Hi, was looking at some of the gene info I have gathered on my boys...ran across this. Don't know if it helps any....
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. 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). All these subjects were heterozygotes and two showed non-classical CF: one patient was affected by CBAVD and the other with pulmonary symptoms of an unknown aetiology. 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. 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. The 688C and 688R primers contain either C or T at their 3' end (nucleotide 2134 in CFTR mRNA), to discriminate between the R and C alleles at amino acid 668 (Fig. 1A). 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). Analysis of the cDNA products in all cases revealed the presence of two transcripts of 449 and 362 bp containing or lacking the exon 12, respectively (Fig. 1B). 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). On the contrary, in normal subjects, the two polymorphic alleles with 668C or 668R produced an equally low amount of leaky splicing (Fig. 1B, lanes 1 - 6). The presence of leaky splicing in normal alleles is consistent with previous data where, in normal individuals, variable amounts of mRNA transcripts lacking exon 12 (5 - 30%) have been reported (16). In order to quantitate the proportion of exon 12+ CFTR mRNA accurately, in vitro transcribed mRNAs, with and without exon 12, were mixed in varying proportions, reverse transcribed and analysed as for the nasal samples (Fig. 1C, left panel). The data were then plotted against the proportion of input RNAs (Fig. 1C, right panel) and, according to the resulting graph, the experimental proportions of CFTR exon 12 transcripts in nasal epithelial cells were corrected. This analysis showed that the mutant D565G and G576A alleles produced about 40 and 22% of exon inclusion, respectively (Table 1). 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.
Sorry, should have put more of a space in there...makes for hard reading.
Take care,
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. 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). All these subjects were heterozygotes and two showed non-classical CF: one patient was affected by CBAVD and the other with pulmonary symptoms of an unknown aetiology. 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. 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. The 688C and 688R primers contain either C or T at their 3' end (nucleotide 2134 in CFTR mRNA), to discriminate between the R and C alleles at amino acid 668 (Fig. 1A). 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). Analysis of the cDNA products in all cases revealed the presence of two transcripts of 449 and 362 bp containing or lacking the exon 12, respectively (Fig. 1B). 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). On the contrary, in normal subjects, the two polymorphic alleles with 668C or 668R produced an equally low amount of leaky splicing (Fig. 1B, lanes 1 - 6). The presence of leaky splicing in normal alleles is consistent with previous data where, in normal individuals, variable amounts of mRNA transcripts lacking exon 12 (5 - 30%) have been reported (16). In order to quantitate the proportion of exon 12+ CFTR mRNA accurately, in vitro transcribed mRNAs, with and without exon 12, were mixed in varying proportions, reverse transcribed and analysed as for the nasal samples (Fig. 1C, left panel). The data were then plotted against the proportion of input RNAs (Fig. 1C, right panel) and, according to the resulting graph, the experimental proportions of CFTR exon 12 transcripts in nasal epithelial cells were corrected. This analysis showed that the mutant D565G and G576A alleles produced about 40 and 22% of exon inclusion, respectively (Table 1). 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.
Sorry, should have put more of a space in there...makes for hard reading.
Take care,