The 384910 kb CT Mutation

The 3849+10 kb C^T is a nucleotide substitution, 10 kb downstream nucleotide 3849, the last nucleotide of CFTR exon 19 (Fig. 1a). This substitution generates a cryptic donor splice site that leads to partial inclusion of 84 bp "exon" between exon 19 and exon 20 (Highsmith et al. 1994). The 84 bp "exon" contains an in-frame stop codon and therefore the aberrantly spliced transcripts lead to the generation of a truncated protein. The 3849+10 kb C^T is the twelfth most common mutation worldwide with frequency of 0.2% (http://www.genet.sickkids.on.ca/cftr/), and is particularly frequent in several subpopulations, including Polish (4%) and Ashkenazi Jews (6%; Kerem et al. 1995; Orgad et al. 2001).

The 3849+10 kb C^T mutation was found to be associated with a relatively mild form of CF. The patients present better nutritional status, older age at diagnosis, and moderately elevated sweat chloride values (Augarten et al. 1993; Highsmith et al. 1994). Nevertheless, there is a marked variability in disease severity among different patients in the same organ, and among different organs of the same patient. Some patients have severe pulmonary disease, while others have normal pulmonary function. About 30% of the patients suffer from pancreatic insufficiency, while others have normal pancreatic function. Interestingly, several of the males carrying the 3849+10

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Fig. 1a-c. The effect of overexpression of splicing factors on the splicing pattern of CFTR exons and on CFTR Cl- efflux, in CFP15a cells carrying the 3849+10 kb C^T splicing mutation (Nissim-Rafinia et al. 2004). a: The effect of the 3849+10 kb C^T mutation on the CFTR splicing. b: The effect of splicing factors on the level of aberrant CFTR transcripts. Gray columns represent a significant effect (P<0.01) using Mann-Whitney u test, adjusted with Bonferroni correction for 8 comparisons. White columns represent no effect. Error bars represent the standard deviations. UT = untransfected. c: The effect of splicing factors on the CFTR Cl-efflux. Black symbols indicate activation of the CFTR Cl- efflux. Empty symbols indicate no effect. An arrow indicates the time of forskolin administration

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kb C^T mutation, are fertile, although the vast majority of male CF patients suffer from CBAVD (Augarten et al. 1993; Dreyfus et al. 1996; Gilbert et al. 1995; Hamosh and Corey 1996; Highsmith et al. 1994).

The 2789+5 G^A Mutation

The 2789+5 G^A mutation is a nucleotide substitution at position +5 of the splice donor site in intron 14b, leading to incomplete skipping of exon 14b, predicted to generate out-of-frame, truncated product (Highsmith et al. 1997). This mutation accounts for 0.1% of CF chromosomes worldwide, and appears with a higher frequency in Greece (4.5%) and in North France (3%; Claustres et al. 2000; Estivill et al. 1997). The 2789+5 G^A mutation is associated with a moderately mild form of CF. Nevertheless, there is variability in disease severity among different patients in their lung function and pancreatic disease; about 60% of the patients suffer from pancreatic insufficiency, while others have normal pancreatic function (Dugueperoux and De Braekeleer 2005; Highsmith et al. 1997).

The 3272-26 A^G Mutation

The 3272-26 A^G mutation leads to the creation of an alternative acceptor splice site that competes with the normal site of exon 17b, but still allows some correctly spliced transcripts to be produced. The aberrantly spliced transcripts include extra 25 nucleotides from intron 17a, which contain a stop codon (Beck et al. 1999). This mutation is widespread in Europe, with the highest frequency in Portugal (2%; Amaral et al. 2001). Patients carrying the 3272-26 A^G mutation show phenotypic variability in age at diagnosis, lung function, and pancreatic disease. The correctly spliced transcripts (~5%) were suggested to account for the milder and variable phenotype found in these patients (Ramalho et al. 2002).

The IVS8-5T Allele

Another well-studied splicing allele is the IVS8-5T, in the acceptor site of exon 9 (Chillon et al. 1995a; Jarvi et al. 1995). This allele was found to generate high levels of exon 9 skipping, due to a short polypyrimidine tract, which consists of only five thymidines. Aberrant transcripts, lacking exon 9, are translated into non-functional proteins (Chu et al. 1993; Strong et al. 1993). The IVS8-5T allele is frequent (20-25%) among infertile men due to CBAVD. This frequency is significantly higher than the frequency of the IVS8-5T allele in the general population (5%; Chillon et al. 1995a; Jarvi et al. 1995; Zielenski and Tsui 1995). Therefore, the IVS8-5T was designated as a mutation causing CBAVD. The IVS8-5T allele was also found in several typical mild CF patients (Kerem et al. 1997; Noone et al. 2000). Thus, the IVS8-5T allele is associated with an extreme variability in clinical CF presentation: from normal healthy fertile individuals or males with CBAVD to typical clinical phenotype of CF.

In addition to the IVS8-5T two other IVS8-T alleles are found in the population, the IVS8-7T and IVS8-9T. Most of the population (~85%) carries the IVS8-7T, and ~10% carry the longest tract of IVS8-9T (Chillon et al. 1995a). All the IVS8 poly-T alleles are shorter than the minimal effective 11 pyrimidine tract required for efficient splicing (Coolidge et al. 1997). Indeed, each of these alleles generates both correctly and aberrantly spliced transcripts (Chu et al. 1993). An inverse correlation is found between the length of the poly-T and the level of exon 9 skipping. The longest allele (IVS8-9T) leads to low levels of exon 9 skipping (<10%), the IVS8-7T allele leads to higher levels of skipping (up to 50%), and the shortest IVS8-5T allele leads to very high levels (up to 90%; Chu et al. 1993; Larriba et al. 1998; Mak et al. 1997; Rave-Harel et al. 1997). It should be noted that although an inverse correlation is found between the length of the poly-T and the level of exon 9 skipping, this level varies considerably among individuals carrying each of the alleles (Chu et al. 1993; Larriba et al. 1998; Mak et al. 1997; Rave-Harel et al. 1997).

An additional cis element, which regulates the splicing pattern of exon 9, is a TG repeat, located in a close proximity upstream to the T tract. The number of the TG repeat varies in the population between 10 and 13. The level of correctly spliced exon 9 transcripts is in correlation with the length of the TG repeat, such that a longer repeat is associated with higher levels of correctly spliced transcripts (Buratti et al. 2001). Taken together, a shorter T tract and a longer TG repeat in the acceptor site of exon 9 lead to high levels of exon 9 skipping.

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