BntWi MeScal Bulletin (1992) VoJ 4«, No 4, pp717-737 C Tbc Bnnsh Council 1992

Milestones in cystic fibrosis M Super Paediatrw Genetics Unit and Cystic Fibrosis Clinic, Royal Manchester Children's Hospital, University of Manchester Medical School, Manchester, UK

The study of cystic fibrosis (CF) provides a fascinating insight into developments in medicine in the 20th century. Milestones include the first clear clinical descriptions in the 1930s, discovery of a sweat electrolyte abnormality, establishing the autosomal recessive mode of inheritance and improvements in treatment. Microdissection experiments on sweat glands allowed the main defect to be delineated as one of chloride transport. Location of the gene to chromosome 7 made prenatal diagnosis feasible and carrier detection in siblings. The CF gene—its product being the cystic fibrosis transmembrane conductance regulator (CFTR), and its major mutation Delta F508 was discovered in 1989. World-wide collaboration has resulted in discovery of more than 150 further mutations. Incorporation of CFTR into non-chloride transporting insect cells by conferring chloride transport, proved it a chloride channel. CFTR incorporated into adenovirus results in correction of the chloride transport defect in airway cells, bringing gene therapy closer.

The study of cystic fibrosis (CF) provides a fascinating insight into advances in medicine in the 20th century. We take for granted the invention of the microscope, classification of bacteria and discovery of methods of culture and identification, electrolyte and biochemical measurement, an understanding of modes of genetic transmission, the discovery of antibiotics and the structure and properties of DNA. McFariane Burnett, writing in the 1950s claimed that all the basic important scientific discoveries had been made by then and that developments thereafter would simply be a question of correct applications of this knowledge. Whether time has proved him right is highly debatable. The Editor, Editorial Board and authors would like to give specific thanks to the Cystic Rbrosis Research Trust without whom most of the work presented in this issue would not have been possible.

718

CYSTIC FIBROSIS

However in acknowledging milestones of discovery in CF, we do need to remain humble and to realise that without the seminal discoveries already in place in medicine, many of the subsequent, exciting advances in our knowledge would not have been possible. Fanconi (Fig. 1) writing from Switzerland in 19361 described a coeliac syndrome with pancreatic changes which he realised was different from coeliac disease. There was controversy between him and Wangensteen who thought the condition might be something unique to the cantons in which Fanconi worked. Dorothy Anderson2 made the disease known in the English speaking world with an outstandingly accurate and detailed description in 1938 in which she discussed the relationship with meconium ileus and described approaches to treatment, including administration of pancreatic enzymes. She got one point wrong, considering that the disorder was one of vitamin A deficiency—'there's a drop of gall in the sweetest wine'. Farber (1950)3 in noting thickened secretions coined the term mucoviscidosis, a term still used to describe CF in some European countries and still part of the logo of the ICF(M)A—International Cystic Fibrosis (Mucoviscidosis) Association. Reference to earlier publications shows that Landsteiner, discoverer of the blood groups, had provided the first clear description of meconium ileus in 19054. It is almost surprising that the great pathologists of the 18th century did not describe the post-mortem changes in detail, perhaps because many children died at home and paediatric pathology was not well developed. Archibald Garrod, who coined the term 'Inborn Errors of Metabolism' described a consanguineous English family in 19105, with a number of children with steatorrhoea who died of bronchopneumonia. He suggested possible autosomal recessive inheritance, which had only been 'rediscovered' by Bateson a few years earlier when he read of Mendel's 1865 work on a train to London. It took until 1952 for this correct assumption about the genetic transmission to be accepted and even after this, articles appeared from time to time suggesting too many affected children in sibships and wishing to invoke two gene theories of partial dominance. Most of the authors had forgotten to make a correction for carrier couples with no affected children, in their calculations. ''Das kind sterbt bald wieder, dessert Stirne beim Kusscn salzig schmeckt.' Roland Busch6 has drawn our attention to this quotation from German Children's Songs and Games from Switzerland—'The child will die soon, whose forehead tastes salty when kissed'. Similar folklore is quoted from France, Germany and Denmark, suggesting a bewitched child and one who ought not to be baptised. Imagine the added anguish of a couple who might have had two or more such

MILESTONES IN CYSTIC FffiROSIS

719

'bewitched' children, for these descriptions are almost certain to have been of cystic fibrosis. Table 1 provides a summary of the advances since Landsteiner's description. Figures 1 to 4 show some of the major figures involved in these milestones. Only the major milestones will be discussed further, under the headings of clinical advances, then advances in our understanding of the pathophysiology and lastly the genetic milestones. Detailed descriptions may be found in specific chapters of this volume. It is far easier to write on these milestones in retrospect, since discovery of the gene and agreement on the transport defect. For a long time CF was fertile only in the profusion of false dawns and in controversy between workers who could not reproduce one another's work. Now we have a fine distillate from what was once a very muddy wine. Some well-known names in CF research are all but forgotten and their work certainly is—where are the theories of Spock, Bowman, Hosli and Nadler now? They all had good ideas. CLINICAL MILESTONES In 1951 there was a heatwave in New York. Perceptive physicians noted the relative excess of children with CF who presented with heat prostration. Di Sant'Agnese (Fig. 2) realised the significance and discovered the excess of sodium and chloride in the sweat7 which became the cornerstone of the diagnostic test and pointed the way to the study of exocrine glands and secretory cells. Di Sant'Agnese pioneered a great deal of research over the next 30 years and wrote major reviews for the New England Journal of Medicine8, summarising research and developments at the time. Di Sant'Agnese stimulated Gibson and Cooke to publish a standardised way of sweat collection by pilocarpine iontophoresis9 which became the gold standard for this important, though exacting diagnostic test Shwachman first published on CF without pancreatic involvement.10 Some paediatricians, physicians and general practitioners still need reminding about the possibility of normal pancreatic function in CF. Shwachman (Fig. 1) and Kulcycki11 published a scoring system still in use today which allocates points for well-being; respiratory symptoms and signs; nutritional symptoms and signs; and the chest radiograph. Their articles also provided the first long-term studies. George and Norman12 published life tables of survival for CF in 1971 followed by Robinson and Norman13 in 1975, which showed the first modest improvements in prognosis.

720

CYSTIC FIBROSIS

TaWe 1 Milestones in cystic fibrosis Name Landsteiner Fanconi Anderson Di Sant'Agnese/ Anderson Faber Carter Di Sant'Agnese

Year 1905 1928, 1936 1938 1946 1950 1952 1953

Watson and Cnck Cystic Fibrosis Foundation formed Shwachman Bishop and Koop Gibson and Cooke

1953 1955

Shwachman and Kulczycki CF Research Trust IGF(M)A Holsclaw and Shwachman Cnspen and Norman Crossley Quinton

1958

Carbarns, Brock

1983

Yacoub Sato and Sato Eiberg Tsui

1984 1984 1985 1985

1956 1957 1958

1964 1964 1968 1974 1979 1983

Williamson, Scambler Wainwright, EsteviU Farrall

1986-1988

Spense Tsui, Riordan, Collins Tsui

1988 1989 1989

KartDer Crystal

1991 1990

1986

1992

Discovery Described meconium ileus First clinical descriptions Outstandingly accurate and comprehensive Fjiglish description S. Aureus main sputum pathogen Corned the term 'Mucoviscidosis' CF is autosomal recessive Excess of sodium and chloride in sweat The structure of DNA CF without pancreatic involvement An lleostomy for meconium ileus A standardised method of sweat sodium and chloride testing A clinical scoring system

Vas deferens and seminiferous tubule blockage An X-ray scoring system Newborn immune reactive trypsin The basic defect is of chloride secretion Microvillar enzymes m amnioOc fluid Heart-lung transplant No secretory response to B agonists Paraoxinase linked to CF The gene for CF is on Chromosome 7 Discovery of many clinically useful gene probes First child born after accurate DNAbased exclusion of CF Uniparental disomy m CF Discovery of the CF gene CFTR International CF Genetic Analysis Consortium founded CFTR is a chloride channel Incorporation of CFTR into adenovirus Creation and cure of secretory defect

Prognosis in meconium ileus had been very poor with over 50% dying at or soon after operation. Bishop and Koop described an Ueostomy

MILESTONES IN CYSTIC FIBROSIS

721

2.

Fig. 1 Dr G Fancom with H Shwachman and M Graub. Fancom pubhshed the first chrucal descriptions ' Fig. 2 Dr Paul Di Sant' Agnese who discovered that S Aureus was the main sputum pathogen in CF.7. 8 Fig. 3 Dr Lap-Cbee Tsui who with John Riordan and Collins discovered the CF gene CFTR 46, 55-57, 60 Fig. 4 Dr Martin Bodian who, in 1952, descnbed focal bibary cirrhosis, still regarded as pathognomonic, in 15 of 62 autopsies from Great Ormond Street.19

722

CYSTIC FIBROSIS

in 1957 14 which reduced handling of the bowel and which resulted in an immediate great improvement in survival. Further advances in neonatal surgery have allowed the Bishop-Koop ileostomy to be largely replaced by end to end anastomosis which is normally possible. Noblett15 in 1969 described administration of (hygroscopic) gastrografin by enema, allowing non-surgical resolution of meconium ileus in some instances. Rickham16 published on a group of survivors born with meconium ileus or peritonitis who turned out not to have CF and this has proved a valuable contribution when genetic analysis has revealed no CF genes in fetuses or newborns who have perished with post-mortem features suggestive of meconium ileus. Sterility which affects the great majority of affected males was discovered by Holsclaw and Shwachman to be due to blockages causing blind-ending channels in the vas deferens and seminiferous tubules.17 A major milestone in CF treatment was undoubtedly the discovery of B-lactamase resistant penicillins, allowing adequate treatment for die first time of staphylococcal chest infection. This must have played a part in improving the long-term prognosis, though strangely there has never been the seminal article published which proves this. Cohen18 described more than 100 pregnancies in women with CF. He struck a gloomy note in presenting the large percentage who were dead within 2 years, although their survival did not differ significantly from women who had never conceived. The occurrence of liver cirrhosis was alluded to by Anderson in her 1938 article and in 1952 Bodian1^ described focal biliary cirrhosis, still regarded as pathognomonic, in 15 of 62 autopsies from Great Ormond Street (Fig. 3). Abnormality of glucose tolerance was included in Anderson's original 1938 article. The first detailed descriptions of diabetes mellitus in CF were given by Schwachman in 195520 and again in 196221. The condition occurs mainly in those with pancreatic insufficiency and since discovery of the gene has only been described where genotypes are associated with pancreatic insufficiency.22 The incidence of diabetes is not specifically age related, nor is it associated with severity of disease. Some of the best British work on CF has come from the Brompton Hospital working with the increasing numbers of adults as survival has improved. Included is work on inhaled antibiotics23, on the assessment of physiotherapy techniques24 and, more recently, on genotype/phenotype correlations.25 Screening for CF, aspects of improvements in nutritional treatment and heart-lung transplantation are well dealt with elsewhere in this publication and few details will be discussed here. Introduction of enteric coated microgranules of pancreatic extracts may be regarded as a legit-

MILESTONES IN CYSTIC FIBROSIS

723

imate milestone, allowing normal stool frequency for the majority and improved nutrition. The existence of increased numbers of carriers of CF among those with raised immune reactive trypsin is interesting and should shed new light on the pathogenesis in CF.26 The emergence of specific organisations, Cystic Fibrosis Foundation (North America 1955), the Cystic Fibrosis Research Trust (UK 1964), The European Working Group on Cystic Fibrosis and the International Cystic Fibrosis (Mucoviscidosis) Association may with justification be regarded as milestones because of the enormous impetus which these organisations have provided by facilitating high quality research and, more recently, patient care. Associations of adults with CF have brought greater awareness of psychosocial issues such as employment, marriage, childbearing and financial issues, including rife insurance. In a 1986 newsletter of the Association of CF Adults in the United Kingdom, 48% were in employment, 17.6% unemployed, 3.6% unable to work through illness, 16% students and 8.8% housewives, with the remainder unspecified. A 63 year old was listed as one of those who had replied to this questionnaire study. Spock discussed psychological characteristics of affected children in 196627 and Gayton wrote on psychological aspects of CF in affected children, their siblings and parents in 1977.28 Mothers may tend towards depression but each group displays their own coping strategies. More recent studies are agreed that despite the risk posed by a chronic disease, children with CF show little by way of adverse psychosocial adjustment Better nutrition and reductions in stool frequency and flatus must help a great deal towards a better body image. There is a high level of general knowledge about CF displayed by the parents and affected individuals; siblings know the least about CF, perhaps part of their coping strategy. PATHOPHYSIOLOGY Quintan's discovery of the specific defect in chloride reabsorption29 signalled a dramatic change in CF research—at last everyone agreed with the correctness of these findings. Patch clamp experiments by Frizell30 and Welsh31 allowed sophisticated studies of secretory cells. Tissue culture techniques, which enabled specialised qualities of cells from various tissues to be retained, has created valuable in vitro models for study,32 but the absence of a laboratory animal model for CF has slowed research. Since the discovery of the gene attempts to create a transgenic model have proved difficult but recent success has been reported.

724

CYSTIC FIBROSIS

GENETIC MILESTONES The autosomal recessive nature of CF was pointed out by Carter in 195233 and a number of publications thereafter dealt with the incidence in various countries and peoples of different edinic backgrounds.34-36 Incidences of between 1 in 2000 and 1 in 5000 were described in peoples of European origin and thus it was realised that CF was the commonest autosomal recessive of Europeans. Kulczycki published an incidence in blacks in Washington of 1 in 17 000 37 corresponding roughly to European admixture, it was thought Genetic analysis, since the discovery of the gene shows real differences in the types and proportions of CF mutations in different populations and some, encountered thus far only in black people have been described {see below). Explanations of the high frequency of CF carriers mainly favour a heterozygous advantage with postulated relative resistance to chloridelosing diarrhoea amongst carriers the most favoured. The genetic discovery allows one to determine the parental origin of the CF carrier state. An excess of carrier sons born to carrier fathers, noted by Kitzis38, suggests meiotic drive as an explanation of the high frequency. Pritchard D J 3 9 who studied the uncles and aunts of individuals with CF came to similar conclusions. A very unexpected result of molecular analysis allowed two instances of uniparental isodisomy to be discovered. In both cases the CF child had received two copies of one of the mother's chromosome number 7, with no contribution from the father and this maternal chromosome carried a CF gene.40- 41 The authors of the second paper claimed that uniparental disomy may be a common phenomenon. It has been described in other genetic disorders such as Prader-Willi and Angelman's syndromes. Before discovery of the gene there were many attempts to define changes in carriers. Discovery of a 'Cystic Fibrosis protein' 42 circulating attached to IgG proved difficult to reproduce, though the existence of the protein as a secondary phenomenon of the basic gene defect is accepted. Carbarns discovered that amniotic fluid from pregnancies with the fetus affected by CF contained reduced amounts of microvillar enzymes.43 Brock developed measurement of 4 such microvillar enzymes into a prenatal test applicable to couples known to be at a 1 in 4 risk of having a further affected child.44 Unfortunately the test has a significant false positive (8%) and false negative (5%) rate and errors in both directions were encountered. The test still does have a limited application in couples where genetic analysis is not fully informative, though whether the same degree of accuracy would be obtained as in

MILESTONES IN CYSTIC FIBROSIS

725

the majority, who are informative and on whom the main proportion of the error rates will have been based, is not certain. With the protein product of the CF gene unknown, studies in families with more than one affected child were undertaken. Using polymorphic markers an attempt was made to show linkage and to demonstrate die gene by reverse genetics, ie to find the gene and then derive the protein product. This approach allowed Eiberg in Copenhagen45 to define a linkage to the enzyme paraoxinase which exists in two forms in the healthy population. These studies showed mat generally when an affected person with CF showed one form, die sibling showed that form too 90% of die time, aldiough parental study had indicated that either form of die polymorphism might have been present in tiieir offspring. John Edwards had drawn up a negative gene map of CF, ie based on all the similar experimentation which had yielded negative results and presented it at the 1984 International Gene Mapping meeting. The chromosomal location of paraoxinase was not known. Lap-Chee Tsui (Fig. 4) in Toronto concentrated on the negative gene map and, in a series of mouse-human hybrid experiments, rapidly demonstrated a marker on human chromosome 7 linked both to paraoxinase and to CF.46 In fact die marker was further away from the CF gene than paraoxinase, cosegregating about 85% of the time. Reference to existing markers known to be on chromosome 7 and described a short time earlier, revealed two which proved extremely closely linked to CF 47 . 48, die Met oncogenes, Met H and Met D developed by Van de Woude in Salt Lake City and an anonymous DNA probe pJ3.11 from Professor Williamson's laboratory in St Mary's Hospital, London. The generosity of diese two groups in sharing the probes with workers, worldwide, soon allowed a great number of families to be analysed showing cosegreation of diese polymorphic markers 98.5% of die time. Testing for earner status in siblings and prenatal diagnosis, widi very low error rates, became feasible. The first successful prenatal diagnosis using diese memods was reported by Farrall in 198749 and 30 such tests from Manchester in die following year.50 A United Kingdom Register of Prenatal Diagnosis of CF using DNA methods is held widi the support of die Cystic Fibrosis Research Trust and by December 1991, 513 pregnancies had been reported to h(see Table 2). Some of the couples who have had such tests say that diey would not have undertaken die pregnancies were it not for the existence of die tests. Collaborating groups were able to agree that CF was located between Met and pJ3.11, a distance of 2.5 Megabases apart.51 The search of this section was carried out by pulse field experiments capable of studying large segments of DNA and dien studies of contiguous sections, allow-

726

CYSTIC FIBROSIS

Table 2 A United Kingdom Register of Prenatal Diagnosis of CF 1986-1991 (Number of Centres = 16) Attempted prenatal diagnoses on DNA derived from chononic villi or ammotic fluid No sample obtained Miscarriage after biopsy Termination after DNA prediction of CF Termination on 50:50 odds of CF Termination after different finding e.g Tnsomy 18 Continuing - prediction no CF genes - prediction earner - prediction 50-50 carrier or no CF genes Prediction affected; awaiting outcome Born affected as predicted Bom affected prediction no CF genes based on findings in foetus terminated for positive microvillar assay (1977) Born affected - prediction unaffected Bom carrier Bom no CF genes 50% carrier/50% affected outcome not reported Other (includes 2 sets of twins)

512 2 26 84 5 5 61 121 11 18 5 1 4 83 71 5 10

Note: UK Register of CF Prenantal Diagnosis This register has functioned since May 1987. The false negatives occurred in the first year, as did the difficulties in obtaining a biopsy specimen The help of participating centres is acknowledged.

ing the map to be completed in greater and greater detail. Polymorphic probes of great clinical usefulness were discovered in the course of this search. Notable amongst these were the probes XV2c 52 and KM1953, both discovered in Professor Williamson's laboratory and again widely distributed, through his generosity. These probes were so close to the CF gene that they participated in a 'false dawn' when it was thought that a probe very near by, Cs7, was the CF gene itself.52 Although this was not so, strong linkage disequilibrium was found with specific Xv2c alleles and KM 19 alleles, found far more commonly on chromosomes bearing the CF gene than on the non-CF bearer, in studies of the healthy parents of offspring with CF. The so-called 'B' haplotype, consisting of the larger Xv2c and smaller KM 19 allele has been best studied. Ethnic differences in the percentages of B versus the other haplotypes were found to exist, suggesting that there would be more than one CF mutation. In our population with a high positive correlation, tests on spouses marrying into families with CF became possible with homozygosity for the B haplotype indicating a 1 in 4 chance of being a carrier, while more favourable haplotypes indicated a much lower carrier likelihood than the general population risk.54 The false idea that Cs7 might be the CF gene slowed down the London group who concentrated on this area of the genome. Collins

MILESTONES IN CYSTIC FIBROSIS

727

employed gene jumping, simply 'jumping over' segments which proved difficult to analyse in the laboratory or unlikely from the base-pair composition to harbour the gene. Working together with Lap-Chee Tsui and John Riordan, they encountered a segment rich in GC sequences, thought to signal the proximity of a gene and found an open reading frame segment which they were able to demonstrate in lower species, thus indicating a conserved important segment; they were able to demonstrate that functional RNA was found specifically in secretory tissues affected in CF and was not expressed in others and, lastly they were able to demonstrate a deletion of uiree base pairs coding for phenylalanine confined to CF sufferers and carriers. They described the gene in 3 seminal papers in Science on 8th September 198955~57 with Rommens, Riordan and B-S Kerem the three first authors. Riordan termed the gene CFTR—Cystic Fibrosis Transmembrane Conductance Regulator, and the mutation which they found in 69% of the Canadian and American CF genes studied they called Delta F508 to indicate a missing phenylalanine at position 508 of the 1480 aminoacid protein. The existence of some mutations involving Delta F508 and another mutation, indicated that compound heterozygosity for differing CF mutations on each chromosome would also cause CF and this has been confirmed with discovery of specific mutations. From computerised simulations of hydropathy plots, ie hydrophobia or hydrophllia of segments of the protein and its likely stereotypic configuration, a model of CFTR was proposed, showing 2 transmembrane regions, 3 intracytoplasmic regions, containing 2 nucleotide binding folds and a regulatory region and lastly an extracellular glycosylated region {see diagram in Higgins' chapter.) The complementary DNA was said to contain 24 exons—subsequently this has been increased to 27. The Delta F508 mutation was situated in the first of the binding folds. Similarity of the protein structure to the p-glycoproteins of multiple drug resistance associated with drug resistance in cancer and of the transport systems of some bacteria was noted by Riordan.. In the third paper genetic findings of 252 Canadian and American CF chromosomes were given. Homozygotes for Delta F508 (about 48% of the total) were invariably pancreatic insufficient, while compound heterozygotes, or those with no Delta F5O8 contained individuals pancreatic sufficient and insufficient Detailed haplotype data was also given, but since many of the polymorphic probes had not been shared with other laboratories, this information could not be compared readily, worldwide. XV2c and KM 19 had been widely distributed and large collaborative studies in Europe58 quickly showed mat the great majority of haplotype B was associated with the Delta F508 mutation but that this mutation did not account for all the B haplotype. This raised the intriguing prospect mat

728

CYSTIC FIBROSIS

perhaps the heterozygote advantage in CF was more associated with this haplotype than with CF and that, perhaps, CF was a hitchhiker. Recent analysis shows a better correlation between KM 19 type 2 genotype (part of haplotype B) and sweat chloride levels, than exists with Delta F508 59 , so movement of chloride into and out of cells may indeed be associated with the heterozygote advantage. Tsui, Riordan and Collins were first recipients of the Paul Di Sant'Agnese Prize, presented at the 1989 North American CF meeting. Table 3

Ammo-acid abbreviations

A = Alanine E = Glutamic Acid H = Histidine L = Leucine P = Proline S = Serine W = Tryptophan

C = Cysteine F = Phenylalanine I = Isoleucioe M = Methionine Q = Glutamine T = Threonine Y = Tyrosine

D = Aspartic Acid G = Glycine K = Lysroe N = Asparagine R = Axginine V = Vahne

X = Stop Codon, Delta = Deletion, Ins = Insertion Number followed by + or - Denotes intron/exon boundary alteration.

Following on the discovery of CFTR, Lap-Chee Tsui quickly set up an international group to search for other gene mutations and to pool knowledge. A worldwide incidence of Delta F508 has been published.60 Our laboratory was fortunate enough to find the second mutation in CF Delta 1507 when we undertook a family study and showed a carrier father and his healthy (carrier) son with weaker than expected bands on testing with the so-called allele-specific oligonucleotide probe designed to demostrate Delta F50861 {see Figs 5 and 6). In the 2 years following the discovery more that 100 further mutations were reported to the consortium and information on about half of these has been published. Less than 20 occur with any frequency worldwide, though there are some very interesting ethnic differernces and in some the XV2c and Km 19 haplotypes differ from the B haplotype found with Delta F508. Table 3 gives the nomenclature used in describing aminoacid alterations. In Europe the incidence has been particularly well studied58. A genetic gradient from south-east to north-west exists in Europe, with the lowest Delta F508 incidence described in Turkey and the highest in Denmark (88%). In Britain the overall incidence is 76%; in the northwest of England this rises to 81%, in a population which we have studied extensively.62

MILESTONES IN CYSTIC FIBROSIS

Hcraozy(jote -

729

Heterozygote

1

Delta F508

Naoative

Fig. 5 Allele specific oligonucleotide autoradiograph demonstrating individuals homo zygous for Delta F5Q8, heterozygous and free of the mutation. The homozygotes have CF proven by this result; heterozygotes could be earners or affected, depending on the clinical picture and whether the other gene is normal or harbours a different CF mutation.

Among the relatively less frequent mutations there are some very interesting ethnic variations: one of the earlier exon 11 mutations to be described G551D, 63 is found particularly in peoples of celtic origin. Of CF genes 5-10% are due to this mutation in Ireland, the West of Britain, Brittany and Czechoslovakia. Figure 7 shows results of a number of samples analysed for G551D using polymerase chain reaction, a technique which has allowed analysis to take less than one day which is extremely valuable when there is clinical urgency. Figure 5 shows one heterozygote for G551D and one homozygote (born to consanguineous Irish tinker parents). The only other mutation found which exceeds Delta F508 in the population studied is W1282X64, a 'stop' mutation from the second nucleotide binding fold, found particularly in Ashkenazi Jews, with incidences of close on 60% described from Israel.65 Table 4 gives the mutations which have been discovered in the British CF population. By applying the commonest of the mutations identified to carrier detection we are able to detect 92% of CF genes in our part of England and 84% of all couples at a 1 in 4 risk (Fig. 6). (In Canada when Delta F508 was found in 68% of CF genes, this meant that 45% of couples at 1 in 4 risk would be identified). Our findings also imply

730

CYSTIC FIBROSIS

>

NOHMAl ASO

Fig. 6 Pedigree and autoradiograph of allele 'specific' oligonucleotide result from a family in which the affected boy is a compund heterozygote for Delta F 5 0 8 and Delta I 307 . The proximity of the mutation to position 508 has resulted in weak annealing in the carrier father and his earner son, giving the faint upper bands Definitive testing was by DNA sequencing, not shown.

that by using DNA analysis as a diagnostic test for CF we would make a definite molecular diagnosis in 84% and would identify one CF gene in another 15% of those with definite CF. Only in 1% are all tests negative with no doubt about the CF diagnosis. Our carrier detection programme concentrates on families with a known history of CF. Table 5 gives the risk which we would provide to couples tested in this way.

MILESTONES IN CYSTIC FIBROSIS

731

Fig. 7 Polaroid photograph of G551D analysis after polymerase chain reaction, using the restriction enzyme EMB01. A homozygote and a heterozygote are shown. The diagnosis of CF in the homozygote is confirmed. Whether the heterozygote is a carrier or has CF depends on the clinical picture and whether the other gene harbours a different CF mutation

GENOTYPE/PHENOTYPE CORRELATION The first claimed genotype/phenotype association has been alluded to-the finding of pancreatic insufficiency invariably in homozygotes for Delta F508. The authors referred to this as the severe phenotype, regarding those showing pancreatic sufficiency as the mild phenotype. While it is generally true that CF with pancreatic sufficiency is mild, so may homozygotes for Delta F508 have mild disease and there are clearly factors important in determining the severity other than simply the genotype. There are a number of factors other than the CF genotype which seem to affect severity—genes nearby but outside the CFTR locus seem to play a part25 and delay in making the diagnosis as well as neglect of treatment can affect the clinical picture. No specific genotype or group of genotypes goes specifically with pancreatic sufficiency. Mutations in the transmembrane portion of the gene might be expected to result in milder disease, being in a portion not specifically involved in nucleotide binding and transport. Only in the case of the mutation R117H is there evidence for this. Dean has published on a mild clinical picture associated witih pancreatic sufficiency in compound heterozygotes for Delta F508 and RU7H. 66 Interest has been focused on patients with premature 'stop' codons, resulting presumably in a truncated CF protein. Homozygotes for G542X have mild disease.67 It is postulated that alternate routes of chloride transport may open up when CFTR does not function at all. Thus a protein conformational change in CFTR resulting in a signif-

732

Table 4

CYSTIC FIBROSIS

CF Mutations encountered in United Kingdom CF chromosomes screened

Mutation

Delta F508 G551D R553 G542X R56OT N1303K DI507 R117H 621+1G-T G85E W1282X E60X R75Q V520F 1717-1 G-A

CF chromosomes with mutation

1 Mutations encountered in North-West England 1062 863 199 37 (non Delta F508) 199 8 199 11 199 6 199 6 199 4 199 5 199 10 199 4 199 2 30 2 15 1 199 3 199 3

Percentage

81.2 3.48 0 75 1.03 0.58 0 58 038 0.47 0.98 0.38 0 19 ? ? 0.29 0.29

Number of mutations seen = 15 89 85% identified: it is assumed that when all non-delta F5O8 chromosomes have been analysed that 92% of CF genes will have been identified.

1154insTC R347P A455E G458V Q493X C524X S549N R1283M Q1291H

2. Mutations encountered elsewhere in UK but not yet m N-W 199 (in the north-west group) 199 199 0 0 0 199 199 0

icant alteration in function appears to be worse than no CFTR being formed at all. There is further evidence for this in the report of patients with frame-shift mutations, which should interfere seriously with the formation of CFTR, resulting in mild disease.68 There has been a single report to the Consortium, of a second mutation of the same CF gene. Interestingly the mutation R553Q, associated with Delta F508 downstream resulted in an apparently milder phenotype than usual (Tummler, personal communication). Could one engineer a mutation elsewhere in the gene and cause clinical improvement? Time might tell. Meconium ileus is an obvious clinical presentation capable of genotype/phenotype study. There is a familial predisposition to recurrence,

MILESTONES IN CYSTIC FIBROSIS

733

Fig. 8 Shows ARMS Multiplex test, in which the four commonest mutations seen in north-west England are tested simultaneously, with results possible on the same day on which the specimen reaches the laboratory. The upper track shows 621+1 G->T, the next G551D, then G542X and the lowest track shows Delta F5O8. Two contiguous tracks belong to each specimen tested, for the absence and presence of the allele Thus the patterns shown are: negative, negative, heterozygous for Delta F508, homozygous for Delta F508, compound heterozygote for 621+1 and Delta F5O8, heterozygous for G551D, and compound heterozygote for G542X and Delta F508, then the size ladder ARMS is a registerd trademark of ICI Diagnostics Table 5 Applications of earner testing to families with a known history of CF. The average prior probability of blood relatives seeking testing is taken to be 1/2 COUPLE TESTING (THE IDEAL, ESPECIALLY PRECONCEPTION) Possible results Risk of CF Action offspring Both earners

1 in 4

Formal Genetic counselling Offer prenatal diagnosis

Relative earner Partner negative (13 mutations)

1 in 1200

Genetic Counselling Reassurance on risk figure No prenatal tests Sweat tests on symptomatic infants or children once bom

Relative negative Partner earner

1 in 2400

Repeat tests (to check no sample nux-up) If confirmed, as above

Relative negative Partner negative

1 in 720,000

Strong reassurance

suggesting a specific genetic influence. Homozygosity for Delta F508 comprises by far the largest proportion of meconium ileus but that proportion is similar to those who present without it Certain nega-

734

CYSTIC FIBROSIS

tive correlations with meconium ileus have been claimed, eg among heterozygotes of Delta F508 and G551D. A positive correlation of the rare mutation G542X/Delta F508 is awaiting confirmation, but our own figure of 5 out of 7 with meconium ileus seems likely to be confirmed. FUNCTION OF CFTR The proposed model of CFTR and its function has provoked lively debate, elaborated on in Higgins' chapter (this issue). Experiments which conferred chloride transport on non-epithelial insect cells by incorporating CFTR prove it to be a chloride channel in its own right.69 Recent expression of CFTR with a Delta F5O8 mutation into monkey kidney fibroblast cells induced reduced activity of cyclic AMP activated chloride channels, showing a role in mediating chloride transport too. 70 The chloride defect has been corrected in airway cells in vitro71 and Crystal's experiments of incorporating the relevant portion of the RNA for normal CFTR into a modified adenovirus and successfully 'infecting' the lung of a cotton rat with it makes gene based treatment feasible, at least for the lung.72 Milestones do not deal with the future but hopefully the most exciting chapters on this challenging disorder will have been written before the millenium. ACKNOWLEDGEMENTS I am grateful to Martin Schwarz and Geraldine Malone for their work in the DNA laboratory for helpful discussions, also to.my colleagues at the CF clinic, Dr Garry Hambleton and the CF clinic team for their support. I thank the Cystic Fibrosis Research Trust for providing us with secretarial support. The help of ICI Diagnostics in developing techniques and providing pnmers and kits is gratefully acknowledged.

REFERENCES 1 Fanconi G, Uehlinger E, Knauer C. Das Coehakiesyndrom bei angeborener zysticher Pankreasfibromatose und Bronkiektasen. Wien Med Wochenschr 1936, 86: 753-756 2 Anderson DH Cystic fibrosis of the pancreas and its relation to cehac disease: clinical and pathological study Am J Dis Child 1938; 56: 344-399 3 Farber S. Some organic digestive disturbances in early life Pathological changes associated with pancreatic insufficiency. Michigan Med Soc 1945, 44 587-594 4 Landsteiner K Darmverschluss durch eingedicktes Meconium. Pankreatitis. Zentrabl Allg Pathol 1905; 16 903-907 5 Garrod AE, Hurley WH. Congenital familial steatorrhoea. Q J Med 1912-3, 6: 242-258 6 Busch R. Historical aspects of cystic fibrosis. Wissenshaftlike zeitschnft der WillemPieck Universitat Rostock, 1986

MILESTONES IN CYSTIC FIBROSIS

735

7 Di Sant'Agnese PA, Darling RC, Perera GA, Shea E. Abnormal electrolyte composition of sweat in cystic fibrosis of the pancreas, its clinical significance and relationship to the disease. Pediatrics 1953; 12: 549-563 8 Di Sant'Agnese PA, Davis PB Res Cystic Fibrosis. 1976; 295: 481-485, 534-541, 597-4502 9 Gibson LE, Cooke RE A test for concentration of electrolytes in cystic fibrosis using pilocarpine by electrophoresis Pediatrics 1959; 23 549-563 10 Shwachman H, Dooley RR, Guilmette F, Patterson PR, Weill C, LeubneT H. Cystic fibrosis of the pancreas with varying degrees of pancreatic insufficiency Am J Dis Child 1956; 92: 347-368 11 Shwachman H, Kulczycln LL. Long term study of 105 patients with cystic fibrosis: studies made over a five to fourteen year period. Am J Dis Child 1958, 96. 6-15 12 George L, Norman AP. Life Tables in Cystic Fibrosis. Arch Dis Child 1971; 46: 139-143 13 Robinson MJ, Norman AP. Life Tables in cystic fibrosis. Arch Dis Child 1975, 50: 835-836 14 Bishop HC, Koop CE Management of meconium ileus, resection, Roux-en-Y anastomosis and lleostomy irrigation with pancreatic enzymes Ann Surg 1957; 145: 410-^14 15 Noblett HR. Treatment of uncomplicated meconium ileus by gastrografin enema: a preliminary report. J Pediatr Surg 1969; 4: 190-197 16 Rickham DP, Boeckman CR. Neonatal meconium obstruction in the absence of mucoviscidosis Am J Surg 1965; 109: 173-177 17 Holsclaw DS, Shwachman H. Genital function in cystic fibrosis. In. D Lawson, ed Proceedings of 5th International cystic fibrosis conference. London 1969, pp308—319 18 Cohen LF, Di Sant'Agnese PA, Fnedlander J Cystic fibrosis and pregnancy a national survey. Lancet 1980, l 842-844 19 Bodian M, ed. Pathology in fibrocystic disease of the pancreas. London William Heinemann 1952, pp67-146 20 Shwachman H, Leubner H Mucoviscidosis Adv Pediatr 1955; 7: 249-323 21 Rosan RC, Shwachman H, Kulczycki LL Diabetes melhtus and cystic fibrosis of the pancreas Laboratory and clinical observations. Am J Dis Child 1962; 104 625-634 22 Krueger LJ, Leroer A, Katz SM, Mack R, Holsclaw DS Jr, Lebenthal E J. Pediatr Gastroenterol Nutr 1991, 13: 209-219 23 Hodson ME, Penketh ARL, Batten JC Aerosol Carbeniccillin and gentamycin treatment of Pseudomonas aemgeinosa infections in patients with cystic fibrosis Lancet 1981,2: 1137-1139 24 Pryor JA, Webber BA, Hodson ME, Batten JC. Evaluation of the use of forced expiration technique as an adjunct to postural drainage in treatment of cystic fibrosis BrMed J 1979; 2: 417^18 25 Sands G, Osborne L, Knight RA, Hodson ME. Independent genetic determinants of pancreatic and pulmonary status in cystic fibrosis Lancet 1990; 336: 1081-1084 26 Laroche D, Travert G Abnormal frequency of delta F508 mutation in neonatal transitory hypertrvpsinaemia. Lancet 1991; 337. 55 27 Spock A, Stedman DJ. Psychologic characteristics of children with cystic fibrosis. NC Med J 1966; 27 426-428 28 Gayton WF, Friedman SB, Tavormina JF, Tucker F Children with cystic fibrosis I Psychological findings of patients, siblings and parents Pediatrics 1977; 59: 888-894 29 Quinton PM Chloride impermeability in cystic fibrosis. Nature 1983; 301: 421-422 30 Fnzell RA, Rekkemmer G, Shoemaker RL. Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 1986; 233: 558-560 31 Welsh MJ, Ming L, McCann JD et al Regulation of apical membrane chloride channels from normal and cystic fibrosis epithelium. Pediatr Pulmonol 1989, (Suppl 4) 105-106 32 Yaskansis JR. Epithelial cell lines for gene expression studies. Pediatr Pulmonol 1989 (Suppl 4): 109-110

736

CYSTIC F1BROSIS

33 Carter CO Familial incidence. In: Bodian, ed Rbrocysnc disease of the pancreas. London Heyneman 1952: 50-64 34 Steinberg AG, Brown DC. On the incidence of cystic fibrosis of the pancreas. Am J Hum Genet I960; 12: 416-424 35 Conneally PM, Merntt AD, Yu P-L. Cystic fibrosis population genetics. Texas Rep Biol Med 1973, 31: 639-650 36 Super Cystic fibrosis in the South-west African Afrikaner an example of population drift possibly with heterozygote advantage S Afr Med J 1975; 49- 818-820 37 Kulczycki LL, Schauff V. Incidence and clinical characteristics of cystic fibrosis in black children in the Greater DC area. Pediatr Res 1972; 6: 429 38 Kitzis A, Chommel JC, Kaplan JC. Unusual segregation of cystic fibrosis alleles to males. Nature 1988; 333 215 39 Pntchard DJ. Why cystic fibrosis is on the increase Nature 1987, 330 319 40 Spense JE, Periccante RG, Greig GM et al Uniparental disomy as a mechanism of human disease. Am J Hum Genet 1988; 42: 217-226 41 Voss R, Ben-Simon E, Avital A et al. Isodisomy of chromosome 7 in a patient with cystic fibrosis: could uniparental disomy be common? Am J Hum Genet 1989, 45 373-380 42 Wilson GB, Fudenbexg HH, Jahn TL Studies on cystic fibrosis using iso-electnc focusing an assay for detecting cystic fibrosis homozygotes and heterozygotes in serum. Pedialr Res 1975; 9 635-640 43 Carbarns JB, Gosden C, Brock DJH. Microvillar peptidase activity in amniotic fluid: possible use in prenatal diagnosis of cystic fibrosis. Lancet 1983, I: 329-331 44 Brock DJH, Bedgood D, Barron L, Haward C Prospective prenatal diagnosis of cystic fibrosis. Lancet 1985; l 1175-1178 45 Eiberg H, Mohr J, Schmiegelow K et al. Linkage relationships of paraoxinase (PON) with other markers: evidence of PON-cystic fibrosis synteny. Clin Genet 1985, 28 265-271 46 Tsui L-C, Buchwald M, Barker D et al. Cystic fibrosis locus defined by a genetically linked polymorphic marker. Science 1985; 230: 1054-1057 47 White R, Woodward S, Leppert M et al. A closely linked marker for cystic fibrosis Nature 1985; 318, 382-384 48 Scambler PJ, Wainwnght BJ, Watson E et al Isolation of a further anonymous sequence from seven, closely linked to cystic fibrosis. Nucleic Acids Res 1986; 14 1951-1956 49 Farrall M, Law H-Y, Rodeck CH et al First trimester prenatal diagnosis of cystic fibrosis with linked DNA probes. Lancet 1986; i 972-973 50 Super M, Ivinson AJ, Schwarz MJ et al Clinic experience of prenatal diagnosis of cystic fibrosis by use of bnked DNA probes Lancet 1987, ir 782-784 51 Scambler PJ, Estivill X, Bell G et al. Physical and genetic mapping of cosmids from the vicinity of the cystic fibrosis locus. Nucleic Acids Res 1987; 15 3639-3652 52 Estivill X, Farrall M, Scambler PJ. A candidate for the cystic fibrosis locus isolated by selection for methylaUon free islands. Nature 1987, 326 840-845 53 Esnvill X, Scambler PJ, Wainwnght BJ et aJ Patterns of polymorphism and linkage disequilibrium for cystic fibrosis Genomics 1987; 1: 257-263 54 Ivinson AJ, Read AP, Harris R et al. Testing for cystic fibrosis using allele association. J Med Genet 1989; 26 426-430 55 Rommens JM, Ionuzzi MC, Kerem B-S et al Identification of the cystic fibrosis gene chromosome walking and jumping. Science 1989; 245. 1059-1065 56 Riordan JR, Rommens JM, Kerem B-S et al Identification of the cystic fibrosis gene cloning and characterisation of complementary DNA Science 1989; 245 1066-1072 57 Kerem B-S, Rommens JM, Buchanan JA et al. Identification of the cystic fibrosis gene, genetic analysis Science 1989; 244- 1353-1356 58 European Working Group on cystic fibrosis Gradient of distribution in Europe of the major CF mutation and its associated haplotype. Hum Genet 1990; 85. 436-441

MILESTONES IN CYSTIC FIBROSIS

737

59 Gasparini P, Pignatti PF, Borgo G, Mastella G. Sweat chlonde concentration in cystic fibrosis patients varies with KM 19 genotype but not with the presence of the common Delta F5O8 deletion. Am J Med Genet 1991; 39: 230-231 60 The Cystic Fibrosis Genetic Analysis Consortium. Woridwide survey of the Delta F508 mutation. Am J Hum Genet 1990; 47: 354-359 61 Schwarz MJ, Summers C, Heptinstall L, Newton C, Markham A, Super M. A deletion mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) locus: Delta 1507 In: L-C Tsui, ed Identification of the CF (Cystic Fibrosis) gene New York, Plenum Press, 1991 62 Super M, Schwarz MJ. Mutations of the cystic fibrosis gene locus within the population of the north-west of England Eur J Pediatr 1992, 151: 108-111 63 Cutting GR, Kasch LM, Rosenstern BJ et al. A cluster of cystic fibrosis mutations m the first nucleodde-bindrng fold of the cystic fibrosis conductance regulator protein. Nature 1990, 346: 366-368 64 Vidaud M, Fanen P, Martin J, Ghanem N, Nicolas S, Goossens M Three mutations of the CFTR gene in French Cystic Fibrosis patients identification by denaturing Hum Genet 1990, 85- 446-449 65 Shosharu T, Bashan A, Augarten A et al. A termination mutation (W1282X) the most common mutation in the Jewish Ashkenazi CF patients in Israel, is assoicated with a severe disease presentation Pediatr Pulmonol 1991; (Suppl 6): 242-243 66 Dean M, White M, Amos J et al Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 1990, 61: 863-870 67 Bonduelle M, Lissens W, Libaers I, Malfroot A, Dab I. MiW cystic fibrosis in child homozygous for G542 nonsense mutation in CF gene. Lancet 1991; 338: 189 68 Dean M, White M, Kapronov N, Amos J, Krueger L. Cystic fibrosis patients with frame-shift mutations are not all severely affected Am J Hum Genet 1990, 47: A213 69 Kartner N, Hanrahan JW, Jensen TJ et al Expression of the cystic fibrosis gene m non-epithelial invertebrate cells produces a regulated anion conductance. Cell 1991; 64:681-691 70 Rich DP, Anderson MP, Gregory RJ Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells Nature 1990; 3467: 347-358 71 Dalemans W, Barbry P, Champigny G et al. Altered chlonde ion channel kinetics associated with the Delta F508 cystic fibrosis mutation Nature 1991: 354 526-528 72 Crystal R. New frontiers in therapy. Pediatr Pulmonol 1991; (Suppl 6): 64-65