Cystic Fibrosis

Mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein have been described to lead to dysfunction of several organs such as lung, sweat glands, genital tract, intestine and pancreas producing the complex cystic fibrosis symptoms (Fig.1). These diseases are commonly referred to as Cystic Fibrosis (CF, OMIM: 219700).

The occurrence rate of CF varies between populations but it is estimated that the incidence of disease is approximately 1/3300 in most Caucasian populations, making it the most frequent childhood genetic disease known to date.

Molecular cause and consequences

The CF gene is constituted by a single locus on human chromosome 7 (region q31). The encoded mRNA is about 6 kb-long and encodes for a membrane-associated glycoprotein with a molecular weight of 169 kD. The CFTR protein consists of five domains: two nucleotide binding folds that bind ATP (NBF1 and NBF2), two hydrophobic transmembrane domains (TMD1 and TMD2), each constituted by six membrane-spanning segments and the regulatory (R) domain, which contains several consensus phosphorylation sequences.

Normally, all of its 27 exons are included in the mature mRNA. However, mutations affecting the CF splicing process have been increasingly reported to be asosciated with disease. Most notably, the occurrence of congenital bilateral absence of vas deferens (CBAVD) and full bown CF disease have been clearly associated with production of an inactive CFTR protein following the loss of exon 9 from the coding mRNA through a process of aberrant alternative splicing (Fig.2) (Groman et al., 2004).

Several genetic studies have thus been aimed in the past at identifying the cis-acting elements on the human CFTR gene in the vicinity of exon 9 that might explain this unusual splicing process. The principal element identified so far include a TG(m)T(n) polymorphic element (Fig.2). Initially, variability in a Tn polymorphic locus located within the 3'-splice site of IVS8 was the first element to be associated with a variable efficiency of exon 9 splicing. In normal populations, the Tn allele generally occurs in four repeat numbers, T9, T7, T5, and T3, with disease normally associated with T5 alleles or lower. Nonetheless, the T5 allele effect has partial penetrance, as it is quite possible to find healthy homozygous carriers. It is now clear that a second polymorphic locus based on (TG)m repeats (ranging from 9 to 13 repeats in humans) localized immediately upstream from the (T)n tract can profoundly influence the efficiency of exon 9 splicing and thus occurrence of disease (Fig.2). The combined effect of these two polymorphic sequences to cause CFTR exon 9 skipping can be clearly reproduced in culture cell lines using a minigene system (Fig.3) (Niksic et al., 1999).


Gene's structure and Alternative Splicing (AS) events

To see a full gene diagram and AS pattern of CFTR please go to: 

Molecular approaches to diagnoses and therapy

In 2001, Francisco Baralles group have identified HIV-1 TAR DNA Binding Protein (TDP-43) as the cellular factor binding to the TG element in CFTR exon 9 pre-mRNA and capable of modulating CFTR exon 9 alternative splicing (Buratti and Baralle, 2008; Buratti et al., 2001). Several analyses aimed at characterizing the molecular mechanisms of this protein have shown that it is capable of binding other hnRNP proteins of the A/B family through its C-terminal tail and through this interaction can inhibit the recognition of CFTR exon 9 acceptor site (Buratti et al., 2005). In addition, cells depleted of TDP-43 display much increased CDK6 expression levels, altered nuclear shape, cell cycle misregulation, and apoptosis (Ayala et al., 2008).

In parallel, Baralle’s lab has demonstrated that knockdown of TDP-43 using siRNA technology in culture and patient cell lines can restore CFTR exon 9 splicing inclusion both in the presence of unfavorable TG(m)T(n) combinations, a finding that provides a likely basis for future therapeutic applications. (Fig.4) (Ayala et al., 2006).

From a biochemical point of view the TDP-43 protein is made up by 414 residues that can be divided in four major regions:, an N-terminal sequence, two RNA recognition motifs named RRM-1 and RRM-2 (RRM-1 is both sufficient and necessary to bind UG-repeated sequences), and a Glycine rich C-terminal tail (Fig.5). The different functions of these regions with regards to regulatory properties is still very much unknown and Fig.5 shows a summary of the properties known to this date.

An additional point of interest regarding TDP-43 is represented by the observation that this protein has been identified as the major protein component of neuronal inclusions in several neurodegenerative diseases such as Frontotemporal dementias (FTDs) and Amyotrophic Lateral Sclerosis (ALS) (Arai et al., 2006; Neumann et al., 2006). In particular, the TDP-43 inclusions are associated with an aberrant phosphorylation pattern of this protein, its ubiquitination, and its cleavage by caspase enzymes of its C-terminal region (Zhang et al., 2007). At the moment, further work is aimed at better characterizing the role of TDP-43 in the pathophysiology of these diseases. Indeed, the recent reports of TDP-43 mutations clustered in the C-terminal region of this region that associate with both sporadic and familial ALS/FTLD cases (Gitcho et al., 2008; Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008) suggests that this protein can potentially play a very active role in the occurrence of disease.

Presently, Francisco Baralle’s lab is involved characterizing the protein sequences that control its nucleocytoplasmic distribution. This is achieved using a variety of tagged wild-type and mutant sequences that are transfected into culture cells and the recombinant proteins are then detected by immunofluorescence. In parallel, these results are all validated by biochemical analyses through the separation of the cytoplasmic and nuclear fraction of the transfected cells and the detection of TDP-43 by Western blots (not shown). Preliminary results (Fig.6) have confirmed the presence of an NLS region in the N-terminal portion of TDP-43. When this region is mutated (mutants NLS#1 and #2) the resulting TDP-43 molecule loses its nuclear-specific localization.

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