The history of Cystic Fibrosis

1929 Flemming A. "On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenza." Br J Exp Pathol 1929; 10: 226–36.
Alexander Flemming (1881-1955) (figure 13) was a Scottish bacteriologist, working at St Mary’s Hospital, London, whose discovery of penicillin (1928) prepared the initial step towards the highly effective practice of antibiotic therapy for infectious diseases.
In 1945 Fleming shared the Nobel Prize for Physiology or Medicine with Ernst Boris Chain (1906-1979) and Howard Walter Florey (1898-1968) who both (from 1939) were responsible for carrying forward Fleming's initial observation by further isolation, purification, testing, and quantity production of penicillin
In 1940 a report was issued describing how penicillin had been found to be a chemotherapeutic agent capable of killing sensitive germs in the living body. Thereafter great efforts were made, with government assistance, to enable sufficient quantities of the drug to be made for use in World War II to treat servicemen with war wounds.
Penicillin became available for a few patients with CF in the USA in 1943 and the results were reported by Paul di Sant’Agnese (1944 below) – prior to this virtually all children with CF died in infancy or early childhood from Staphylococcal pneumonia and malnutrition.

It is noteworthy that Flemming failed to develop his 1929 discovery – this was done over 10 years later by Florey and Chain. Nor was Flemming the first to observe the antibacterial effects of moulds. Ernest Duchesne (1874 –1912) (figure 14) was a French physician who noted that certain moulds kill bacteria. He made this discovery thirty-two years before Alexander Fleming observed the antibiotic properties of penicillin, a substance derived from those moulds, but his research went unnoticed. Duchesne entered l'Ecole du Service de Santé Militaire de Lyon (the Military Health Service School of Lyon) in 1894. Duchesne's thesis, “Contribution à l’étude de la concurrence vitale chez les micro-organismes: antagonisme entre les moisissures et les microbes” (Contribution to the study of vital competition in micro-organisms: antagonism between moulds and microbes), that he submitted in 1897 to get his doctorate degree, was the first study to consider the therapeutic capabilities of moulds resulting from their anti-microbial activity. Duchesne had made his breakthrough by observing how the Arab stable boys at the army hospital kept their saddles in a dark and damp room to encourage mould to grow on them. When he asked why, they told him that the mould helped to heal the saddle sores on the horses. Intrigued, Duchesne prepared a solution of the mould and injected it into a series of diseased guinea pigs. All recovered.
In a series of meticulous experiments, Duchesne studied the interaction between Escherichia coli and Penicillium glaucum, showing that the latter was able to completely eliminate the former in a culture containing only these two organisms. He also showed that an animal inoculated with a normally lethal dose of typhoid bacilli would be free of the disease if the animal was also inoculated with Penicillium glaucum. Unfortunately, as he was only 23 years old and unknown, the Institut Pasteur did not even acknowledge receipt of his dissertation! He urged more research but unfortunately his army service, after getting his degree, prevented him from doing any further work. Considerable attention has been allotted to penicillin as before the availability of penicillin few infants with CF survived infancy. It was the introduction of penicillin and later other antibiotics which was the main development that permitted survival beyond infancy.

1922 Banting FG, Best CH. Internal secretion of the pancreas. J Lab Clin Med 1922; VII: 251-266.

The work reported in this classic paper eventually led to a Nobel Prize in 1923 for Banting and Macleod, in whose Toronto laboratory the work was done. Frederick Banting (1891-1941) (figure 11) was a Canadian orthopaedic surgeon who became an assistant in physiology in Ontario where he worked in Richard McLeod’s laboratory with Charles Best, a medical student. It was here, after many ups and downs and arguments and with the help of Bertram Collip, a highly trained biochemist, to extract the insulin, that they eventually produced and tried the insulin on a diabetic patient in 1922.
The paper begins –“The hypothesis underlying this series of experiments was first formulated by one of us in November 1920 (Banting was then assistant in Physiology at Western University, London, Ontario) while reading an article dealing with the relation of the isles of Langerhans to diabetes (Baron: Surg Gynec Obstetr xxxi No 5 p437). From the passage in the article which gives a resume of degenerative changes in the acini of the pancreas following ligation of the ducts, the idea presented itself that since the acinous but not the island tissue degenerates after this operation, advantage might be taken of this fact to prepare an active extract of the islet tissue. The subsidiary hypothesis was that trypsinogen or its derivatives was antagonistic to the internal secretion of the gland. The failures of other investigators in this much worked field were thus accounted for”.
The authors concluded from their experiments that –“intravenous injections of extract from dog’s pancreas, removed from 7 to 10 weeks after ligation of the ducts, invariably exercises a reducing influence upon the percentage sugar of the blood and the amount of sugar excreted in the urine”.
This medical classic is a “good read” and one of the most significant medical papers of the 20th century. It is included here not only for its historical interest but also for its relevance to CF as the majority of those affected will eventually develop CF related diabetes in adult life. Although the Islets of Langerhans in CF are functioning adequately through childhood in most patients, despite their severe exocrine pancreatic insufficiency, they are eventually destroyed resulting in the majority of adults with CF developing diabetes mellitus. Efforts to prevent the slow destruction of the pancreas and prevent or delay the onset of CF related diabetes will surely become an area of research as more people survive to develop this complication which has an adverse effect on their prognosis.

1936 Fanconi G, Uehlinger E, Knauer C. Das Coeliakie-syndrom bei angeborener zystischer Pankreasfibromatose und Bronchiektasien. Wien Med Wchnschr 1936; 86:753-756. (Celiac syndrome with congenital cystic fibromatosis of the pancreas and bronchiectasis).
Guido Fanconi (1892-1979), was Professor of Paediatrics in Zurich from 1929-1965 and a pioneer of modern scientific paediatrics (figure 2). Although this publication is considered by some Europeans to be the first clear description of CF, it is a brief report of only two children who died aged 10 months and three years with ‘coeliac syndrome’, purulent bronchitis and bronchiectasis. The pancreatic changes were quite typical of those later described in cystic fibrosis. “The changes in the lungs and pancreas, two vital organs, are so profound that their failure appears understandable”.

Martin Bodian quotes a 1928 publication of Fanconi’s (1928 Beih. Jb. Kinderhlk 21) as noting cases of coeliac disease starting in early infancy and often associated with bronchitis but surprisingly Fanconi did not mention this in his 1936 paper which is so frequently quoted in Europe as the first description of cystic fibrosis.
Speaking in 2008, Walter Hitzig, the distinguished immunologist from Zurich, recalls (referring to this paper) that “My teacher Guido Fanconi, in his usual enthusiasm, presented a “new disease” to the Swiss paediatricians in 1936. However, one of the doctors questioned its importance in his daily practice. Fanconi’s answer: “Oh certainly it is important – I have seen already 3 cases!” of course earned him a big laughter in the audience! And today the syndrome described as Pankreas-Fibrose, now called cystic fibrosis, is considered to be the most frequent hereditary disease in the Caucasian population, and no doubt every paediatrician must diagnose it”.
Lynn Taussig describes meeting Knauer, the third author, after this paper was published. Apparently the two children described in Knauer’s doctoral thesis in 1935 were the two described in this paper. The next year Fanconi, Uehlinger and Knauer published this paper. He notes that the first name on the paper was now Fanconi’s. Maurice Super mentions that there was controversy between Fanconi and Wangensteen “who thought the condition might be something unique to the cantons in which Fanconi worked”. The hereditary genetic nature of CF was not suspected at this time.
Fanconi published a number of papers on various aspects of intestinal malabsorption and other aspects of paediatrics between 1921 and 1956 mostly in German or French. He was referred to in one article as a “Jack of all trades” – as were many of the paediatricians of the time. Reviewed objectively, none of these publications, with the possible exception of this 1936 paper, made any major or original contribution to the understanding or definition of cystic fibrosis. However, Fanconi made other very important contributions; in 1929 he described hereditary panmyelopathy known as Fanconi’s anaemia, in 1941 he deduced that polio was spread via the gastrointestinal route rather than by droplets and apparently predicted that Down’s syndrome was due to a chromosome abnormality 20 years before trisomy 21 was discovered.

1938 Andersen DH, Cystic fibrosis of the pancreas and its relation to celiac disease: a clinical and pathological study. Am J Dis Child 1938; 56:344-399.
Although many infants had already been reported who, in hindsight, obviously had cystic fibrosis, this was the first clear detailed clinical and pathological description of a large series of affected infants in English. Dorothy Andersen reported 49 patients - 20 from her hospital and others from colleagues and the literature. She described the neonatal intestinal obstruction, intestinal and respiratory complications and many other features – but particularly the characteristic pancreatic histology (figures:3 and 4). Salient points from the original description are as follows - “In 45 of the cases the pancreas presented a microscopic picture which is described by the term cystic fibrosis. The acini contain secretions of various sizes, and the acinar cells were flattened to form a thin epithelial wall around them. The smaller concretions were surrounded by relatively normal cells, which occasionally contained eosinophil granules…….The size of the cysts varied in each case but large ones were not often noted in the youngest infants. Surrounding the acini and also the lobules there were moderate to large amounts of fibrous tissue, the quantity varying roughly with the age of the child...….The islets of Langerhans were usually normal in number and appearance.”

Andersen likened much of the epithelial histology to that found in vitamin A deficiency which for some years she regarded as the primary cause of the systemic features of the condition; this causation was never substantiated although she continued to support this theory for some years.
The paper contains an excellent tabulated review of the previously reported cases and describing her paper Martin Bodian (1952 below) writes - “such a clear account of the symptoms that it enabled many cases to be recognised that had hitherto been missed, and aroused such interest that it was followed by a shoal of case reports and confirmatory reviews” which appeared during the Forties (many are described below).

Dorothy Andersen (1901-1963) (figure 5) was the pathologist at the Babies Hospital at the Columbia Presbyterian Medical Center in New York. She had a wide range of interests and made contributions in many other areas of medicine. She was said to be a rugged individualist, a paediatric clinician, pathologist, a research chemist – also apparently a roofer and carpenter, happy to make her own home improvements! Eventually over 600 children with CF were referred to her in New York; she involved Dr. Paul di Sant’Agnese, eventually another leader in the CF field, to provide paediatric care for her patients as she was primarily a pathologist. Dr Phillip Farrell of Wisconsin lists 4 reasons for the referral of so many children to her in New York. First was this seminal 1938 publication, second a combination of her reputation and her arrogance (she is alleged to have said she was the only person in the world who knew about cystic fibrosis in the late Thirties and Forties!), third the desperation of parents and fourth New York was the one place people could reach easily in those days. Described as "windblown" by friends and detractors alike, Dorothy Andersen was considered quite a character. She is said to have kept a particularly untidy lab, holding semi-annual "glüg" parties there, in honour of her Scandinavian heritage. She was a niece of Hans Christian Andersen. She was a bright compassionate and sensitive scientist who was also a chain smoker and died of lung cancer at the age of 62 years.

In 1950 Milton Graub, a paediatrician who eventually became President of the US CF Foundation, suspected his two year old son had CF and describes how he called Dr Andersen on a Sunday morning and found her working in her laboratory. She listened to the medical history and saw his son the next day. The diagnosis was much more difficult and was made primarily on the clinical features plus analysis of the duodenal secretions. These were done and the diagnosis of cystic fibrosis of the pancreas was established. Since Dr Andersen was a pathologist she referred his son Lee for further clinical care to Dr Paul di Sant’Agnese. "He was a pediatric specialist and a delight – soft spoken, reassuring and supportive" (from Doershuk CF, 2001. below).

1946 Di Sant’Agnese PA, Andersen DH. Celiac Syndrome IV Chemotherapy in infections of the respiratory tract associated with cystic fibrosis of the pancreas; observations with penicillin and drugs of the sulphonamide group, with special reference to penicillin aerosol. Am J Dis Child 1946; 72:17-61.
This is the first report of the use of penicillin in cystic fibrosis. In 1943 a small quantity of penicillin had become available from the US Army to treat three children with CF and a further 2 in 1944 with intramuscular penicillin; the results were variable. Bryson and co-workers at the Carnegie Foundation were first to investigate the use nebulised penicillin (Bryson et al, Science 1944; 100:33). In 1944 Alvan Barach of the Presbyterian Hospital was using a penicillin aerosol to treat asthma and bronchiectasis (Barach et al, Ann Int Med 1945; 22:485). Similar apparatus was used by di Sant 'Agnese for these children with CF; it consisted of a rubber mask with re-breathing bag and nebuliser with a flow of oxygen into the nebuliser (figure 10).

Figure 10: Nebuliser used for administration of penicillin.

Figure 11: Dr Paul di Sant'Agnese. From Doershuk CF. Cystic fibrosis in the 20th Century. AM Publishing, Ltd. Cleveland, Ohio, 2001. With permission of Dr Carl Doershuk.

This is an interesting first report describing early treatment in CF and in particular the first use of nebulised penicillin 20,000 units seven times daily with or without intramuscular penicillin 160,000 units daily. According to di Sant’Agnese, dramatic response occurred in 15 infants and young children with CF who eventually received the treatment but little success was achieved in infants less than a year old.
From 1947 there were numerous publications on the use of the recently available penicillin in other conditions by various routes both oral, by aerosol and even powdered inhalations – no less than 18 such publications were reviewed in The 1948 Year Book of Pediatrics. (Poncher HG (ed). Year Book Publishers. Chicago). Undoubtedly a new era of treatment of infection had begun.

Di Sant’Agnese (1914 - 2005) (figure 11) wrote that “Penicillin is the first drug known to affect the course of fibrocystic disease after cyanosis has marked the existence of suppurative Staphylococcal bronchitis”. Later, in 2001, he recalled “In most patients the results (of penicillin treatment) were dramatic. From death’s door, slowly dying from chronic pulmonary disease while we watched helplessly, patients revived in a few days”. Sulphonamides were said to be useful in prophylaxis and for intercurrent infections but not after the stage of suppurative bronchitis.

Dr Lynn Taussig, who worked with di Sant’Agnese, later recalls that Paul di Sant’Agnese and Harry Shwachman were definite rivals but had considerable respect for each other. Di Sant’Agnese came from a noble family in Italy. His father was an obstetrician and radiotherapy expert who was physician to the Italian Royal Family. Paul went to Rome medical school and then came to USA in 1939 to study medicine in New York. He was chief resident in paediatrics and published his first paper on tick paralysis - as initially he worked in infectious disease and immunology; he studied the effect of immunisations in early life. He went to work with Dorothy Andersen at the Presbyterian Hospital in New York. In 1950 he described glycogen storage disease of the heart (Pompe’s disease) and as a gastroenterologist in the early 1950s he wrote on coeliac disease.
In 1953 his observation of abnormal sweat electrolytes in CF was one of the most important observations in the history of CF and provided the basis of the sweat test (Di Sant’Agnese et al, 1953 below). He wrote over 140 papers mostly on CF - physicochemical differences, mucoproteins, calcium in secretions, and with West the first report of pulmonary function tests, pneumothorax, ventilation, pancreas, duodenal contents, and measurements of absorption. He was the first to describe the liver changes - a few months before Shwachman’s group. He had an interest in every aspect of the condition. But the patient always came first and he was an outstanding clinician as well as a gifted researcher. In 1955 he was involved with the medical aspects of the CF Foundation. In 1960 in Europe he met Archie Norman, David Lawson, and the International Cystic Fibrosis and Mucoviscidosis Association (ICFMA) had 28 delegates and guests from 14 countries. Di Sant’Agnese said he was most proud of training so many associates and colleagues in CF care and research. “If your plan is for one year, plant rice, if for 10 years plant trees, if for life – educate!”
In 1946 this present paper on the use of penicillin for fibrocystic disease, with special reference to aerolised penicillin, was the first publication on the use of this new antibiotic in cystic fibrosis.

1946 Andersen DH, Hodges RC. Celiac syndrome V. Genetics of cystic fibrosis of the pancreas with consideration of the etiology. Am J Dis Child 1946; 72:62-80.
Investigating 47 of their own families and 56 from the literature, the authors concluded that the familial incidence indicated a hereditary disorder with a recessive mode of inheritance, but which required more than one factor for its expression. Although the incidence in siblings approximates to the 25% expected of a Mendelian recessive trait, an hereditary condition requires more than one factor for its expression. This was a quite different explanation to that given by Fanconi et al, 1944 but in line with that of Bodian, 1952. Andersen continued to believe that “the pulmonary infection is the result of the nutritional deficiency” – obviously she considered this to be the additonal factor required.
This is the first clear statement, backed by clinical evidence, that CF is inherited in a Mendelian recessive manner. Others, including David Lawson, would also speculate on the relative contribution of the basic defect and the secondary effects of the defect (such as chest infections and malnutrition) to the ultimate outlook for the patient. i.e. if the secondary effects could be prevented would the outlook be much better? Subsequent progress showed that this was indeed the case.

1951 Kessler WR, Andersen DH. Heat prostration in fibrocystic disease of the pancreas and other condition. Pediatrics 1951; 8:648.
One of the most important papers up to that time from New York. Walter Kessler, the senior resident at the time, and Dorothy Andersen reported 12 children with heat prostration. Ten were admitted during a New York heat wave in 1948 and no less than 7 were known to have cystic fibrosis. These were the days before air conditioning was generally available in New York and 1948 was a particularly hot summer. Paul di Sant’Agnese, was working with Dorothy Andersen at the time and looking after her patients, later said that he treated these particular infants as Andersen was away vacationing in Europe when they were admitted! The authors of this present report queried whether the sweat glands, as well as the glands of the pancreas and other organs, were inadequate in function or alternatively a low grade infection lowered the margin of tolerance to increased temperatures. At the time of this report there was no explanation as to why infants with CF were particularly susceptible to heat prostration and salt depletion – fortunately Paul di Sant’Agnese decided to find out! This was the first report that children with CF were particularly susceptible to heat. It was this original incident that eventually led Paul di Sant’Agnese, who was working with Dorothy Andersen, to search for the reason for salt depletion in many of these CF infants and eventually to his recognising the abnormally high sweat sodium and chloride, and to a lesser extent potassium. This was undoubtedly the first and most important major advance in the understanding of the causation of CF up to that time (see di Sant’Agnese et al, 1953 below).

1953 Darling RC, di Sant’Agnese PA, Perera GA, Andersen DH. Electrolyte abnormalities of the sweat in fibrocystic disease of pancreas. Am J M Sc 1953; 225:67-70.
The first report of elevated sweat electrolytes in cystic fibrosis. di Sant’Agnese collaborated with Bob Darling who was the head of the Rehabilitation Department of the Columbia Presbyterian Hospital. In this department there was a constant temperature room and a method of collecting sweat. Initially two teenagers with CF and two controls were selected and although the sweating rate was similar the level of electrolytes was much higher in the patients with cystic fibrosis. Subsequently sweat from 9 CF children and 8 controls showed chloride more than three times higher in the people with CF than in the controls.
This was an unexpected finding quite unrelated to any previously recognised abnormalities in the condition (di Sant’Agnese et al, 1953 below) but it was the most important observation since the clear identification of CF as a specific entity by Dorothy Andersen in 1938.

1953 di Sant’Agnese PA, Darling RC, Perera GA, Shea E. Sweat electrolyte disturbances associated with childhood pancreatic disease. Am J Med 1953; 15:777-784.
In this study there were 50 patients with CF, 9 with other pancreatic diseases and 50 controls. All the CF patients had similar elevations in the sweat electrolytes. Their adrenal and renal function was normal. The authors considered the findings to justify abandoning the term “mucoviscidosis” and returning to the unsatisfactory term “cystic fibrosis of the pancreas” until a better one was proposed.

1953 di Sant’ Agnese PA, Darling RC, Perera GA, Shea E. Abnormal electrolyte composition of the sweat in cystic fibrosis: Clinical significance and relationship to the disease. Pediatrics 1953; 12: 549-563.
This is the main paper that di Sant’Agnese himself quotes as describing the sweat electrolyte abnormality expanding on the paper of Darling et al, 1953 (above). Di Sant’Agnese mentions the original report of Kessler and Andersen 1951 (above) and also that the susceptibility of patients with CF to heat in the summers was noted also during subsequent summers after 1948. Paul Quinton more recently recalls that di Sant’Agnese told him that the development of heat tolerance among troops sent to North Africa was attributed to adaptations to sweating, so di Sant’Agnese pursued excessive salt loss in the sweat as the most likely origin of volume depletion during the high heat stress (Quinton, 1999 below). Bob Darling was head of Rehabilitation at Columbia Presbyterian Hospital and had a constant temperature room. Di Sant’Agnese decided “as a shot in the dark to see if sweating function was impaired in CF patients that would lead to a smaller than normal volume of sweat, or whether there was something wrong with the sweat electrolyte concentration”. di Sant’Agnese continued - “In April 1952 two teenage children with CF and two controls were put in the constant temperature room and their sweat then analysed for electrolytes. To my surprise and excitement the answer was right there. There was a tremendous difference in the sweat electrolyte concentration between the two groups”. “In contrast the sweating rate was similar in the two groups”. (Described in detail by Paul di Sant’Agnese. Experiences of a Pioneer Researcher. In: Doershuk CF (ed.) Cystic Fibrosis in the 20th Century 2001; Fanos JH. 2008; 17-35.). Sant’Agnese and colleagues showed the sweat abnormality was unrelated to renal or adrenal disease and was definitely related to sweat losses.

For this present paper they examined 43 people with CF, 9 patients with other pancreatic diseases and 50 controls in a room at 32.2 C for 1-2 hours. Sweat was collected onto dry gauze under adhesive waterproof plaster. Sweat chloride in the CF patients was 106 (60-160) meq/l, in controls and other pancreatic diseases only 32meq/l (4-80) and in CF the Na 133 (80-190), and in controls 59 (10-120).

This was undoubtedly the most important advance in the understanding of CF up to that time. However, astonishingly, di Sant’Agnese recalls the paper received a very cool reception at the 1953 meeting of the American Pediatric Society with not a single question! Also when presented before Jas Kuno, apparently a distinguished sweat physiologist, Kuno uttered one word -“impossible”- and walked out of the room! It is also said that even di Sant' Agnes's close colleague Dorothy Andersen was at first reluctant to accept the findings. However, and perhaps predictably, Harry Shwachman soon visited di Sant’Agnese in New York; he was impressed and with his usual alacrity and energy was able to present a large supportive series by October 1954 much to di Sant’Agnese’s delight!

1981 Knowles MR, Gatzy JT, Boucher RC. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N Eng J Med 1981; 305:1489-1495. [PubMed]
Undoubtedly this was a major landmark paper. Michael Knowles from North Carolina describes how they developed a technique that measured a single parameter of epithelial function – the transepithelial potential difference (PD); this they used to define salt (ion) transport properties of nasal and lower respiratory epithelium in normal humans in vivo and then in patients with CF whose transepithelial PD they showed was markedly higher than normal – a feature apparently present within hours of birth suggesting a primary genetic epithelial defect rather than due to any circulating “CF factor” or effect from infection.
Knowles and colleagues eventually identified a defect in the ability of Cl to move across CF cells, the Cl permeability of the airway epithelium was not activated by beta-agonists and there was a very rapid absorption of salt and water. This led to the theory that CF airway epithelium has an excessive rate of NaCl and water absorption leading to dehydration of the secretions, reduced clearance of mucus predisposing to chronic airway infection – a theory which increased in popularity and was championed particularly by Knowles’s colleague Richard Boucher.
Michael Knowles (figure 1) was honoured with an award from the CF Foundation at the 2008 Annual North American CF Conference. He and Richard Boucher are certainly two of the outstanding North American CF clinicians and researchers of the era.

1983 Quinton PM. Chloride impermeability in cystic fibrosis. Nature 1983; 301:421-422. [PubMed]
Another landmark paper in the understanding the CF defect by Paul Quinton (figure 2) , who himself has CF. He later recalls (Quinton, 1999 below) that Mike Knowles (1981 above) reported a significantly larger than normal electronegative potential across the nasal epithelium, along with the fact that NaCl absorption was inhibited in the CF sweat ducts and also that sodium was relatively more absorbed than chloride. This gave Quinton the idea that the basic defect in the CF duct had to be due to an anion impermeability and not defective anion exchange.
Using a series of microperfusion experiments of sweat glands (it is said from his own forearms) he measured the electrolytes by “energy dispersive X-ray analysis”. The chloride impermeability he had shown in sweat glands was the basis for the raised sweat electrolytes and provided a physiological explanation for the high salt content of CF sweat and also “provided the first description of a basic cellular defect that has since proven to be uniformly inherent in all CF affected cells”.
Paul Quinton holds the Nancy Olmsted Chair of Pediatric Pulmonology UC San Diego and is Professor of Biomedical Sciences at UC, Riverside.

1985 Eiberg H, Mohr J, Schmiegelow K, Neilsen LS. Linkage relationships of paraoxinase (PON) with other markers: evidence of PON-cystic fibrosis synteny? Clin Genet 1985; 28:265-271. [PubMed]
This was the first positive move towards narrowing down the identification of the CF gene. The linkage relationships of the serum arylesterase paraoxonase (PON) was examined in normal Danish families and in Danish and English CF families. The highest correlation was found between the inheritance of paraoxinase and cystic fibrosis. Linkage studies for PON against 64 other polymorphic marker systems did not give such a close relationship. By the present screening about 2/3 of the genome could tentatively be excluded as the region of PON and cystic fibrosis. (Synteny = the presence together on the same chromosome of two or more gene loci).

1985 Tsui L, Buchwald M, Barker D, Braman JC, Knowlton R, Schumm JW, Eiberg H, Mohr J, Kennedy D, Plavsic N, et al. Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science 1985; 230:1054-1057. [PubMed]
Lap Chi Tsui and co-workers used a restriction fragment to localise the gene to the long arm of chromosome 7. In a set of 39 families, a polymorphic DNA marker was genetically linked to the autosomal recessive gene that causes cystic fibrosis. The DNA marker (called D0CRI-917) was also linked to the paraoxinase locus, which by independent evidence is linked to the CF locus (Eiberg et al, 1985 above). The location of the CF gene was now narrowed to about 1 percent of the human genome (about 30 million base pairs). This was the first step in molecular analysis of the CF gene. This was a major step forward localising the CF gene to chromosome 7.
Apparently Lap Chi Tsui encountered several problems when working with the pharmaceutical firm, Collaborative Research Inc., that had already invested $10 million in the project; they could see the commercial opportunities of developing markers for antenatal and carrier diagnosis. The problems are well described by Leslie Roberts (Science 1988; 240:141-144 & 282-285). Essentially Collaborative Research Inc. wanted to delay publication of the location of the gene on chromosome 7 until more markers could be identified and patents could be applied for. Meanwhile other groups, Williamson’s in London (Wainwright et al, 1985 below) and Ray White’s in Utah (White et al, 1985 below) were said to know of the findings that the CF gene was on chromosome 7 and had identified other markers very close to the CF gene.

1989 Kerem B-S, Rommens JM, Buchanan JA, Markiewicz D. Cox TK. Chakravarti A. Buchwald M. Tsui LC. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245:1073-1080. [PubMed]
Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level had important implications for genetic diagnosis.

1989 Riordan JR, Rommens JM, Kerem B-S, Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou JL. et al. Identification of the cystic fibrosis gene: cloning and characterization of the complementary DNA. Science 1989; 245:1066-1073. [PubMed]
Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative CF locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.

1989 Rommens JM, Iannuzzi MC, Kerem B-S, Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou JL. et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989; 245:1059-1065. [PubMed]
An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.