Journal of the Royal Society of Medicine Supplement No. 18 Volume 84 1991

Cystic fibrosis: the



D J H Brock PhD FRCPE A E Shrimpton PhD C Jones PhD Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU

I McIntosh PhD

Keywords: cystic fibrosis; mutation frequencies, prenatal diagnosis, heterozygote screening

Introduction The phrase 'new genetics' was first used in 1979 to describe a novel and theoretical method of identifying genes of unknown structure and function. The technology depends on the existence within the human genome of large numbers of restriction fragment length polymorphisms (RFLPs) and hypervariable regions which can be used as markers to track the segregation of disease-causing genes in disorders with a clear Mendelian mode of inheritance. In contrast to the classical genetic approach, where a gene is cloned and identified through knowledge of its expressed protein product, the new genetics works in reverse and uses information on gene structure to infer the amino acid sequence and function of the protein it controls. For this reason the phrases 'new genetics' and 'reverse genetics' have become largely synonymous. An outline of the major steps in reverse genetics is shown in Figure 1. Although it was apparent in 1979 that a technology existed which should in principal allow the identification of genes responsible for most Mendelian disorders, it was 10 years before a human gene was cloned by a pure reverse genetic approach without REVERSE GENETICS Mendelian disorder with a clear mode of inheritance (dominants and recessives) Search for DNA markers which cosegregate with the disease locus

Establish firm genetic linkage to a marker Use DNA marker to localise disease gene locus to a specific region of a chromosome

Continue search for more closely-linked markers Chromosome walking and jumping from marker to disease gene locus Clone gene and determine nucleotide sequence

Determine nucleotide sequence in DNA of affected individuals

Infer amino acid structure of protein product of normal and disease genes

Isolate protein product of normal and disease genes

Figure 1. An outline of the major steps in the reverse genetic approach to cloning a gene

any shortcuts. That gene was the cystic fibrosis (CF) gene'.3. The important stages in cloning the CF gene are shown in Figure 2. They include the establishment of genetic linkage and cytogenetic localization on the long:arm of chromosome 7 in 1985, the bracketing of the CF gene in 1,986, the finding of a set of veryclose markers in 1987, and the ultimate cloning -of the CF gene in 1989.-It should not be forgotten that in the period 1980 to 1985 an enormous volume ofwork was poured into the firit stage of the process, the search for DNA markers which unambiguously cosegregated with the CF gene. The triumph of reverse, genetics in cloning the CF gene has ramifications far beyond the disorder itself. Most importantly it has demonstrated that the procedures outlined in 1979 have the power to -identify genes which are currently only defined by. a gross disease phenotype. There is little doubt that in the next decade hundreds of human genes will be cloned and sequenced using these and related methodologies. Such considerations are beyond the scope of this review. Here we will concentrate on the immediate implications of the cloning of the CF gene, and in particular on the possibilities that arise for improved diagnosis, prenatal diagnosis and population heterozygote screening.

The CF gene The CF gene is a comparatively large one, spanning about 250 kilobases (kb) of genomic DNA and containing 27 exons'. A transcript of 6.5 kb has been detected in lung, pancreas, colon, cultured sweat glands and nasal polyps, placenta, liver, kidney and parotid gland, but not in brain, adrenals, cultured skin fibroblasts or lymphoblast cell lines2. Analysis of the sequence of overlapping complementary DNA clones predicted a polypeptide product of 1480 amino acids with a molecular mass of 168 kiloDaltons. This protein has been named the cystic fibrosis transmembrane conductance regulator (CFTR). It has not yet been isolated but on the basis of homology data and molecular modelling is thought to be an ATP-dependent transport protein rather than a chloride (or other) ion channel4. It seems unlikely that CFTR will be found to be expressed in easilyaccessible tissues such as blood components. One of the key pieces of evidence that the cloned DNA sequence was indeed the CF gene was the finding of the same specific mutation on 70% of CF chromosomes3. The mutation deleted a three base-pair (bp) sequence in exon 10 of the gene, and a phenylalanine residue at position 508 of the polypeptide product. This mutant allele, known as AF508, can be readily detected after amplification of exon 10 by the polymerase chain reaction (PCR), and

Journal of the Royal Society of Medicine Supplement No. 18 Volume 84 1991


Two DNA markers, J3.11 and met, show tight linkage to the CF gene and localise it on 7q

CF/J3.1 1 /met

7q 1986:

J3.11 and met bracket the CF gene at 7q21-31

l met *- CF J3.11 7q


Further DNA markers are even closer to the CF gene, and set stage for chromosome walking and jumping 11



The CF gene is cloned and a cDNA sequence described. Consistent mutation found on CF chromosomes. The amino acid sequence of the gene product is deduced

CF 7q

Figure 2. The principal steps in cloning the CF gene Table 1. Geographic distribution of the AF508 mutation'


Figure 3. Polyacrylamide analysis ofPCR-amplified products of exon 10 of the CF gene. N, homozygous normal; A, homozygous ,F508 affected; H, heterozygote for normal and AF5. alleles. Note the heteroduplex bands in heterozygous samples

inspection ofthe product on polyacrylamide or agarose gels (Figure 3). A recently-published worldwide survey5 of the frequency of this allele shows considerable variation in different ethnic and population groups. Data for adequately surveyed European populations are shown in Table 1. Th-e highest frequencies are found in north European countries and lower frequencies in Italy, Portugal, Spain and Greece. In the British Isles the fiequency for England, Scotland and the Republic of Ireland is about 75%, but that for Northern Ireland reported at 54%. Early expectation that the remaining CF mutant alleles would comprise a limited group of mutations has now faded. Confidential reports to an international CF genetic analysis consortium listed more than 60 distinct mutations by October 1990. These included missense, nonsense and RNA splice mutations, as well as deletions and insertions. Many have been found to be segregating in only one or a handful of families, while others seem specific to particular ethnic groups.

Belgium Bulgaria Czechoslovakia Denmark France Germany Greece

Italy Netherlands Portugal Spain Switzerland England

Scotland Northern Ireland Republic of Ireland Canada USA

CF chromosomes No. screened iFo 334 110 354 423 1445 1360 194 1128 401 84 608 334 1548 238 201 120 466 439


236 64 240 373 1058 970 105 535 310 45 331 232 1184

(71) (58) (68) (88) (73) (71) (54) (47) (77) (54) (54) (69) (76)



111 91

(54)(76) (71) (76)

330 333

It has become painfully apparent that with the exception of the predominant AF508 allele, CF is a genetically heterogeneous condition. In an attempt to ascertain the more important CF alleles, we have recently surveyed over 400 CF chromosomes for the presence or absence of 16 specific mutations in a largely Scottish population. Wherever possible both parents were examined as well as the index affected patient, though obviously each CF chromosome in the nuclear family was only



Journal of the Royal Society of Medicine Supplement No. 18 Volume 84 1991

Table 2. CF alleles in the Scottish population

CF allele


G551D G542X R117H 1717-1G-A intron R560T W1282X 621 + 1G-T intron A455E Unpublished (5) Not detected D11OH R347P

1507 S549N S549I R553X 2566insAT

Frequency Reference

Exon 10 11 11 4 10 11 20 4 9

0.728 0.063 0.051 0.018 0.015 0.007 0.007 0.004 0.004 0.026 0.923

6 9 8 7 8 8 11 8 8

4 7 10 11 11 11 13

scored once. The data is shown in Table 2. Apart from AF508, only four alleles were found on more than 1% of CF chromosomes. These are two missense mutations, G551D and R117H, a nonsense mutation G542X, and an RNA splice mutation 1717-1G--+A. Another nine different mutations were detected but at very low frequencies, while the remaining seven alleles surveyed were not encountered.

Detection of mutant CF alleles As indicated in Figure 3 the predominant AF508 CF allele is easily detectable by simple electrophoresis of PCR-amplified DNA. The test can be applied to blood samples, buccal cell scrapings or chorionic villus biopsies. Amongst the Caucasian population in the UK about 55% of patients with CF are homozygous for AF508 and will show a single fastmigrating band on acrylamide gels. Another 38% of patients with CF will be compound heterozygotes for AF508 and another CF allele. On the type of gel shown in Figure 3 they will be indistinguishable from unaffected CF heterozygotes. The remaining 7% of patients with CF will appear on such gels as normal homozygotes. In tracking CF alleles in either families with an affected child or in the general population we are thus faced with the problem that AF508 analysis is a substantially incomplete test. As shown in Table 2 the next most common alleles are G551D at 6.3% and G542X at 5.1%. Each of these mutations is in exon 11, and thus PCR amplification of this region of the CF gene, followed by either restriction enzyme digestion of the product with HincdI and MboI (G551D) or allele-specific hybridization with appropriate oligonucleotides (G542XL) can increase the heterozygote detection rate to about 85%. There are several other less frequent CF alleles in exon 10 and 11, so that amplification of these two regions of the gene followed by a full analysis of products will raise the heterozygote detection rate to about 90%. This corresponds to a detection rate of patients with CF of about 80%. Thereafter, fiurther searching for minor CF alleles becomes less and less productive. Considerations of costs suggest that detecting 90% of CF alleles is as high as is practical using current methodologies.

Applications Diagnosis Although pilocarpine iontophoresis and sweat chloride determination remains the primary laboratory test for CF, it is not an easy assay and may give problems in newborns and again in older patients. There is of course no primary yardstick by which the reliability of sweat testing can be judged, and the quoted figure of 95% must be viewed with suspicion. Laboratories are therefore sometimes asked whether the new molecular genetic techniques can contribute to confirmation or exclusion of diagnosis in doubtful cases. Obviously if such a case is either a homozygote or a compound heterozygote for defined CF alleles, the diagnosis of CF is confirmed and beyond dispute. However, the absence of two mutant CF alleles in such a case does not exclude a diagnosis of CF. The individual may carry CF alleles which have yet to be defined, or defined CF alleles which the laboratory is unable or unwilling to screen for. It is not yet possible to place any reliable figure on the probability of a diagnosis of CF in an individual who has had a range of defined CF alleles excluded. The reason for this is the possibility that in doubtful CF cases (ie mild symptoms, ambiguous sweat tests) there may be a higher-than-expected frequency of undefined CF alleles. Thus until virtually all CF alleles are characterized direct testing for mutations can only be used to confirm diagnoses. Prognosis One of the intriguing features of the first publication of the CF gene was the apparent association between patients with pancreatic insufficiency (PI) and the AF508 allele. About half of the PI patients were homozygous for the deletion, while no pancreatic sufficient (PS) patients had this genotype. Kerem et al.3 proposed that non-AFs08 alleles could be divided into the severe (S) and the mild (M). PI patients would have genotypes AF5N/AF5F AF5N/S or S/S, while PS patients would be AF508/M, SIM or MIM. The observed distribution of PI and PS patients in the Canadian cohort could be fitted to a Hardy-Weinberg equilibrium where the gene frequencies for AF508, S and M were 0.68, 0.24 and 0.08, respectively3. However, analysis of a German cohort of patients did not confirm this hypothesis6, and there are now some doubts about whether homozygosity for AF508 is a useful prognosticator of a more severe course of disease. Santis et al.7 divided a group of 59 CF patients into those with severe and those with mild lung disease and found similar proportions of AF508 homozygosity and heterozygosity in each set. There is as yet little data on clinical correlations with the less common CF alleles. Kerem et al.8 have suggested that A455E and P574H belong to their classification of mild alleles, while Dean et aL9 found R117H in two families with exceptionally mild disease. These preliminary data await confirmation in larger series of patients. Prenatal diagnosis It has been possible to carry out prenatal diagnosis of CF since 1983 when the second-trimester amniotic fluid microvillar enzyme test was introduced. However, the sensitivity of this test is only 95% while the specificity is 92%, so that microvillar enzyme assay must now be regarded as a last resort'0. The discovery in 1985 of DNA markers tightly linked to the CF gene

Journal of the Royal Society of Medicine Supplement No. 18 Volume 84 1991 Table 3. Predictability of microvillar enzyme testing in pregnancies with low prior odds Control



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Cystic fibrosis: the new genetics.

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