SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Cystic Fibrosis
  4. CFTR Gene Structure and Organisation
  5. CFTR Mutations
  6. Genotype/Phenotype Correlation
  7. Concluding Remarks
  8. References

Cystic fibrosis is a common autosomal recessive disorder that primarily affects the epithelial cells in the intestine, respiratory system, pancreas, gall bladder and sweat glands. Over one thousand mutations have currently been identified in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene that are associated with CF disease. There have been many studies on the correlation of the CFTR genotype and CF disease phenotype; however, this relationship is still not well understood. A connection between CFTR genotype and disease manifested in the pancreas has been well described, but pulmonary disease appears to be highly variable even between individuals with the same genotype. This review describes the current classification of CFTR mutation classes and resulting CF disease phenotypes. Complex disease alleles and modifier genes are discussed along with alternative disorders, such as disseminated bronchiectasis and pancreatitis, which are also thought to result from CFTR mutations.


Cystic Fibrosis

  1. Top of page
  2. Summary
  3. Cystic Fibrosis
  4. CFTR Gene Structure and Organisation
  5. CFTR Mutations
  6. Genotype/Phenotype Correlation
  7. Concluding Remarks
  8. References

Cystic fibrosis (CF) is an autosomal recessive disorder that affects approximately one in 2500 births among most Caucasian populations, though its frequency may vary in specific groups. The disorder primarily involves epithelial cells in the intestine, respiratory system, pancreas, gall bladder and sweat glands. Poor mucociliary clearance with excessive mucus production causes obstructive lung disease and chronic bacterial infections leading to respiratory failure, which is the major cause of mortality. More than 95% of patients also fail to produce digestive enzymes in the pancreas, resulting in pancreatic insufficiency (PI). Population studies of CF patients suggest that most of the PI patients were descendants from a single mutational event at the CF locus, whereas the pancreatic sufficient (PS) phenotype resulted from multiple different mutations (Kerem et al. 1989; Estivill et al. 1995; Casals et al. 1997; Mateu, 2002). There is a high level of male infertility (>95%) caused by absence or obstruction of the vas deferens, and many females also suffer from reduced fertility, possibly due to altered cervical mucus properties. Life expectancy of CF patients has increased dramatically over the past four decades, with an expectancy of less than one year in 1960, whereas the median survival age in 2002 is around 40 years. The sweat gland duct is affected in CF and the concentration of salt in the sweat of CF patients is greatly elevated (3–5 times normal); this is used as a diagnostic test for the disease. Neonatal meconium ileus (MI), the obstruction of the distal intestine due to accumulation of thickened meconium, occurs in 10–20% of CF newborns. This is very rare in non-CF patients and is therefore almost diagnostic of CF. The absence of MI in 80–90% of CF patients suggests the existence of modifier genes that are involved in modulating the phenotype and severity of the CF disease within CF individuals with the same genotype (Zielenski et al. 1999 and discussed further below).

CFTR Gene Structure and Organisation

  1. Top of page
  2. Summary
  3. Cystic Fibrosis
  4. CFTR Gene Structure and Organisation
  5. CFTR Mutations
  6. Genotype/Phenotype Correlation
  7. Concluding Remarks
  8. References

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene located on chromosome 7q31.2, which when mutated causes CF, spans approximately 190 kb of genomic DNA (Ellsworth et al. 2000). Several research groups constructed 1.5Mb Yeast Artificial Chromosome (YAC) contigs encompassing the CFTR genomic locus. A number of YACs were isolated that contained the entire CFTR gene plus flanking DNA sequence (Green & Olson, 1990; Anand et al. 1991). Restriction analysis of these YACs allowed the determination of exonic regions within the genomic context.

The CFTR gene consists of 27 exons and encodes a mature transcript of 6.5 kb that is translated into a 1480 amino acid protein of approximately 168 kDa (Riordan et al. 1989) (Figure 1). The cDNA sequence of CFTR was determined in 1991 along with the locations of intron/exon boundaries (Zielenski et al. 1991).

image

Figure 1. A) Diagram of the CFTR gene transcript and resulting protein. Adapted from Chu et al. 1991 by permission of Oxford University Press. B) CFTR gene to scale – exons are denoted by vertical black rectangles. C) CFTR genomic locus shown to scale with the neighbouring genes identified as horizontal black rectangles.

Download figure to PowerPoint

CFTR Mutations

  1. Top of page
  2. Summary
  3. Cystic Fibrosis
  4. CFTR Gene Structure and Organisation
  5. CFTR Mutations
  6. Genotype/Phenotype Correlation
  7. Concluding Remarks
  8. References

Spectrum of CFTR Mutations

Over one thousand mutations have been defined in the CFTR gene to date (CF Genetic Analysis Consortium http://www.genet.sickkids.on.ca/cftr/). These mutations are situated throughout the entire coding region of the gene and also the promoter region, although there are regions where mutations are more common, such as the nucleotide binding domains (NBD) and regulatory (R) domain. These regions are likely to indicate functionally or structurally important domains and amino acids within the protein. The ΔF508 mutation accounts for approximately 70% of chromosomes in CF patients. The majority of the other CFTR mutations are very rare with only four other mutations (G542X, N1303K, G551D and W1282X) having overall frequencies above 1%. Many of the other mutations are unique to a particular individual or family or have been found in only a handful of cases across the world (Cystic Fibrosis Genetic Analysis Consortium, 1994). There are relatively few genomic rearrangements (1%) such as large deletions or insertions within the CFTR gene, e.g. Δexons 4–10, Δ95.7 kb starting in intron 1, Δexon 14b-18 (20 kb) (see CF Genetic Analysis Consortium http://www.genet.sickkids.on.ca/cftr/).

CFTR Protein

The CFTR protein is a member of the ATP-binding cassette (ABC) membrane transporter superfamily that includes proteins such as the multiple drug resistance protein (MDR) and bacterial periplasmic permeases (reviewed by Higgins, 1992). The CFTR protein contains 1480 amino acids comprising two homologous halves (Riordan et al. 1989). It consists of two membrane-spanning regions, each comprising 6 subunits, two nucleotide binding domains (NBD) and a cytoplasmic regulatory domain, unique within the ABC family, which contains many potential substrate sites for phosphorylation by protein kinases (Figure 2) (reviewed by Akabas, 2000). CFTR functions as a cyclic adenosine monophosphate- (cAMP) dependent chloride channel that plays an important role in chloride transport across apical epithelial surfaces (Rich et al. 1990). CFTR may also have other functions, for example in bicarbonate transport (Choi et al. 2001), and the protein interacts directly with other molecules, including the sodium epithelial channel ENaC, suggesting a regulatory role for CFTR (reviewed by Schwiebert et al. 1999).

image

Figure 2. Proposed domain structure of the CFTR protein within the cell membrane. Y represents possible glycosylation sites at asparagine residues. Shaded circles denote regions that may be involved in putative interaction between the transmembrane domains and NBDs.

Download figure to PowerPoint

Effects of Mutations

Mutations in the CFTR gene have been classified into five different groups according to the mechanism by which they disrupt CFTR function; however, these classes are not mutually exclusive (Figure 3). The classes were recently modified to include previous Group V mutations with Group I since all these mutations cause an alteration in level of functional CFTR mRNA (Welsh, 2001).

image

Figure 3. CFTR mutation classes represented as protein production by a cell. Adapted from Cell73, Welsh & Smith, Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis, pp. 1251–94 (1993), and reproduced with permission of Elsevier Science.

Download figure to PowerPoint

Class I Mutations affecting biosynthesis About half of the CFTR mutations are expected to prevent proper synthesis of full-length, normal CFTR protein because of nonsense (14%), frameshift (22%), or aberrant splicing of mRNA (8%) (reviewed by Zielenski & Tsui, 1995). Class I mutations in the CFTR gene include the most severe CF phenotypes due to no protein being synthesised. These mutations, the most common being G542X, prevent the synthesis of a stable protein or result in the production of a truncated protein due to the creation of a premature termination codon. The truncated proteins are usually unstable, are recognised by chaperone proteins in the endoplasmic reticulum (ER) and are rapidly degraded. Nonsense mediated decay (the degradation of transcripts harbouring codons signalling the premature termination of translation (Hentze and Kulozik, 1999)) is further implicated in mutations in this class. Class I also contains the least severe phenotypes (Welsh, 2001) (previously known as Class V mutations) which occur due to mis-splicing mutations (e.g. aberrant splicing of exon 9 discussed below) producing a small amount of CFTR transcript and low levels of functional protein that is translocated to the cell membrane. Predicted promoter mutations are likely to have similar effects, by reducing the level of transcription, as well as ‘stop’ mutations that reduce the level of full-length mRNA by splicing-out of the exon containing the mutation (Hull et al. 1993). As a small amount of full-length mRNA is often still synthesised in these cases, they would be predicted to result in a mild phenotype, as was demonstrated for the 3849 + 10kb C [RIGHTWARDS ARROW] T mutation (Highsmith et al. 1994).

One way to correct a class I mutation at the CFTR level would be gene replacement via gene therapy techniques. Alternatively, recent studies showed that premature stop codons, such as G542X and R553X, were suppressed by the addition of aminoglycoside antibiotics (e.g. gentamicin or G418) that are known to stimulate the suppression of stop codons in various organisms by near-cognate mis-pairing of an aminoacyl-tRNA with the premature stop codon (Howard et al. 1996). The authors observed a dose-dependent increase in full-length CFTR produced by cells transfected with CFTR mRNAs containing stop codons and these were shown to function as cAMP-activated ion channels. Systematic treatment of CF patients with gentamicin resulted in small increases in CFTR Cl conductance in vivo (Clancy et al. 2001).

Class II Mutations affecting protein maturation Class II mutations result in the misprocessing of CFTR, producing a lack of functional protein at the cell membrane. The ΔF508 mutation, a deletion of three bases encoding a phenylalanine residue at position 508 within the first NBD, results in mis-folding of the CFTR protein and hence mislocalisation of the mature protein. ΔF508 was classified as a class II mutation based on analysis in a heterologous expression system which demonstrated that ΔF508 CFTR was synthesised, but failed to mature or to proceed beyond the endoplasmic reticulum (ER) (Cheng et al. 1990). The ΔF508 protein was also shown to be mis-localised in CF sweat gland duct epithelial cells (Kartner et al. 1992). However, in more recent immunocytochemical studies of intestinal, respiratory and hepatobiliary epithelia of ΔF508 homozygous CF patients, a proportion of CFTR protein was shown to be targeted to the apical membranes (Kalin et al. 1999). Further, measurements of Cl conductance of intestine and respiratory tissues of ΔF508 homozygote CF patients suggested that, in vivo, at least some ΔF508 CFTR can reach the plasma membrane (Bronsveld et al. 2001).

Therapy for class II mutations would require pharmacological methods to increase the levels of functional protein at the cell membrane, by increasing the efficiency of protein folding or suppressing the protein degradation processes.

Class III Mutations affecting Cl channel regulation/gating Mutations of the CFTR gene in class III produce protein that is trafficked to the cell membrane but then does not respond to cAMP stimulation. All mutations so far attributed to this class are located within the nucleotide-binding folds, and are likely to affect the binding of ATP or the coupling of ATP binding to activation of the channel, such as by preventing transmission of a conformational change. The missense mutation G551D is an example of a class III mutation. Therapy for mutations in classes III and IV would require increased activation of the functional CFTR present at the cell membrane.

Class IV Mutations affecting Cl conductance Class IV mutations include cases where the CFTR gene encodes a protein that is correctly trafficked to the cell membrane and responds to stimuli but generates a reduced Cl current (for example R117H). Most of the class IV mutations analysed to date are located within the membrane-spanning domains. Expression of several such mutants in a heterologous system resulted in the production of a chloride current that was activated by cAMP (Sheppard et al. 1993). Regulation by ATP appeared to be normal; however, the current was much reduced due to a decrease in amplitude of a single-channel current and also a lower open state probability. More recent studies of Class IV CFTR mutations located within exon 13 that encodes the regulatory domain have demonstrated that these mutations can have different effects on the levels of chloride conductance (Vankeerberghen et al. 1998a). Three mutant CFTR proteins, G622D, R792G and E822K, that were transiently expressed in COS cells showed lower chloride channel activities when compared to wild-type CFTR, whereas mutants H620Q and A800G showed increased activities. Furthermore, mutants T665S and E826K showed no difference from the wild-type channel conductance. Several mutations within exon 18, which encodes transmembrane helix 12 and the subsequent intracytoplasmic loop, were also shown to fall into Class IV with M1137V, I1139V, ΔM1140, D1152H and D1154G mutants exhibiting significantly reduced cAMP-activated chloride currents (Vankeerberghen et al. 1998b).

Mutations affecting protein stability An additional class of CFTR mutation has recently been proposed by Haardt et al. and discussed by Zielenski as Class VI1 (Zielenski 2000). This novel class includes protein stability mutants (which result in inherent lability of the CFTR protein) lacking the last 70–98 residues of the CFTR C-terminus (Haardt et al. 1999). Although the C-terminus is not required for the biogenesis and chloride channel function of CFTR, the authors report that it is indispensable for maintaining the stability of the complex-glycosylated CFTR. The shortest truncation reported that caused CF with pancreatic insufficiency and recurrent pulmonary infection is Q1412X, that lacks 70 amino acids (CF Genetic Analysis Consortium). Pancreatic insufficiency is a marker of severe CF disease and similar severe CF phenotypes are caused by frameshifts resulting in the deletion of the last 81, 97 and 101 amino acids (4326delTC, 4279insA and 4271delC respectively). Truncated CFTRs were transiently and stably expressed in heterologous expression systems and all proteins showed identical localisation to the wild type protein (Haardt et al. 1999). Furthermore, the cAMP-stimulated current of the truncated proteins resembled the characteristics of wild type CFTR, demonstrating that deletion of the last 26 amino acids had no effect on CFTR channel function. Analysis of biosynthetic processing showed that the last 82 amino acids of CFTR were not essential for post-translational folding, but the half-life of complex-glycosylated truncated CFTR was reduced 5- to 6-fold. This suggests that the biological stability of truncated CFTR molecules is dramatically reduced, potentially leading to severe CF phenotypes.

Aberrant splicing of exon 9 Exon 9 of the CFTR gene encodes the first 21% of NBD1 and is critical for CFTR function (Riordan et al. 1989). RT-PCR analysis of bronchial epithelial cells from 12 non-CF individuals revealed that all samples contained some CFTR mRNA transcripts that were lacking exon 9 (exon 9-) (Chu et al. 1991). The amount of exon 9-transcript varied in non-CF individuals between 0–92% (Chu et al. 1993). The fact that up to 92% of CFTR mRNA transcripts can lack exon 9 without causing CF defined a ‘threshold’ level for the amount of functional CFTR mRNA necessary to maintain a clinically normal phenotype. This ‘threshold’ level was modified by Rave-Harel in 1998 to 25%, in a study of CFTR transcripts in nasal and epididymis epithelium (discussed further below) (Rave-Harel et al. 1997). It should be noted, however, that these results were gained by semi-quantitative RT-PCR whereas the figures reported by Chu et al were derived from non-quantitative RT-PCR and so may be slightly inaccurate due to the increased efficiency of amplification of smaller DNA fragments. A dinucleotide repeat (TG)m was identified at the intron 8 – exon 9 junction with 10–12 repeats and a thymidine nucleotide tract ((T)n) varying between 5 to 9 nucleotides. Notably, the two alleles analysed with T5 correlated with expression of high amounts of exon 9-CFTR mRNA (Chu et al. 1991). This was supported by a study of 124 individuals in which it was found that the shorter the poly-T tract the greater the relative amount of exon 9-CFTR mRNA transcripts present in respiratory epithelium (Chu et al. 1993). More recent evidence supports the role of exon skipping for the T5 allele and also shows that the (TG)m repeat can further modulate alternate splicing, but only when activated by the T5 allele (Niksic et al. 1999). The authors found that in transient transfections of minigenes containing the T5 allele linked to the (TG)m repeats, the longer the (TG)m tract the higher the proportion of exon 9-CFTR mRNA transcripts.

The molecular basis for the skipping of exon 9 was investigated using an in vivo minigene model system with differing numbers of the (TG)m and Tn repeats (Pagani et al. 2000). The authors found that alternative splicing of human exon 9 was negatively regulated by the intracellular concentration of different trans-acting factors, including SR (serine-arginine-rich) proteins and hnRNPs (heterogeneous nuclear ribonucleoproteins). This effect was modulated by cis-acting elements (TG)m, Tn, an exonic splicing enhancer (ESE) located in exon 9, and an intronic splicing silencer (ISS) located in intron 9. Increasing the intracellular concentrations of both hnRNPA1 and SR proteins was shown to repress correct splicing of exon 9, the latter working via an interaction with the ISS in intron 9. The nuclear factor TDP-43 (HIV-1 TAR DNA-binding protein) was identified as the factor interacting with the (TG)m element in CFTR mRNA (Buratti et al. 2001). Overexpression of this protein resulted in the inhibition of exon 9 splicing whereas inhibition of TDP-43 expression resulted in an increase in exon 9 recognition.

Another mechanism of exon 9 skipping has been investigated by mutating the 5′ splice sites both up- and down-stream of exon 9 (Hefferon et al. 2002). Alteration of the sequence of the downstream 5′ splice site to the consensus sequence resulted in a substantial reduction in exon 9 skipping. These results, obtained from experiments carried out in minigene constructs, are supported by in vivo observations in both sheep and mouse. The sheep CFTR gene contains a long polypyrimidine tract (T14) and also a short (TG)m repeat within intron 8. Although this would previously have been predicted to improve retention of exon 9, transcripts lacking exon 9 were evident in all sheep tissues analysed, possibly due to the non-consensus downstream 5′ splice site. In the mouse, even though the polypyrimidine tract in intron 8 is short (Y5), exon 9 is not excluded because the 5′ splice site in intron 10 is close to consensus. These results suggest that there may be multiple factors involved in the splicing of CFTR exon 9, including divergence from consensus splice site sequences, intronic splicing silencers and exonic splicing enhancers.

Geographical and Ethnic Variations

The incidence of CF varies markedly among different populations. In Northern European populations, such as Northern Ireland and Sweden, the incidence of disease varies from 1 in 1700 live births to 1 in 7700 respectively (Tsui, 1992). This equates to carrier frequencies of 1 in 20 in Northern Ireland to 1 in 44 in Sweden. The geographical distribution of CFTR mutations also varies worldwide. Across Europe there is a northwest to southeast gradient in the frequency of ΔF508, with the highest frequency in Denmark (90%), and the lowest frequencies in Turkey, where analysis of 122 unrelated chromosomes from 73 families revealed the prevalence of ΔF508 to be 18.8% (Onay et al. 1998), and Romania, where the proportion of CF patients carrying ΔF508 was determined to be 25% (Popa et al. 1997). Ashkenazi Jews have a low incidence of ΔF508 but have an increased frequency (60%) of the nonsense mutation W1282X (Shoshani et al. 1992). These variations are likely to be due to founder effects as the various groups migrated and settled in different areas. Such information is important in order to be able to design suitable screening programmes for different populations.

DNA Polymorphisms

Over 200 DNA polymorphisms that are not disease associated have been identified within the CFTR gene (http://www.genet.sickkids.on.ca/cftr). Over half of these polymorphisms occur in CFTR coding regions and around half result in amino acid substitutions. DNA polymorphisms have also been detected within the CFTR locus in non-CF individuals that could also potentially result in alterations in CFTR gene expression (Rowntree & Harris, 2002). These polymorphisms, located within regions suggested to play a regulatory role in the expression of the CFTR gene, result in an alteration of the binding of transcription factors to these sites, and so could have an effect on the severity of the CF phenotype. Healthy individuals would tolerate such polymorphisms due to the presence of a wild-type allele producing functional CFTR protein. However, if an individual carried a CF-causing mutation on one allele resulting in non-functional CFTR molecules, and a relevant polymorphism on the other allele, this could lead to a reduction in transcriptional activity of the non-mutant allele. An associated fall in the amount of functional CFTR protein produced to below the required ‘threshold’ could cause a mild or atypical CF phenotype. Additionally, point mutations that are assumed to be single nucleotide polymorphisms (SNPs) play a role in CF disease by interfering with splicing signals (see aberrant splicing of exon 9) and causing mis-splicing of the gene, such as 1717–9T [RIGHTWARDS ARROW] C-D565G in exon 12 (Tzetis et al. 2001), as reviewed by Cartegni et al. 2002.

Theories of Heterozygote Advantage

The carrier frequency of CF alleles is approximately 1 in 25 in the UK and similar in other European countries. It has been proposed that in heterozygotes, mutations of the CFTR gene provide increased resistance to infectious diseases, thereby maintaining the mutant alleles at high frequency in selected populations. Cholera toxin (CT) mediates its action by an irreversible elevation of cAMP levels that leads to the activation of cAMP-regulated Cl channels and subsequent fluid secretion. This can result in potentially lethal chloride-secretory diarrhoea. Cl secretion in response to CT was shown to be proportional to amounts of CFTR protein in cftr null mice (Gabriel et al. 1994). Heterozygous mice secreted 50% of the normal fluid and chloride ions in response to CT. Hence individuals with only one functional copy of the CFTR gene might not achieve normal levels of fluid secretion in response to CT due to lower cAMP-activated chloride channel activity. Therefore, whilst showing no symptoms of CF, carriers would be more resistant to the lethal dehydrating effects of cholera.

However, these theories were disputed by Högenauer et al. who measured the in vivo basal and prostaglandin-stimulated jejunal chloride secretion in normal subjects, CF heterozygotes, and patients with CF (Hogenauer et al. 2000). The results from this study showed that individuals who were heterozygous for CF mutations secreted chloride at the same rate as people without a CF mutation, contradicting the earlier data gained from mouse models.

Pier et al. investigated whether increased resistance to typhoid fever could be another factor that maintained a high frequency of ΔF508 in the population. Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. S. typhi, but not other Salmonella species, was found to use CFTR for entry into epithelial cells (Pier et al. 1998). Murine epithelial cells expressing ΔF508 CFTR internalised significantly less S. typhi than wild type CFTR-expressing cells. Furthermore, translocation of S. typhi into the intestinal submucosa in transgenic mice heterozygous for ΔF508 was 86% less than in wild type mice. However, this theory remains to be widely accepted.

Genotype/Phenotype Correlation

  1. Top of page
  2. Summary
  3. Cystic Fibrosis
  4. CFTR Gene Structure and Organisation
  5. CFTR Mutations
  6. Genotype/Phenotype Correlation
  7. Concluding Remarks
  8. References

The CF phenotype is highly heterogeneous among individual patients, even between siblings carrying identical CFTR mutations. This indicates the contribution of other factors, such as alternative genomic loci containing modifier genes, and also environmental effects, in the determination of disease severity. So-called ‘severe’ mutations are those where the absence of functional CFTR correlates well with pancreatic insufficiency (>95% cases), liver disease (in a subset of 3–5% of patients), young age at diagnosis (usually <1 year), high sweat chloride levels (>80 meq/l) and meconium ileus (∼20% of cases). ‘Mild’ mutations that may still produce a small amount of functional CFTR are generally associated with pancreatic sufficiency (70–80% cases) and a later age at diagnosis (usually >10 years), lower sweat chloride levels, no meconium ileus and milder pulmonary disease.

Pancreatic versus Pulmonary Disease

Of the various clinical symptoms of CF only the pancreatic function has been shown to correlate well with CFTR genotype. Patients with pancreatic sufficiency have at least one ‘mild’ allele (i.e. Class IV, partial mis-splicing mutations of class I or protein stability mutations) whereas pancreatic insufficient patients are homozygous or compound heterozygous for two ‘severe’ mutations (i.e. Class I, II or III). Mutations R117H, R334W and R347P are usually associated with less severely impaired pancreatic function (reviewed by Tsui, 1992). Affected individuals within the same family have a similar degree of pancreatic function. Conversely, pulmonary disease is usually highly variable, even among sibs who have identical mutations. This was supported by a study of a pair of dizygotic twins with a compound CFTR genotype ΔF508/1717-1G [RIGHTWARDS ARROW] A (Borgo et al. 1993). Although both twins were pancreatic insufficient, the severity of pulmonary disease varied considerably. Whilst one twin had chronic symptoms of Pseudomonas infection, the other twin showed no abnormal lung function nor a need for persistent antibiotic treatment.

More recent reports, however, have demonstrated a correlation with certain mutations and the severity of pulmonary disease. The splicing variant 5T allele in intron 8 of the CFTR gene (described earlier) was shown to be associated with partial penetrance of CF disease (Rave-Harel et al. 1997). A significant correlation between the level of normal (exon 9+) CFTR transcripts and the severity of lung disease was observed in nasal epithelium. No individuals with normal lung function and minimal or no lung disease had less than 25% of normal transcripts. Furthermore, analysis of four infertile patients with congenital bilateral absence of vas deferens (CBAVD, discussed further below) showed that they possessed between 6–24% normal CFTR transcripts. These results suggest that variability in splicing mechanisms of these CF individuals is associated with the variable penetrance of the disease. The splicing mutation 3849 + 10 kb C [RIGHTWARDS ARROW] T also exhibits correlation of CF pulmonary phenotype with the amount of normal CFTR transcripts (Chiba-Falek et al. 1998). 3849 + 10 kb C [RIGHTWARDS ARROW] T results in the creation of a partially active splice site in intron 19 that leads to the insertion of a new 84bp cryptic exon containing a premature in-frame stop codon; thus both correctly spliced and aberrant transcripts can be produced simultaneously. When levels of aberrantly spliced CFTR transcripts were lower than 3% individuals showed no lung disease and normal lung function, whereas higher levels (9–28%) caused moderate or severe lung disease in these individuals (Chiba-Falek et al. 1998). Variable levels of aberrantly spliced CFTR mRNA were found among patients with the same genotype, consistent with the variable nature of phenotypes caused by CFTR mutations, again suggestive of variation in the splicing factors rather than cis elements within the CFTR loci. Another example of correlation between a CFTR allele and pulmonary disease is the A455E mutation, which was associated with milder lung and pancreatic disease in A455E compound heterozygotes than ΔF508 homozygotes in Quebec (De Braekeleer et al. 1997).

Complex Alleles

Although most cases of complex alleles (two CF-associated mutations carried on one chromosome) may represent association of a benign sequence variation with an actual disease-causing mutation in the same gene, there are examples where the second site mutation can modulate the effect of the principal mutation. The first complex allele to be described was in 1991 where R553Q was detected on the same allele as ΔF508 of a CF patient also carrying the R553X mutation (Dork et al. 1991). This patient exhibited pancreatic insufficiency and severe pulmonary disease but a borderline sweat test, suggesting that the R553Q mutation is somehow modulating the severity of CF disease normally associated with ΔF508. Analysis of the crystal structure of the related ABC protein, HisP, showed that these two mutations are predicted to lie in adjacent α-helices and that R553Q lies in a region essential for the integrity of protein folding (Hung et al. 1998). Therefore it is possible that the substitution of a glutamine residue at position 553 improves the protein folding enough to compensate for the lack of the phenylalanine at position 508, resulting in a more functional CFTR molecule and less severe disease phenotype.

The promoter mutation –102T [RIGHTWARDS ARROW] A has been found in association with S549R (T [RIGHTWARDS ARROW] G) (Romey et al. 1999). Two individuals who had genotypes 102T [RIGHTWARDS ARROW] A + S549R(T [RIGHTWARDS ARROW] G)/ΔF508 and 102T [RIGHTWARDS ARROW] A + S549R(T [RIGHTWARDS ARROW] G)/S945L both had mild CF disease and were pancreatic sufficient. As the S549R mutation has previously been described as a ‘severe’ allele, associated with pancreatic insufficiency, it appears that cis-mutations can modulate the clinical phenotype. In vitro analysis demonstrated that the 102T [RIGHTWARDS ARROW] A mutation resulted in an up-regulation of CFTR expression by the formation of a Yin Yang 1 (YY1) transcription factor-binding site in the CFTR promoter region (Romey et al. 2000).

In addition to ‘severe’ alleles combining to result in a less severe phenotype, a number of complex alleles have been identified in which more than one defined ‘mild’ mutation occur together in cis resulting in a more severe CF phenotype. Clain et al. investigated the complex allele with two mild mutations (R347H-D979A) previously identified in pancreatic sufficient CF patients and CBAVD patients respectively (Clain et al. 2001). R347H was found to be associated with moderately defective Cl channel activity whereas D979A led to misprocessing of CFTR. The double mutant, R347H-D979A, combined both defects and dramatically decreased the Cl current. The complex allele described contained three missense mutations, D443Y, G576A and R668C (Abramowicz et al. 2000). Each of these mutations had previously been observed independently in CF patients and those suffering from CBAVD. The complex allele G576A-R668C had been reported in an individual with a normal sweat test, although the affected members of the family exhibited a mild CF phenotype. It is therefore possible that the inclusion of the D443Y mutation resulted in the CF phenotype in these patients.

The R117H mutation is usually responsible for mild CF disease due to production of a partially functional protein that is localised to the apical cell membranes. However, this mutation appears to be modulated by the presence of the T7 allele in intron 8 of the CFTR gene, with a CF phenotype only occurring if R117H is present with the T5 allele (Kiesewetter et al. 1993). In eight individuals with CBAVD and one asymptomatic individual (R117H/ΔF508) the R117H allele was found in association with the T7 allele. Furthermore, as discussed previously, the T7 allele is associated with normal splicing of exon 9 resulting in normal levels of CFTR mRNA. Therefore, R117H/T7 individuals would produce normal levels of partially functional CFTR resulting in a mild CF phenotype. This is in contrast to R117H/T5 individuals where the amount of full-length CFTR mRNA is reduced, resulting in a more severe phenotype due to a decreased amount of R117H CFTR protein at the cell membrane.

Modifier Genes

The heterogeneity of the CF phenotype suggests that there are more factors involved in the determination of phenotype than CFTR genotype alone, for example environmental factors and other genetic determinants. Twin studies currently underway may provide some insight into the relative contribution of these factors to the phenotype. The observation that cftr mice crossed with different background strains showed prolonged survival indicated the involvement of additional genetic components in a model system where these could be studied (Rozmahel et al. 1996). Partial correction of Cl and Na+ ion transport was shown to be due to an up-regulation of calcium-activated Cl conductance. Using breeding experiments with inbred mouse strains the authors were able to show that prolonged survival of these mice was conferred by certain mouse strains (CD1, C57BL/6J or BALB/cJ) but not by others (e.g. DBA/2J). Genetic linkage analysis revealed a region of mouse chromosome 7 that showed significant deviation from the expected random segregation of CD1, C57BL/6J and BALB/cJ alleles. These results strongly suggested the presence of a genetic modifier locus on the proximal region of mouse chromosome 7, a region corresponding to human chromosome 19q13. Zielenski et al. investigated the presence of a genetic modifier gene in this region in 136 sibpairs and parents, and confirmed linkage to this locus for meconium ileus but not CF pulmonary disease (Zielenski et al. 1999). The region defined by this study was >7Mb, therefore candidates for the modifier gene were too numerous (141) to analyse without further refinement of the locus (Drumm, 2001).

Several groups have investigated genes involved in innate and adaptive immunological defence and inflammation with respect to pulmonary CF disease modifiers. A polymorphism in the promoter region of the tumour necrosis factor α (TNF-α) gene, the TNF2 allele, encoding a pro-inflammatory cytokine, is associated with increased levels of TNF-α transcription and has also been shown to be associated with more severe CF pulmonary disease (Hull & Thomson, 1998). Polymorphisms in the transforming growth factor β (TGF-β) gene, associated with proliferation of fibroblasts, have also been investigated in modifying CF pulmonary disease (Arkwright et al. 2000). This study showed that a mutation in exon 10 of TGF-β was associated with a more rapid decline in lung function. Conversely, an enhancer polymorphism in the α1-antitrypsin (AAT) gene, a protease inhibitor that modulates elastase activity, has been associated with less severe pulmonary disease (Mahadeva et al. 1998; Henry et al. 2001; Sobczyqska-Tomaszeska 2002). Other reports have questioned the validity of the TGF-β and AAT results (reviewed by Salvatore et al. 2002), and stress the need for large samples sizes in these studies. Variant alleles of mannose-binding lectin (MBL, a serum protein involved in innate immunity) have also been shown to affect the severity of CF lung disease (Garred et al. 1999). The presence of variant MBL alleles that reduce serum MBL concentration is associated with poor prognosis and early death in CF patients, perhaps due to defective protection against bacterial and viral infections.

Associated Disorders

CBAVD Individuals with congenital bilateral absence of the vas deferens (CBAVD) frequently carry mutations in the CFTR gene but show no classical CF clinical phenotype. CBAVD prevents the transport of spermatozoa from testicular or epididymal structures to the vas deferens, resulting in infertility. Approximately 70% of CBAVD patients have one known CFTR mutation and 10% of patients possess two. The proportion of CBAVD patients carrying the T5 allele in CFTR intron 8 is 4 to 6-fold higher than among the normal or CF population (Zielenski et al. 1995; Zielenski & Tsui, 1995). CBAVD patients are frequently compound heterozygotes for a known disease-associated CFTR mutation and the T5 allele. In some cases, the proportion of exon 9CFTR transcripts is increased in the adult vas deferens compared to nasal epithelial cells of the same individual (Teng et al. 1997). A mild CFTR mutation may then become a disease-causing mutation in the vas deferens of CBVAD patients due to the overall decrease in full-length CFTR mRNA. However, CBAVD patients have no pulmonary disease, possibly due to amounts of functional CFTR mRNA exceeding the necessary ‘threshold’ transcript level for a non-CF phenotype in these tissues. Cuppens et al. investigated the prevalence of natural polymorphisms in the CFTR gene in combination with the T5 allele, and suggested that the presence of a combination of particular alleles at several polymorphic loci affects the quantity/quality of CFTR, resulting in a partial CF phenotype (Cuppens et al. 1998).

Disseminated bronchiectasis Mutations in the CFTR gene are also thought to be involved in other obstructive pulmonary diseases besides CF. In a study of 16 patients with disseminated bronchiectasis (an obstructive pulmonary disorder involving morphological abnormalities that is associated with childhood lung infections and with some genetic disorders and immunodeficiencies), the frequency of the intron 8 T5 allele was significantly increased when compared to a control population (Pignatti et al. 1996). 56% of patients analysed carried the T5 allele or an alternative CFTR mutation. In a similar study 32 disseminated bronchiectasis patients were analysed and 13 CFTR mutations were detected (Girodon et al. 1997).

Pancreatitis This disorder involves atrophy of pancreatic acinar tissue, fibrosis and inflammation. A number of studies have shown an increased incidence of CFTR mutations in patients with pancreatitis. In a study of 134 patients with chronic pancreatitis, ∼14% carried CFTR mutations on one allele and 10% had the T5 allele (twice the expected frequency) (Sharer et al. 1998). A recent study, however, showed that the frequency of the T5 allele was not observed to be higher in patients with pancreatitis than in the general population (Malats et al. 2001). Therefore the relevance of the T5 polymorphism in this disorder remains unclear and it is likely that both environmental and genetic factors are involved. However, of 27 patients suffering from idiopathic chronic pancreatitis (ICP), 37% carried at least one abnormal CFTR allele (Cohn et al. 1998). Furthermore, in two recent studies of 39 and 20 ICP patients (Audrezet et al. 2002 and Ockenga et al. 2000 respectively) at least 30% of patients carried CFTR mutations, demonstrating that CFTR mutations are associated with idiopathic chronic pancreatitis.

Other disorders Chronic rhinosinusitis (CRS), the inflammation of the sinus epithelium, is a consistent feature of CF. A panel of 147 individuals with CRS were screened for 16 common CFTR mutations and 10 individuals (7%) were identified with mutations in this gene, significantly higher than the 2% found in control individuals (Wang et al. 2000). 9 out of 10 CRS individuals with CFTR mutations also carried the M470V variant that results in a chloride channel with reduced activity, suggesting that these individuals may be predisposed to CRS. The amino acid variant 470M/V (methionine or valine at position 470) is found in both CF patients and non-CF individuals. Studies have shown that the M470 variant has a two-fold increased chloride channel activity compared to V470. 15 missense mutations in the CFTR gene were also detected in a study of 144 patients with asthma (Lazaro et al. 1999). The percentage (8.3%) of asthma patients with CFTR mutations was higher than in the control sample of 184 individuals from the general population (6%), but it was not statistically significant. The M470 variant was also detected in 90% of asthma patients with CFTR mutations but only 63% without.

Concluding Remarks

  1. Top of page
  2. Summary
  3. Cystic Fibrosis
  4. CFTR Gene Structure and Organisation
  5. CFTR Mutations
  6. Genotype/Phenotype Correlation
  7. Concluding Remarks
  8. References

CF is a disorder that results in a complex spectrum of disease phenotypes. The amount of CFTR required by each organ involved in the disease to remain phenotypically ‘normal’ varies and, similarly, the extent to which each organ contributes to the CF phenotype varies substantially. The type of mutation in the CFTR gene is clearly directly linked to CF phenotype in the case of pancreatic function, whereas CFTR mutations have a more variable impact on pulmonary phenotype where modifier genes and environmental effects also contribute to disease severity. The identification of a gene involved in modifying the effects of CFTR mutations with respect to meconium ileus on mouse chromosome 7 led to the detection of a region located on human chromosome 19 containing potential modifier genes. Further analysis of this locus, and perhaps others, is required to identify the genes responsible for such modulation. The identification of environmental modifiers of CF is a more difficult task due to the potentially large number of factors. Smoking in the home and patient to patient transmission of virulent bacterial pathogens are just two such factors that have been suggested to affect the severity of CF disease. Much more remains to be elucidated before we will know the full story regarding the interaction of CFTR, other genes and the environment, and therefore be better equipped to treat the disease.

Note in proof.  The authors wish to point out that this Review was accepted for publication in August 2002 and thus does not take into account data published since then.

Footnotes
  1. 1Class VI may be re-named Class V following the re-classification by Welsh (2001).

References

  1. Top of page
  2. Summary
  3. Cystic Fibrosis
  4. CFTR Gene Structure and Organisation
  5. CFTR Mutations
  6. Genotype/Phenotype Correlation
  7. Concluding Remarks
  8. References
  • Abramowicz, M. J., Dessars, B., Sevens, C., Goossens, M. & Boulandet, E. G. 2000. Fetal bowel hyperechogenicity may indicate mild atypical cystic fibrosis: a case associated with a complex CFTR allele. J Med Genet 37, E15.
  • Akabas, M. H. 2000. Cystic fibrosis transmembrane conductance regulator. Structure and function of an epithelial chloride channel [In Process Citation]. J Biol Chem 275, 372932.
  • Anand, R., Ogilvie, D. J., R. Butler, Riley, J. H., Finniear, R. S., Powell, S. J., Smith, J. C. & Markham, A. F. 1991. A yeast artificial chromosome contig encompassing the cystic fibrosis locus. Genomics 9, 12430.
  • Arkwright, P. D., Laurie, S., Super, M., Pravica, V., Schwarz, M. J., Webb, A. K. & Hutchinson, I. V. 2000. TGF-beta(1) genotype and accelerated decline in lung function of patients with cystic fibrosis. Thorax 55, 45962.
  • Audrezet, M. P., Chen, J. M., Le Marechal, C., Ruszniewski, P., Robaszkiewicz, M., Raguenes, O., Quere, I., Scotet, V. & Ferec, C. 2002. Determination of the relative contribution of three genes-the cystic fibrosis transmembrane conductance regulator gene, the cationic trypsinogen gene, and the pancreatic secretory trypsin inhibitor gene-to the etiology of idiopathic chronic pancreatitis. Eur J Hum Genet 10, 1006.
  • Borgo, G., Cabrini, G., Mastella, G., Ronchetto, P., Devoto, M. & Romeo, G. 1993. Phenotypic intrafamilial heterogeneity in cystic fibrosis. Clin Genet 44, 489.
  • Bronsveld, I., Mekus, F., Bijman, J., Ballmann, M., De Jonge, H. R., Laabs, U., Halley, D. J., Ellemunter, H., Mastella, G., Thomas, S., Veeze, H. J. & Tummler, B. 2001. Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings. J Clin Invest 108, 170515.
  • Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M. & Baralle, F. E. 2001. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. Embo J 20, 177484.
  • Cartegni, L., Chew, S. L. & Krainer, A. R..2002. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3, 28598.
  • Casals, T., Ramos, M. D., Gimenez, J., Larriba, S., Nunes, V. & Estivill, X. 1997. High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes. Hum Genet 101, 36570.
  • Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O'Riordan, C. R. & Smith, A. E. 1990. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 82734.
  • Chiba-Falek, O., Kerem, E., Shoshani, T., Aviram, M., Augarten, A., Bentur, L., Tal, A., Tullis, E., Rahat, A. & Kerem, B. 1998. The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10 kb C [RIGHTWARDS ARROW] T mutation. Genomics 53, 27683.
  • Choi, J. Y., Muallem, D., Kiselyov, K., Lee, M. G., Thomas, P. J. & Muallem, S. 2001. Aberrant CFTR-dependent HCO3-transport in mutations associated with cystic fibrosis. Nature 410, 947.
  • Chu, C. S., Trapnell, B. C., Curristin, S., Cutting, G. R. & Crystal, R. G. 1993. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet 3, 1516.
  • Chu, C. S., Trapnell, B. C., Murtagh, J. J., Moss, J. Jr., Dalemans, W. Jallat, S., Mercenier, A., Pavirani, A., Lecocq, J. P., Cutting, G. R. & et al 1991. Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium. Embo J 10, 135563.
  • Clain, J., Fritsch, J., Lehmann-Che, J., Bali, M., Arous, N., Goossens, M., Edelman, A. & Fanen, P. 2001. Two mild cystic fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue important for cystic fibrosis transmembrane conductance regulator processing and function. J Biol Chem 276, 90459.
  • Clancy, J. P., Bebok, Z., Ruiz, F., King, C., Jones, J., Walker, L., Greer, H., Hong, J., Wing, L., Macaluso, M., Lyrene, R., Sorscher, E. J. & Bedwell, D. M. 2001. Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis. Am J Respir Crit Care Med 163, 168392.
  • Cohn, J. A., Friedman, K. J., Noone, P. G., Knowles, M. R., Silverman, L. M. & Jowell, P. S. 1998. Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis. N Engl J Med 339, 6538.
  • Cuppens, H., Lin, W., Jaspers, M., Costes, B., Teng, H., Vankeerberghen, A., Jorissen, M., Droogmans, G., Reynaert, I., Goossens, M., Nilius, B. & Cassiman, J. J. 1998. Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J Clin Invest 101, 48796.
  • Cystic Fibrosis Genetic Analysis Consortium 1994. Population variation of common cystic fibrosis mutations. Hum Mut 16777
  • De Braekeleer, M., Allard, C., Leblanc, J. P., Simard, F. & Aubin, G. 1997. Genotype-phenotype correlation in cystic fibrosis patients compound heterozygous for the A455E mutation. Hum Genet 101, 20811.
  • Dork, T., Wulbrand, U., Richter, T., Neumann, T., Wolfes, H., Wulf, B., Maass, G. & Tummler, B. 1991. Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene. Hum Genet 87, 4416.
  • Drumm, M. 2001. Modifier genes and variation in cystic fibrosis. Respir Res 2, 125128.
  • Ellsworth, R. E., Jamison, D. C., Touchman, J. W., Chissoe, S. L., Braden Maduro, V. V., Bouffard, G. G., Dietrich, N. L., Beckstrom-Sternberg, S. M., Iyer, L. M., Weintraub, L. A., Cotton, M., Courtney, L., Edwards, J., Maupin, R., Ozersky, P., Rohlfing, T., Wohldmann, P., Miner, T., Kemp, K., Kramer, J., Korf, I., Pepin, K., Antonacci-Fulton, L., Fulton, R. S., Green, D. & et al 2000. Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes. Proc Natl Acad Sci U S A 97, 11727.
  • Estivill, X., Ortigosa, L., Perez-Frias, J., Dapena, J., Ferrer, J., Pena, L., Llevadot, R., Gimenez, J., Nunes, V. & et al 1995. Clinical characteristics of 16 cystic fibrosis patients with the missense mutation R334W, a pancreatic insufficiency mutation with variable age of onset and interfamilial clinical differences. Hum Genet 95, 3316.
  • Gabriel, S. E., Brigman, K. N., Koller, B. H., Boucher, R. C. & Stutts, M. J. 1994. Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. Science 266, 1079.
  • Garred, P., Pressler, T., Madsen, H. O., Frederiksen, B., Svejgaard, A., Hoiby, N., Schwartz, M. & Koch, C. 1999. Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis. J Clin Invest 104, 4317.
  • Girodon, E., Cazeneuve, C., Lebargy, F., Chinet, T., Costes, B., Ghanem, N., Martin, J., Lemay, S., Scheid, P., Housset, B., Bignon, J. & Goossens, M. 1997. CFTR gene mutations in adults with disseminated bronchiectasis. Eur J Hum Genet 5, 14955.
  • Green, E. D. & Olson, M. V. 1990. Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genome mapping. Science 250, 948.
  • Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N. & Lukacs, G. L. 1999. C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation. J Biol Chem 274, 218737.
  • Hefferon, T. W., Broackes-Carter, F. C., Harris, A. & Cutting, G. R. 2002. Atypical 5' Splice Sites Cause CFTR Exon 9 To Be Vulnerable to Skipping. Am J Hum Genet 71, 294303.
  • Henry, M. T., Cave, S., Rendall, J., O'Connor, C. M., Morgan, K., FitzGerald, M. X. & Kalsheker, N. 2001. An alpha1-antitrypsin enhancer polymorphism is a genetic modifier of pulmonary outcome in cystic fibrosis. Eur J Hum Genet 9, 2738.
  • Hentze, M. W. & Kulozik, A. E. 1999. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 96, 30710.
  • Higgins, C. F. 1992. ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8, 67113.
  • Highsmith, W. E., Burch, L. H., Zhou, Z., Olsen, J. C., Boat, T. E., Spock, A., Gorvoy, J. D., Quittel, L., Friedman, K. J., Silverman, L. M. & et al 1994 A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N Engl J Med 331, 97480.
  • Hogenauer, C., Santa Ana, C. A., Porter, J. L., Millard, M., Gelfand, A., Rosenblatt, R. L., Prestidge, C. B. & Fordtran, J. S. 2000. Active intestinal chloride secretion in human carriers of cystic fibrosis mutations: an evaluation of the hypothesis that heterozygotes have subnormal active intestinal chloride secretion. Am J Hum Genet 67, 14227.
  • Howard, M., Frizzell, R. A. & Bedwell, D. M. 1996. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nat Med 2, 4679.
  • Hull, J., Shackleton, S. & Harris, A. 1993. Abnormal mRNA splicing resulting from three different mutations in the CFTR gene. Hum Mol Genet 2, 68992.
  • Hull, J. & Thomson, A. H. 1998. Contribution of genetic factors other than CFTR to disease severity in cystic fibrosis. Thorax 53, 101821.
  • Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F. & Kim, S. H. 1998. Crystal structure of the ATP-binding subunit of an ABC transporter [see comments]. Nature 396, 7037.
  • Kalin, N., Claass, A., Sommer, M., Puchelle, E. & Tummler, B. 1999. DeltaF508 CFTR protein expression in tissues from patients with cystic fibrosis. J Clin Invest 103, 137989.
  • Kartner, N., Augustinas, O., Jensen, T. J., Naismith, A. L. & Riordan, J. R. 1992. Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland. Nat Genet 1, 3217.
  • Kerem, B., Rommens, J. M., Buchanan, J. A., Markiewicz, D., Cox, K., Chakravarti, A., Buchwald, M. & Tsui, L.C. 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245, 107380.
  • Kiesewetter, S., Macek, M., Davis, C. Jr, Curristin, S. M., Chu, C. S., Graham, C., Shrimpton, A. E., Cashman, S. M., Tsui, L. C., Mickle, J. & et al. 1993. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet 5, 2748.
  • Lazaro, C., De Cid, R., Sunyer, J., Soriano, J., Gimenez, J., Alvarez, M., Casals, T., Anto, J. M. & Estivill, X. 1999. Missense mutations in the cystic fibrosis gene in adult patients with asthma. Hum Mutat 14, 5109.
  • Mahadeva, R., Westerbeek, R. C., Perry, D. J., Lovegrove, J. U., Whitehouse, D. B., Carroll, N. R., Ross-Russell, R. I, Webb, A. K., Bilton, D. & Lomas, D. A. 1998. Alpha1-antitrypsin deficiency alleles and the Taq-I G [RIGHTWARDS ARROW] A allele in cystic fibrosis lung disease. Eur Respir J 11, 8739.
  • Malats, N., Casals, T., Porta, M., Guarner, L., Estivill, X. & Real, F. X. 2001. Cystic fibrosis transmembrane regulator (CFTR) DeltaF508 mutation and 5T allele in patients with chronic pancreatitis and exocrine pancreatic cancer. PANKRAS II Study Group. Gut 48, 704.
  • Mateu, E. C. F., Ramos, M. D., Casals, T. & Bertranpetit, J. 2002. Can a place of origin of the main cystic fibrosis mutations be identified? Am J Hum Genet 70, 25764.
  • Niksic, M., Romano, M., Buratti, E., Pagani, F. & Baralle, F. E. 1999. Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9 [In Process Citation]. Hum Mol Genet 8, 233949.
  • Ockenga, J., Stuhrmann, M., Ballmann, M., Teich, N., Keim, V., Dork, T. & Manns, M. P. 2000. Mutations of the cystic fibrosis gene, but not cationic trypsinogen gene, are associated with recurrent or chronic idiopathic pancreatitis. Am J Gastroenterol 95, 20617.
    Direct Link:
  • Onay, T., Topaloglu, O., Zielenski, J., Gokgoz, N., Kayserili, H., Camcioglu, Y., Cokugras, H., Akcakaya, N., Apak, M., Tsui, L. C. & Kirdar, B. 1998. Analysis of the CFTR gene in Turkish cystic fibrosis patients: identification of three novel mutations (3172delAC, P1013L and M1028I). Hum Genet 102, 22430.
  • Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D. & Baralle, F. E. 2000. Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element. J Biol Chem 275, 210417.
  • Pier, G. B., Grout, M., Zaidi, T., Meluleni, G., Mueschenborn, S. S., Banting, G., Ratcliff, R., Evans, M. J. & Colledge, W. H. 1998. Salmonella typhi uses CFTR to enter intestinal epithelial cells. Nature 393, 7982.
  • Pignatti, P. F., Bombieri, C., Benetazzo, M., Casartelli, A., Trabetti, E., Gile, L. S., Martinati, L. C., Boner, A. L. & Luisetti, M. 1996. CFTR gene variant IVS8–5T in disseminated bronchiectasis. Am J Hum Genet 58, 889892.
  • Popa, I., Pop, L., Popa, Z., Schwarz, M. J., Hambleton, G., Malone, G. M., Haworth, A. & Super, M. 1997. Cystic fibrosis mutations in Romania. Eur J Pediatr 156, 2123.
  • Rave-Harel, N., Kerem, E., Nissim-Rafinia, M., Madjar, I., Goshen, R., Augarten, A., Rahat, A., Hurwitz, A., Darvasi, A. & Kerem, B. 1997. The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. Am J Hum Genet 60, 8794.
  • Rich, D. P., Anderson, M. P., Gregory, R. J., Cheng, S. H., Paul, S., Jefferson, D. M., McCann, J. D., Klinger, K. W., Smith, A. E. & Welsh, M. J. 1990. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells [see comments]. Nature 347, 35863.
  • Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J. L. & et al 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA [published erratum appears in Science 1989 Sep 29;245(4925):1437]. Science 245, 106673.
  • Romey, M. C., Guittard, C., Carles, S., Demaille, J., Claustres, M. & Ramsay, M. 1999. First putative sequence alterations in the minimal CFTR promoter region [letter]. J Med Genet 36, 2634.
  • Romey, M. C., Pallares-Ruiz, N., Mange, A., Mettling, C., Peytavi, R., Demaille, J. & Claustres, M. 2000. A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression. J Biol Chem 275, 35617.
  • Rowntree, R. & Harris. A. 2002. DNA polymorphisms in potential regulatory elements of the CFTR gene alter transcription factor binding. Hum Genet 111, 6674.
  • Rozmahel, R., Wilschanski, M., Matin, A., Plyte, S., Oliver, M., Auerbach, W., Moore, A., Forstner, J., Durie, P., Nadeau, J., Bear, C. & Tsui, L. C. 1996. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nat Genet 12, 2807.
  • Salvatore, F., Scudiero, O. & Castaldo, G. 2002. Genotype-phenotype correlation in cystic fibrosis: The role of modifier genes. Am J Med Genet 111, 8895.
  • Schwiebert, E. M., Benos, D. J., Egan, M. E., Stutts, M. J. & Guggino, W. B. 1999. CFTR is a conductance regulator as well as a chloride channel. Physiol Rev 79, S14566.
  • Sharer, N., Schwarz, M., Malone, G., Howarth, A., Painter, J., Super, M. & Braganza, J. 1998. Mutations of the cystic fibrosis gene in patients with chronic pancreatitis. N Engl J Med 339, 64552.
  • Sheppard, D. N., Rich, D. P., Ostedgaard, L. S., Gregory, R. J., Smith, A. E. & Welsh, M. J. 1993. Mutations in CFTR associated with mild-disease-form Cl- channels with altered pore properties. Nature 362, 1604.
  • Shoshani, T., Augarten, A., Gazit, E., Bashan, N., Yahav, Y., Rivlin, Y., Tal, A., Seret, H., Yaar, L., Kerem, E. & et al. 1992. Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease. Am J Hum Genet 50, 2228.
  • Sobczyqska-Tomaszeska, A., Sands, D., Nowakowska, A. & Bal, J. 2002. The 1237G-A enhancer polymorphism of alpha-1 antitrypsin gene as modifier of pulmonary disease in cystic fibrosis patients. Journal of Cystic Fibrosis 1, S8687.
  • Teng, H., Jorissen, M., Van Poppel, H., Legius, E., Cassiman, J. J. & Cuppens, H. 1997. Increased proportion of exon 9 alternatively spliced CFTR transcripts in vas deferens compared with nasal epithelial cells. Hum Mol Genet 6, 8590.
  • Tsui, L. C. 1992. The spectrum of cystic fibrosis mutations. Trends Genet 8, 3928.
  • Tzetis, M., Efthymiadou, A., Doudounakis, S. & Kanavakis, E. 2001. Qualitative and quantitative analysis of mRNA associated with four putative splicing mutations (621+3A [RIGHTWARDS ARROW] G, 2751+2T [RIGHTWARDS ARROW] A, 296+1G [RIGHTWARDS ARROW] C, 1717–9T [RIGHTWARDS ARROW] C-D565G) and one nonsense mutation (E822X) in the CFTR gene. Hum Genet 109, 592601.
  • Vankeerberghen, A., Wei, L., Jaspers, M., Cassiman, J. J., Nilius, B. & Cuppens, H. 1998a. Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator. Hum Mol Genet 7, 17619.
  • Vankeerberghen, A., Wei, L., Teng, H., Jaspers, M., Cassiman, J. J., Nilius, B. & Cuppens, H. 1998b. Characterization of mutations located in exon 18 of the CFTR gene. FEBS Lett 437, 14.
  • Wang, X., Moylan, B., Leopold, D. A., Kim, J., Rubenstein, R. C., Togias, A., Proud, D., Zeitlin, P. L. & Cutting, G. R. 2000. Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population. Jama 284, 18149.
  • Welsh, M., Ramsey, B. W., Accurso, F. & Cutting, G. R. 2001. Cystic Fibrosis. In The Molecular and Metabolic Basis of Inherited Disease (ed. A. B.CL Scriver, W. S.Sly & D.Valle), pp. 51215188. McGraw-Hill, New York .
  • Zielenski, J. 2000. Genotype and phenotype in cystic fibrosis. Respiration 67, 11733.
  • Zielenski, J., Corey, M., Rozmahel, R., Markiewicz, D., Aznarez, I., Casals, T., Larriba, S., Mercier, B., Cutting, G. R., Krebsova, A., Macek, M. Jr, Langfelder-Schwind, E., Marshall, B. C., DeCelie-Germana, J., Claustres, M., Palacio, A., Bal, J., Nowakowska, A., Ferec, C., Estivill, X., Durie, P. & Tsui, L. C. 1999. Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13 [letter]. Nat Genet 22, 1289.
  • Zielenski, J., Patrizio, P., Corey, M., Handelin, B., Markiewicz, D., Asch, R. & Tsui, L. C. 1995. CFTR gene variant for patients with congenital absence of vas deferens. Am J Hum Genet 57, 95860.
  • Zielenski, J., Rozmahel, R., Bozon, D., Kerem, B., Grzelczak, Z., Riordan, J. R., Rommens, J. & Tsui, L. C. 1991. Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics 10, 21428.
  • Zielenski, J. & Tsui, L. C. 1995. Cystic fibrosis: genotypic and phenotypic variations. Annu Rev Genet 29, 777807.