BmM Medical Bulletin (1992) Vol 48, No 4, pp738-753 O The Brmih Camcfl 1992

Cystic fibrosis gene A Harris Paediatric Molecular Genetics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

The cystic fibrosis gene, located at 7q31, spans about 230 kb of genomic DNA and contains 27 exons. The cDNA of 6.2kb would predict an 1480 amino acid protein, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR has a high degree of homology with members of the ABC-transporter super family. The predicted protein structure consists of two membrane-spanning domains, each of 6 sub-units, anchoring CFTR in the apical membrane of specialized epithelial cells, 2 nucleotide binding folds (NBF) and a regulatory (R) domain. Disease-associated mutations in the CF gene are mainly clustered in the nucleotide-binding folds. The most common mutation, occurring in 70% of CF genes in Northern Europe and North America, is the deletion of amino acid phenylalanine at position 508 in the first NBF (ie AF508).

The isolation of the Cystic Fibrosis (CF) gene in 1989 by the groups of Lap-Chee Tsui in Toronto, Canada, and Francis Collins in Michigan, USA was the culmination of more than 5 years work by various research groups to move from linked DNA markers to the locus itself.1"3 The final success of this tour de force of reverse genetics relied on the availability of a cellular expression system for the CF gene (namely sweat gland duct epithelial cells) and on the combined techniques of chromosome walking and jumping. Isolation of the gene opened a new and exciting chapter in CF research. It was now possible to start asking and answering fundamental questions about the molecular basis of the disease. STRUCTURE OF THE GENE The CF gene is large, spanning about 230kb of genomic DNA at chromosome 7q31. The locus has been extensively analysed by chromosome walking and jumping from flanking, linked polymorphic markers on



genomic DNA1- 2 by pulsed-field gel mapping,4-6 and also by isolation of yeast artificial chromosomes (YACS).7> 8 Coding region The coding region of Ch'lR is made up of 27 exons, numbered 1 to 24, but including 6a and 6b, 14a and 14b, 17a and 17b (Fig. 1). Most of these are between 50 and 250bp {see Table 1) with the exception of exon 13 which spans 723bp of genomic DNA.

Tabte 1 CFTR exon sizes Exon


1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 17a 17b 18 19 20 21 22 23 24

121 186 297 406 622 712 876 1002 1249 1342 1524 1717 1812 1899 2622 2752 2789 3041 3121 3271 3500 3599 3850 4006 4096 4269 4375

Size bp

65 111 109 116 90 164 126 247 93 182 193 95 87 723 130 37 52 80 150 229 99 151 156 90 173 106 198

The CF gene product has been named the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), on the basis of its predicted structure. The 1480 amino acid protein predicted from the 6.2kb cDNA sequence would appear to be a member of the ABC-transporter superfamily {see Higgins, this issue). It is made up (Fig. 2) of 2 very similar halves that each contain a nucleotide-binding fold (NBF), and

12 3


5 6a 6b




10 1112


14a14b 15 1617a 17b 18


20 2 1 2 2 23 24



UU Membrane spanning

I U UU UU ATPbinding


Membrane spanning


Fig. 1 Organization of CFTR cDNA to show location of exons relative to predicted functional domains



a membrane-spanning domain, the two halves being separated by the 'regulatory (R) domain'. The R domain, coded for largely by a single exon, (exon 13) contains multiple predicted substrate binding sites for protein kinase A and C, and is a unique feature of CFTR that is not found in the other ABC transporters.




Fig. 2 Diagram to show predicted structure of CFTR protein MSD = membrane spanning domain NBF = nucelotide binding fold

Flanking regions The 5' and 3' untranslated regions are 134 and 1556bp respectively. Sequence analysis of the lkb of DNA immediately 5' to the CF gene shows that it includes a GC-rich region but does not contain a TATA element. Further, there are several putative regulatory signals including 2 consensus GC boxes (GGGCGG) and 3 potential API sites, that may be sites of regulation by phorbol esters.9 In this respect the CF gene shows certain features of house-keeping genes, that is genes that are uniformly expressed at similar levels in all cell types.9 However, the CF gene is clearly not a house-keeping gene by any functional criteria since its expression is highly tissue specific {see below).



There appear to be at least 3 transcription initiation sites, two positive and one negative regulatory elements 5' to the gene.10- M LOCALIZATION OF EXPRESSION The data on expression of CFTR has accumulated both by detection of CFTR mRNA and CFTR protein. The mRNA detection methods include in situ hybridization, Northern blot analysis and RT-PCR (in which mRNA is converted to cDNA by reverse transcription and the cDNA then amplified by the polymerase chain reaction, PCR). CFTR protein has been localized by a variety of polyclonal antisera and monoclonal antibodies raised against CFTR, either by using CFTR peptides or CFTR fusion proteins as immunogens. Expression of the CF gene in readily detectable amounts appears to be restricted to epithelia lining certain organs in vivo. The majority of these sites are ductal, for example the pancreatic ducts, the sweat gland ducts, the male genital ducts and the kidney tubules, both proximal and distal.2" x2< 13 CFTR gene expression is also found in parts of the lung epithelium and in the jejunum and the colon. Interestingly localization studies with antibodies to CFTR suggest that expression of the protein in the respiratory epithelium may be low.12 However, since there is undoubtedly CFTR transcription in parts of the lung epithelium2' 13 it is possible that failure to see the protein is due to lack of antibody binding rather than lack of the protein itself. Alternatively certain restricted cell types alone may express CFTR protein within the lung. In human fetal development CFTR mRNA is already expressed at substantial levels in the pancreas, genital ducts, lung and gut by 18 weeks.13 The data on CFTR localization in human tissues has been added to by an elegant in situ analysis of CFTR expression in the rat14 that shows essentially similar results. However, variation in levels of CFTR expression in the uterine wall during the rat oestrous cycle14 and in the testis during spermatogenesis15, has added to unanswered questions in human tissues. GENE FUNCTION From the results of recent experiments in which full length CFTR cDNAs were introduced into both insect and mammalian cells that did not normally express apical membrane chloride ion channels, it seems probable that CFTR is a specific small chloride ion channel.16- 17 Reconstitution experiments, placing CFTR protein in a lipid bilayer and assaying its function confirmed this. 17a This channel had previously been well defined in rat and human pancreatic duct epithelial cells, 18 - 20



that are known to express high levels of Chi K.13- 21 It remains possible that CFTR has an additional function in vivo (see Higgins, this issue). Mutations One of the best examples of international collaboration in human genetic disease research is the Cystic Fibrosis Genetic Analysis Consortium, chaired by Dr Lap-Chee Tsui. Members of the consortium are individuals/laboratories who are making a contribution either to the definition of novel mutations in the CF gene OF to the population genetics of different CF mutations. To date more than 200 have been described (July 1992). The vast majority of these have only been defined in 1 or a few patients, which represents a screening headache. Most types of mutations have been detected within the gene: missense mutations, single base substitutions resulting in alterations of a single amino acid; nonsense mutations leading to 'stop' signals; frame-shift mutations; splice mutations; and small insertions or deletions. However, to date no major deletions of large parts of the gene have been described. At this stage, many of the predicted mutations have only been detected at the DNA level. It is possible that some of them may be polymorphisms rather than disease-causing mutations. In addition some genuine mutations may have been missed due to the method by which many laboratories are defining mutations. In other words, once the common mutations have been screened for in a population, the uncharacterized chromosomes are then subjected to analysis of individual exons until an error is found. The chromosome is then excluded from further study. In addition some mutations may not cause the actual effect on the mRNA or protein that would be predicted from the change at the DNA level. In the following review of mutations that have been defined in CFTR an attempt will be made to provide a functionally relevant overview against a background of continually accumulating mutation data. Only mutations that have been reported in more than 50 independent chromosomes will be discussed (data from the Cystic Fibrosis Genetic Analysis Consortium). METHODS OF DETECTING MUTATIONS IN THE CF GENE As with mutation detection in all other disease-associated genes a range of techniques are being employed, either analysing genomic DNA or mRNA. Since the CF gene encompasses at least 230kb of genomic DNA7 the task of mutation definition is enormous. Clearly it is not feasible to



analyse 230kb of genomic DNA for each mutant gene, particularly for a locus that does not exhibit frequent major deletions or structural rearrangements (as are seen in many X-linked genes such as dystrophin and the COL4A5 component of basement membrane collagen). Loci, such as these, are amenable to analysis by pulsed field gel electrophoresis. It may still be useful to apply this technique to a small subset of mutant CF genes. Mutations in the CF gene have primarily been analysed by the polymerase chain reaction (PCR) amplification22 of exons and 100-200bp of flanking DNA, followed by use of a method of detecting any mutations within the amplified segment The most laborious means of detecting mutations is to sequence the whole amplified segment. However, many techniques have been developed to attempt to find errors at the level of single base changes within the PCR product, by more rapid means. Single-stranded conformational polymorphism (SSCP) analysis This technique relies on the slightly different electrophoretic mobilities of 2 fragments of DNA that are of exactly the same size but differ in base composition at one or more sites.23 PCR amplification of the fragment of interest is carried out using one or more radioactive nucleotides. This yields a PCR product that can be visualized by autoradiography following denaturation and electrophoresis on an acrylamide gel. Several mutations in CFTR have been found by this method.24 Denaturing gradient gel electrophoresis (DGGE) DGGE exploits the different melting properties of fragments of DNA that have a variant base composition.25 As for SSCP, this method is able to detect single base changes in PCR products from CF patient DNA. Usually, one of the two PCR primers carries a GC clamp, that is a GC-rich domain, at its 5' end to create a high-temperature melting domain adjacent to the region being analysed. This ensures that the test region is the first melting domain. This technique has been successfully applied to the detection of mutations in CFTR by several groups.26- 27 Chemical mismatch Here detection of mutations relies on the formation of a hybrid between control DNA and patient DNA, followed by chemical modification of any single stranded bases arising from mutations in patient DNA causing a mismatch in the hybrid.28 Chemically modified bases (C residues being modified by hydroxylamine and T residues by osmium tetroxide)



are then cleaved by piperidine and fragments separated by acrylamide gel electrophoresis. The technique involves end-labelling a PCR product from control or patient DNAs in order to be able to visualize products of modification. It is sometimes referred to as amplification and mismatch detection (AMD).29 Here again AMD has proven useful in detecting mutations in CFTR.30' 31 In all cases where single base changes or other mutations have been detected in CFTR by one of the three methods described above, they are subsequently confirmed by sequence analysis. In this respect the chemical mismatch method has a major advantage over SSCP and DGGE since it provides accurate information on the location of the mutation, rather than necessitating sequence analysis of the whole PCR product under test. Analysis of CFTR mRNA to detect mutations One of the problems in using CFTR mRNA as a resource for mutation detection is the lack of availability of tissues that are expressing significant amounts of CFTR.2' 12- l 3 The gene is transcribed at a readily detectable level in nasal epithelial cells and since these are frequently obtainable by nasal brushing, several groups have begun to define mutations in the CFTR mRNA in these cells. Clearly the success of this approach is dependent on the relative abundance and stability of mutant CFTR mRNA, particularly in the case of compound heterozygotes where the presence of a more abundant or more stable transcript from the other chromosome might mask the mutant transcript Nasal epithelial cell CFTR mRNA has contributed to the definition of mutations in CFTR.32 Another potential resource for analysing CFTR mRNA is peripheral blood lymphocytes. Though CFTR is not expressed in these cells at any physiological levels, by exploiting the phenomenon of 'ectopic' transcription of genes in lymphocytes, CFTR mRNA can be detected. This is achieved by nested PCR, using two sets of PCR primers, the second set being located inside the first, to amplify CFTR cDNA that has been reverse transcribed from total RNA. This phenomenon has been exploited successfully for several X-linked genes33' 3* and CFTR.35 However in the case of an autosomal locus such as the CF gene, the potential problems of mutant mRNA abundance and stability in compound heterozygotes are even more acute than in the case of nasal epithelial cells. This is due to the numerous cycles of PCR required to obtain adequate amounts of DNA for analysis.



MUTATIONS IN THE FIRST MEMBRANE SPANNING DOMAIN As is shown in Figure 1, the first 8 exons of the CFTR cDNA include the first membrane spanning domain (coded for by parts of exons 3, 4, 6a and 7). A review of mutations in this region24- ^ reveals clustering of mutations in exons 4 and 7, that is now unlikely to represent ascertainment bias. These include all types of mutations discussed above. In addition several predicted splicing mutations have been recorded at the exon/intron boundary at the 3' end of exons 4, 5 and 7. One of these, at the end of exon 4, 621+1G-T—> is a relatively frequently occurring mutation.36 The actual biological effect of these mutations remains to be established, partly due to the difficulty of assessing phenotypic effects and also due to the rarity of patients who are homozygous for the most mutations.30 However, in the case of mutations that would be predicted to alter the charge of amino acids within the membrane spanning domains, these might be expected to alter the selectivity of the chloride ion channel function of CFTR. Experiments designed at assessing the effect of mutations on CFTR function have been carried out in heterologous expression systems in which the mutant gene is greatly over-expressed. In these systems mutations associated with charge changes at certain residues within the membrane spanning domains did indeed alter selectivity of the chloride ion channel.37 MUTATIONS IN THE FIRST NUCLEOTIDE-BINDING FOLD The largest cluster of mutations within CFTR occurs in the first nucleotide binding fold (exons 9-12) 38 . 38a, 39 , particularly in exons 10 and 11. 70% of disease-associated mutations in CFTR in Northern Europe and North America involve the deletion of a phenylanaline residue at position 508 within exon 10.3 This mutation is referred to as AF508. From its location within the first NBF, it might be expected, on the basis of modelling studies,40 not to interfere with ATP-binding or hydrolysis. This has been confirmed by studies that suggest the AF5O8-bearing CFTR protein can indeed function as a small chloride ion channel41. 42 , though the characteristics of this channel, in terms of opening times, are altered. In experimental transfection systems43- ** a large proportion of AF508 CFTR protein remains intracellular, building up in the Golgi/endoplasmic reticulum. However, it is probable that at least part of this may be largely due to the massive overexpression of the protein. Recent data on AF5O8 epithelial cells would support this.45



Other relatively frequent mutations in the first NBF include deletion of the isoleucine residue adjacent to F508 (AI507)38- 38a {see Fig 4 in chapter by Super, this issue); substitution of the glycine at amino acid 551 by aspartic acid (G551D)39; 'stop' mutations at arginine 553 39 and glycine M2 4 6 substitutions of serine 549 by various residues38- 38a> 39 and a predicted splicing mutation at the start of exon 11 (1717-1 G->A).38 MUTATIONS IN THE REGULATORY DOMAIN The 723 bp exon 13, which codes for the R domain contains relatively few mutations compared to the rest of the CFTR cDNA and none of them are common. Of those described to date a high proportion are frameshift mutations47. ^ (CF genetic Analysis Consortium). MUTATIONS IN THE SECOND MEMBRANE-SPANNING DOMAIN The second membrane-spanning domain of CFTR is encoded by exons 14a and 14b, 15, 16, 17a and 17b and 18. With the exception of exon 17b very few mutations have been defined in this domain. Further the location of the exon 17b mutations do not correspond to any cluster of mutations in the equivalent position within the first membrane spanning domain. MUTATIONS IN THE SECOND NUCLEOTTDE-BINDING FOLD Like the first nucleotide-binding fold, the second NBF (exons 19—22) is also a frequent site for disease-associated mutations. In particular multiple mutations have been localized to exons 19 and 20. One of these is sufficiently common to warrant specific mention, it is a 'stop' mutation at amino acid 1282 (W1282X).46 Another common mutation in this region is found in exon 21, the substitution of aspargine 1303 by lysine (N1303K)/» IMPLICATIONS OF DISTRIBUTION OF MUTATIONS Clearly the analysis of mutations within CFTR has potential power in shedding light on functionally important regions of the CFTR protein. Such information may be implicated by naturally occurring mutations though clearly recreation of mutations in in vitro systems is also required. This is discussed further in the chapter by Higgins (this issue).



From the distribution of naturally occurring mutations within CFTR it would appear that most predicted disease-causing alterations that are compatible with survival are housed within the two nucleotide bindingfolds of the protein. This would indicate the functional importance of these regions in CFTR. In addition it is clear from cross-species comparisons50-54 that the two nucleotide-binding folds are the most highly conserved portions of the protein, further supporting their critical role in CFTR function. However, though the R domain shows the highest degree of divergence between mammalian species, most of the predicted substrate-binding sites for protein kinase A and C are conserved. Even in the dogfish homologue of CFTR54 there is substantial conservation of protein kinase A substrate binding sites (as well as most sites in the NBFs that when mutated in human CFTR cause disease).

PHENOTYPE: GENOTYPE CORRELATIONS? At this stage of population analyses it is a little early to make predictions of disease severity on the basis of the particular mutations carried by an individual with CF. However, with a few exceptions there does seem to be quite a good correlation between the AF508 mutation in a homozygous state and pancreatic insufficiency.55- 5(i Indeed this mutation is generally associated with relatively severe CF though the correlation is not complete and there are clearly many other factors involved. It has been reported that the G542X mutation when present in a homozygous form may lead to a milder form of CF than AF508 homozygotes or heterozygotes AF508/G542X.57 Similarly other patients with predicted 'stop' mutations in both CF genes may be less severely affected than AF508 homozygotes.58 In other words if the 'stop' mutation actually abolishes production of CFTR this may be less deleterious than having a malfunctioning protein such as the A508 protein. However this is becoming a controversial point as more mutants are examined.59- 6° Genetic counselling and CF mutations While the majority of CF mutations can be directly detected by simple DNA tests, there remain in all populations analysed a group of patients with undefined mutations. These families are counselled on the basis of either linked DNA markers or by using intra-genic repeat polymorphisms. Prior to the cloning of the CF gene most families segregating for CF were typed for polymorphic markers mat were closely linked to the CF gene.61 However, clearly this has the disadvantage of the potential recombination that may occur between the CF locus and the polymorphic site.



Further, some families remain uninformative at all potential linked polymorphic sites. However, the discovery of certain blocks of repeat units within introns of the CF gene, due to their high degree of informativeness, provides a powerful tool for following inheritance of mutant CF chromosomes even in the absence of precise mutation information. Such clusters of highly polymorphic dinucleotide repeats have been found in introns 6a62, 8 and 9 s 3 and 17b64, with the latter being particularly useful. At least 24 alleles with sizes ranging from 7 to 56 units of a TA repeat have been identified. POPULATION GENETICS AND CF MUTATIONS When the CF gene was first isolated 1 - 3 it was observed that the AF508 mutation was present on about 70% of CF chromosomes in northern Europe and North America.3- ^ Subsequent analysis of the European population showed a gradient of distribution, moving south and east across Europe.66 The frequency of the delta F508 mutation being much higher in Scandinavian countries for example than in Italy and Turkey. As mutation analysis in the CF gene has progressed it has become clear that certain genetic populations have a high frequency of mutations that are much rarer in other populations. This is clearly of importance when devising CF screening strategies for different genetic groups. Of particular note are the Ashkenazi Jewish population, which have a low frequency of the AF508 mutation but a much higher of the G542X37 and W1282X46' 6° mutations. In fact, by screening for these 3 mutations alone somewhat over 90% of CF mutations can be detected in this population.60 Other mutations that show a higher frequency in particular areas are R1162X6? (in exon 19), in Southern Europe^ and R553X« (located in exon 11 in the first NBF) in Germany.69 UNCERTAIN DIAGNOSIS OF CYSTIC FIBROSIS It is not unusual for a diagnosis of ?CF to be made in cases where an individual does not fulfil all the diagnostic criteria of this disease. In particular, this arises in cases where though lung symptoms are indicative of CF, sweat electrolytes are normal. Many of these individuals are analysed at the DNA level and several interesting results have been obtained. In one case two siblings with mild pulmonary disease and normal sweat electrolyte concentrations were found to be homozygous for a glycine to serine substitution at amino acid 551 in the first NBF of CFTR.31 Other patients with this phenotype were found to carry a



mutation in intron 19 that caused the insertion of 83bp into the CFTR mRNA32 or to be compound heterozygotes for other denned mutations in CFTR.™ Another interesting phenotype that may be associated with mutations in CFTR is that of congenital absence of the vas deferens. Though this syndrome is clearly distinct from CF, several affected individuals have been found to be heterozygous for the F5O8 deletion in CFTR.?1 Other mutations in CFTR have also been recorded in this syndrome. It is probable diat the clinical definition of cystic fibrosis may well have missed a percentage of patients who will remain undiagnosed in the absence of molecular analysis of their CF locus. REFERENCES 1 Rommens JM, lannnm MC, Kerem B-S et al. Identification of tbe cystic fibrosis gene chromosome walking and jumping Science 1989; 245. 1059—1065 2 Riordan JR, Rommens JM, Kerem B-S et al. Identificaiotn of the cystic fibrosis gene cloning and characterisation of complementary DNA. Science 1989; 245 1066-1073 3 Kerem B-S, Rommens JM, Buchanan JA et al. Identification of the cystic fibrosis gene genetic analysis. Science 1989, 245: 1073-1080 4 Drumm ML, Smith CL, Dean M, Cole JL, Iannuzzi MC, Collins FS. Physical mapping of the cystic fibrosis region by pulsed-field gel electrophoresis Genomics 1988, 2: 346-354 5. Fulton TR, Bowcock AM, Smith DR et al. A 12 megabase restriction map at the cystic fibrosis locus Nucehc Acids Res 1989, 17: 271-284 6 Poustka AM, Lehrach H, Williamson R, Bates G. A long-range restriction map encompassing the cystic fibrosis locus and its closely linked genetic markers. Genomics 1988; 2:337-345 7 Anand R, Ogilvie DJ, Butler R et al. A yeast artificial chromosome contig encompassing the cystic fibrosis locus. Genomics 1991; 9 124-130 8 Green ED, Olson MV Chromosomal region of the cystic fibrosis gene in yeast artificial chromosomes: a model for human genomic mapping Science 1990; 250: 94-98 9 Yoshimura K, Nakamura H, Trapnell BC et al The cystic fibrosis gene has a 'housekeeping'-type promoter and is expressed at low levels in cells of epithelial origin. J Biol Chem 1991; 266 9140-9144 10 Chou J-L, Rozmahel R, Tsui L-C Characterization of the promoter region of the cystic fibrosis transrnembrane conductance regulator gene J Biol Chem 1991, 266 24471-24476 11 Koh J, Sferra J, Collins FS. Analysis of the CFTR promoter region. Paediarr Pulmonol 1991; S6: 227 12 Crawford IC, Maloney PC, Zeitlin PL et al Immunocytochemical localization of the cystic fibrosis gene product CFTR. Proc Natl Acad Sci USA 1991; 88: 9262-9266 13 Hams A, Chalkley G, Goodman S, Coleman L Expression of the cystic fibrosis gene in human development Development 1991, 113: 305-310 14 Trezise AEO, Buchwald M In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator Nature 1991, 353 434—437 15 Trezise A, Under CC, Gnswold MD, Buchwald M. In vivo cell-specific expression of CFTR. Late breaking science Abstract. North American CF meeting, Dallas TX, USA, 1991



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Cystic fibrosis gene.

The cystic fibrosis gene, located at 7q31, spans about 230 kb of genomic DNA and contains 27 exons. The cDNA of 6.2kb would predict an 1480 amino acid...
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