12 to 18, which includes D565G at position 15, and a central GGATAC sequence from position 47 to 52 which contains G576A and Y577F of position 48 and 51, respectively (Fig. 4). Login to comment 156 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly The mild CF phenotypes of the D565G and G576A patients may be explained considering that the variant CFTR protein is functional and that the defect is the consequence of exon 12 skipping induced by the nucleotide change. Login to comment 157 ABCC7 p.Gly576Ala ABCC7 p.Tyr577Phe It is interesting to note that changes in the codon following G576A result in the classical CF mutation Y577F that produces a non-functional protein, but splicing is not significantly altered. Login to comment 162 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly Thus, according to tissue concentration of regulatory splicing factors, and their variations from individual to individual, the D565G and G576A may produce different quantities of mRNAs lacking the exon leading to phenotype variability. Login to comment 187 ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly MATERIALS AND METHODS Patients and DNA mutation analysis Nasal epithelial cells were collected from six individuals carriers of mutation D565G (A>G at 1826 in CFTR cDNA), from one CBAVD patient with G576A and from four non-CF control individuals heterozygotes for the polymorphism R668C. Login to comment 189 ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly Clinical data and CFTR genotypes of all the D565G and G576A carriers are shown in Table 2. Login to comment 193 ABCC7 p.Arg668Cys ABCC7 p.Asp565Gly The phase of linkage for missense mutations D565G and G556A and polymorphism R668C was deduced from family studies and confirmed by sequencing of allele specific cDNAs. Login to comment 225 ABCC7 p.Gly576Ala ABCC7 p.Gly576Ala ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly ABCC7 p.Asp565Gly Data on individual carriers of D565G and G576A mutations Patient code CFTR genotypea Age (years) Sex Reason for CF testing Sweat test (mEq/l) Pancreatic status 1PM D565G/À 16 F Nasal polyposis, Sa 65, 70 PS 2DF D565G 7 1717À9T>C/À 7 F Recurrent episodes of pneumonia 41.4 PS 3MA D565G/À Adult M Carrier status nt nt 4KA D565G/À Adult F Carrier status nt nt 5KP D565G/À Adult M Carrier status nt nt 6PRA D565G/À Adult F Carrier status nt nt 7ORAb D565G/À Adult M CBAVD <40 PS 8 G576A/À Adult M Testicular azoospermia nt nt PS, pancreatic sufficiency; Sa, Staphylococcus; nt, non-tested; CBAVD, congenital bilateral absence of vas deferens. Login to comment 226 ABCC7 p.Arg668Cys All patients are heterozygous for the R668C allele. Login to comment