Biochemical and molecular genetics of cystic fibrosis.

Chapter 4

Biochemical and Molecular Genetics of Cystic Fibrosis Lap-Chee Tsui and Manuel Buchwald Department of Genetics Research Institute The Hospital for Sick Children and Department of Molecular and Medical Genetics University of Toronto Toronto, Ontario, Canada M5G lX8

INTRODUCTION Cystic fibrosis (CF) is the most common severe recessive genetic disorder in the Caucasian population. In 1938, D. H. Anderson provided the first comprehensive description of the disease and also introduced the name "cystic fibrosis of the pancreas." Patients with CF suffer from excessive mucus accumulation resulting in severe clinical consequences in the respiratory, gastrointestinal, and genitourinary tracts (see Table I). All these symptoms are consistent with defects of exocrine glands, as suggested by S. Farber in 1945; he called the disease "mucoviscidosis," a name still popular in some parts of continental Europe. CF patients also have elevated electrolyte levels in their sweat, an observation which, first described by di Sant'Agnese et al. (1953), became the hallmark for CF diagnosis. The recessive mode of inheritance of CF in families was noted by D. H. Anderson and R. G. Hodge in 1946. The frequency of CF is about 1 in 2500 live births in the Caucasian population and this number has generally been used to derive a carrier frequency of 0.04 and a mutant gene frequency of Advances in Human Genetics, Volume 20, edited by Harry Harris and Kurt Hirschhorn, Plenum Press, New York, 1991. 153

H. Harris et al. (eds.), Advances in Human Genetics © Plenum Press, New York 1991

Lap·Chee Tsui and Manuel Buchwald

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TABLE I. Clinical Symptoms of Patients with Cystic FibrosisO Common Chronic obstructive lung disease Persistent cough Recurrent or refractory lung infections Pancreatic insufficiency Steatorrhea Meconium ileus (10-15010 of cases) Failure to thrive Male infertility Less common Bronchiolitis/asthma Nasal polyposis Rectal prolapse Recurrent abdominal pain Biliary cirrhosis Recurrent pancreatitis Female infertility Hypochloremic or hyponatremic alkalosis °Adapted from Boat et al. (1989).

0.02. These values vary greatly among different geographical locations, however [see review by Boat et al. (1989)]. The major clinical symptoms of CF include chronic obstructive pulmonary disease, pancreatic enzyme insufficiency, and elevated sweat electrolyte levels [see review by Boat et al. (1989)]. A list of the symptoms is shown in Table I. If untreated, affected children usually die at an early age because of severe lung infection, but, as a result of advances in clinical management, the lifespan of patients has increased markedly and many of them now live to adulthood. Although male patients are generally sterile, there are many examples of pregnancies in female patients. Progress is also being made in understanding the basic defect in CF. Electrophysiological studies show that the excessive mucus accumulation in CF patients is probably related to the abnormal regulation of ion transport in the secretory epithelia of the affected organs. A brief discussion of this subject is given below (pp. 186-191). The simple autosomal recessive mode of inheritance of CF suggested that it should be possible to identify the genetic locus (the presumed gene) responsible for this disease by linkage analysis. Although the initial attempts with conventional blood group and cell surface markers were unsuccessful, the introduction of the concept of using restriction fragment length polymorphisms (RFLPs) as genetic markers in the late 1970s and

Chapter 4: Cystic Fibrosis

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early 1980s rejuvenated the linkage analysis approach. As a result, the gene responsible for this disease was localized to chromosome 7 and subsequently isolated through molecular cloning. The major mutation causing CF has also been defined at the DNA sequence level. These studies are summarized below (pp. 191-220). The identification of the gene thus serves as a new entry point to modern CF research. In this review, we try to summarize our current knowledge about the CF gene. Our discussion on the molecular biology of the disease is rather limited, however, as not much is known about the structure and function of the encoded protein at the present time. In view of this, we have included a discussion on many of the early biochemical findings in CF in the next section, as we thought they might shed some light on understanding the basic defect and the symptoms in this disease. Finally, we have decided to organize this review from a historical perspective, so that some of the "classic" observations would not seem to be out of place. The sequence of events that led to the identification of the CF gene also serves as an example of the cloning of a disease gene on the basis of its chromosome localization. Earlier reviews on the molecular genetics of cystic fibrosis have been provided by R. White (1986), Williamson (1987), Dean (1988), Kane (1988), Wainwright and Scambler (1988), Geddes and Alton (1989), Tsui (1989), and Iannuzzi and Collins (1990).

EARLY ATTEMPTS TO IDENTIFY BIOCHEMICAL MARKERS FOR CF The recognition of the genetic basis of CF led to many early biochemical attempts at identifying the underlying basic defect. Two principal objectives were contemplated: use of the information about the nature of the defective gene product (1) for management of the disease (prognosis and treatment) and (2) as a marker for diagnosis. More than 40 years of research into the basic defect in CF without any clear-cut answer showed the difficulty of converting knowledge of the symptoms in patients into the identification of a defective biochemical step (Davis and di Sant'Agnese, 1980). Throughout this period, research was focused on various hypotheses derived from the major clinical observations made on CF patients, namely, (1) both the lung and pancreatic diseases are the result of obstructive phenomena, a consequence of the presence of excess mucus (Wood et al.,


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1976), and (2) CF patients have elevated levels of sweat electrolytes (di Sant'Agnese et al., 1953). These hypotheses could be grouped into four aspects of cellular biology; the CF defect could result from (1) derangement of membrane function, (2) abnormal complex carbohydrate metabolism, (3) the presence of a humoral circulating factor, or (4) an abnormality in the autonomic nervous system (Spock, 1969). A detailed presentation of the evidence leading to these hypotheses and the results obtained in examining them is outside the scope of this review. We will, however, present those studies that led to the testing of biochemical markers for the disease (see Table II) and discuss the evidence in favor of and against these proposed disease markers. The ability of assays to distinguish heterozygotes from CF patients and both of these from controls has relevance for studies of the basic defect. Since CF is an autosomal recessive disorder, an assay that can detect heterozygotes implies that its biochemical basis is close to the basic defect. Phenotypes useful as genetic markers need to be reliable, preferably with defined specificities and sensitivities, easy to perform, and noninvasive. Assays that meet these criteria are most likely to be performed on fluids (such as serum, urine, or amniotic fluid) or easily available cells (lymphocytes, fibroblasts, or amniocytes) derived from patients and controls. This set of requirements has complicated the development of biochemical markers since the disease appears to be manifested primarily by epithelial tissues that are relatively inaccessible. As a consequence, research into the basic defect in CF has, until the past few years, relied almost exclusively on fluids and cells where the expression of the CF gene is not known with certainty.

TABLE II. Phenotypic Markers for Cystic Fibrosis Differences observedo Marker Cellular metachromasia Cystic fibrosis factors Cellular drug resistance Protease activity Lysosomal hydrolases Microvillar enzymes Calcium and mitochondria Beta-adrenergic responses

CF vs. N

+ + + + + + + +

°CF, CF patients; H, heterozygotes; N, normals.

CF. vs. H

+ + + + + +

HET vs. N

+ + + + + + +


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Table II summarizes the principal assays that have been reported in the literature during the past 25 years and their ability to differentiate the three genotypes.

Cellular Metachromasia Cellular Staining

In the 1960s it became evident that many genetic disorders could be detected through the analysis of cultured skin fibroblasts even though patients did not have skin abnormalities. Cellular metachromasia, observed by staining cells with Toluidine Blue 0, is the consequence of intracellular accumulation of negatively charged macromolecules. Such staining had been observed in the study of lysosomal storage disorders (Neufeld et al., 1975). This technique was applied to CF in view of the increased amounts of mucous secretions in the disease. Danes and Beam (1968) reported that cytoplasmic intravesicular metachromasia could be detected in CF fibroblasts. Unlike that seen in mucopolysaccharide storage disorders (MPS), the staining material was contained within vacuoles or vesicles and was not distributed evenly throughout the cytoplasm. The cellular marker behaved as a dominant trait, since cells from CF obligate heterozygotes were also metachromatic. In the initial report, cells from 7 affected patients and 13/14 parents stained positive, whereas those from 26 healthy controls and 11 disease controls were negative. Cells from 2 out of 4 grandparents were also positive. In a subsequent study of 103 individuals in 16 families two different patterns of staining were observed to be segregating: vesicular (4 families) and generalized (12 families). This latter pattern of staining was similar to that seen in MPS. Metachromasia was observed in cells grown in bovine, but not in human, serum. The proportion of positive cells in each culture varied from 10 to 1001170 in different individuals and all parents had positive cultures. It was hoped that this cellular marker could be used to unambiguously identify heterozygotes since this would aid in linkage studies and in genetic counseling (Danes and Beam, 1969). Correlation with Other Markers It was then reported that CF fibroblasts could be better categorized by combining observations of metachromasia with two other measurements:

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presence of the cystic fibrosis factor activity (CFF; see below, p. 160) and metabolic cooperation with normal cells. This latter phenotype was observed as the disappearance of metachromatic granules in positive CF cultures when grown under conditions where cell-to-cell contact with normal cells occurred. If positive CF cultures were similarly grown with ametachromatic CF cells, no correction was observed (Danes, 1973). Under this classification, class I cells were metachromatic, produced CFF in the culture medium, and demonstrated metabolic cooperation with normal fibroblasts. Class II cells were ametachromatic, did not produce CFF, and showed no metabolic cooperation with class I cells. The hypothesis that these two types of cells represented distinct genetic groups within CF was tested by analyzing four families (Danes et at., 1975). The three cell-culture phenotypes could be reliably detected in fibroblasts and segregated in a consistent pattern. Attempts were made to correlate the phenotypes with the clinical course of the disease. A group of 46 adult patients showed that 37 out of 40 patients with a typical disease were class I, the other 3 being class II. Among six atypical patients (milder phenotype), four were class II and the other two were class I. During the course of the 4-year research period all three of the class II typical patients died. Three of the four atypical class II patients had one class I parent and one class II parent, suggesting that they could be genetic compounds (Danes et at., 1976). This hypothesis was further delineated in a study of 49 members of one such family. The maternal side segregated a class I pattern and the paternal side a class II pattern, consistent with the view that this patient represented a compound phenotype (Danes et at., 1977). Other groups have not consistently found that metachromasia is a specific marker for CF. Taysi et at. (1970) used a pediatric patient population as controls and found that 270/0 yielded positive cultures. The disorders with positive metachromasia included chromosome anomalies, inborn errors of metabolism, and miscellaneous conditions such as idiopathic mental retardation, coarctation of aorta, and multiple anomalies. They concluded that metachromasia was unlikely to be useful as a diagnostic tool. Nadler et at. (1969) assessed the usefulness of metachromasia for prenatal diagnosis, since amniocytes share many features with skin fibroblasts. They found that 38 out of 45 CF patients yielded positive fibroblast cultures, confirming the observations of Danes and Bearn (1968). However, four out of six amniotic fluid cultures of mothers at risk of having a child with CF showed metachromasia and two of these led to


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newborns with CF and the other two to non-CF babies. Of the two negative amniotic fluid cultures, only one resulted in a child with CF. Given that this method also identified heterozygotes, it is difficult to assess the significance of the false positives, since all non-CF children could be heterozygotes and therefore be expected to yield positive cultures. Reed et al. (1970) reported that while the method could distinguish non-CF from CF children, the scatter in the numbers of cells with or without metachromasia was too large to yield a useful test. Among cells from 9 non-CF children, the percentage of cells showing metachromasia ranged from 0 to 2 (mean 0.2), while in 11 CF cultures the range was 0 to 19.3 (mean 2.2). The degree of metachromasia seen in eight parents was similar to that of the non-CF children. These values for metachromasia were much lower than those reported by Danes and Beam (1968). Furthermore, Reed et al. (1970) also showed that successive analyses of the culture of a single individual also led to large variation. In nine assays over a period of 60 days, the values ranged from o to 11070 with no obvious trend. In retrospect, this test lacked the necessary specificity to diagnose CF, since metachromasia is also characteristic of many other diseases, notably MPS (Matalon, 1969), and varies with tissue culture conditions (Spicer et aI., 1980). A more troublesome problem was that the study of metachromasia did not provide strong enough clues to the basic defect in CF, and cellular metachromasia was abandoned as a subject of CF research in the middle 1970s. It is intriguing that there is preliminary evidence of elevated levels of macromolecular sulfation in CF epithelial cells (Cheng et at., 1989), since metachromasia has been considered to be due to the presence of negatively charged macromolecules in cells and has, in fact, been artificially induced in fibroblasts by loading them with sulfated polymers (Fluharty et at., 1970). Thus, it is possible that the cellular metachromasia that was observed in CF fibroblasts reflected an increased level of macromolecular sulfation, but that this increase in CF cells was not sufficiently large (or specific) to allow an indirect test (metachromasia) to distinguish CF from normal genotypes.

Cystic Fibrosis Factors Ciliary Dyskinesia Factor

In the course of studying the effect of sera from a variety of patients on ciliary motility, Spock et at. (1967) found that only those from CF patients

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disorganized the rhythmic beating of cilia in explanted rabbit respiratory epithelium and suggested that this bioassay could be used to distinguish normal from affected individuals. Application of the sera from all 75 CF patients (but from none of 75 controls) to the explants led to disorganized beating within 5-10 min. The serum component responsible for the dyskinesia was heat-labile and nondialyzable, eluting in two fractions, with the macro globulins and between gamma globulin and albumin. The activity could also be detected in parents, but only in 7 out of 25 cases. Concentration of the euglobulin fraction by 2.5-fold allowed the activity to be detected in the other 18 parents. Similar concentration of control sera did not lead to the observation of dyskinesia. While it would have been tempting to suggest that the effect of CF serum on ciliary beating had a physiological counterpart in the patients themselves, Spock et al. (1967) found that cilia in nasal polyps from CF patients had a normal rhythm. Thus, the significance of the effect of CF serum on rabbit tracheal explants was not known. Tests using nonmammalian tissues were also developed within a short time. Besley et al. (1969) showed that the motility of gill cilia of the freshwater mussel Dreissensia was inhibited by plasma of CF patients and their parents. This assay, however, was not widely used. On the other hand, the assay with the gill of the Gulf of Mexico oyster Crassostrea virginica has received extensive use. Bowman et al. (1969) were able to show that the sera of 47 homozygotes and 19 heterozygotes inhibited the rhythmic beating of the gill cilia within 40 min, while 62 out of 64 controls did not. Similar observations were made with saliva and urine. The effect of the factor was to stimulate the secretion of mucinlike material from the exposed tissue and the disorganization of the beating was likely due to interference by the macromolecular exudate.

Cystic Fibrosis Factor (CFF)

The factor causing ciliary dyskinesia was also observed in cultured CF cells, a result consistent with the view that the activity was a direct consequence of the CF gene rather than to treatment of the disease. Danes and Beam (1972) used the oyster gill assay to show that both sera and culture media from CF fibroblasts could lead to dyskinesia. They found that material from 18 adult and 18 child controls did not inhibit ciliary beating in less than 75 min. of exposure. In contrast, 24 CF homozygote


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and 24 CF heterozygote metachromatic cell cultures inhibited beating by 25 min. Interestingly, 12 CF homozygote and 12 CF heterozygote ametachromatic cultures did not inhibit beating, consistent with the suggestion of genetic heterogeneity in the disease (Danes et al., 1975). The production of the CF factor (CFF), as the ciliary dyskinesia activity became known, by cultured cells allowed it to be studied in more detail. Danes et al. (1973) found that the activity could not be detected in serum-free media that had been in contact with the cells for less than 48 hr, confirming that the factor was produced by the cells in vitro. The factor was heat-labile, pH-sensitive, and became attached to glass, since it could only be detected when cells in serum-free medium were cultured on plastic. When the medium was dialyzed with a membrane that permitted the passage of molecules smaller than 5 kilodaltons (kD), the factor was found on both sides of the membrane. Addition of IgO to the medium had no effect on its activity, but precipitation of the IgO with anti-IgO antibodies removed the activity from the cell medium, indicating that the factor was bound to the immunoglobulin. These observations were extended in a series of studies using the rabbit tracheal assay. Conover et al. (1973a) were able to improve the original assay (Spock et al., 1967) by better tissue selection and by performing the assay at 37°C and found that the activity could be detected in heterozygotes without the need for concentration of the serum. Factor activity was detected in 12/12 CF patients, 35/35 heterozygotes, and 3/4 sibs, but only 2129 controls. Ciliary dyskinesia was observed within 3-6 min of exposure and was distinguished from ciliotoxicity (immobility of cilia). Only the former was the parameter used to indicate a positive response. Conover et al. (1973b) found that sera from two heterozygotes and one homozygote with metachromatic negative cells led to ciliotoxicity in addition to dyskinesia. Further studies from this same group also showed that the factor could be detected in culture supernatants of PHA-stimulated leukocytes and of Epstein-Barr virus-transformed lymphoid cell cultures. The activity could be removed by rabbit anti-human IgO antibodies, indicating that it was bound to immunoglobulins. The activity was found in 3 patients and 2 parents, but not in 4 controls 72 hr after the start of PHA cultures. Beratis et al. (1973) found that cell culture supernatants of skin fibroblasts cultures from 13 CF patients and 6 parents had factor activity only if human IgO was added. No such activity was found in 15/16 controls. The activity was removed from the medium plus IgO with anti-IgO antibodies. The media from a culture of amniotic fluid

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cells of a pregnant CF woman was positive, but 10/11 controls were negative in the assay. Thus, cells with one or two copies of a mutant CF gene could produce a small « 5 kD) molecule that disorganized the beating of cilia. This activity required the binding of the factor to IgG in the rabbit tracheal assay, but not in the oyster gill assay. The chromosomal location of the gene coding for this factor was studied with somatic cell techniques. B. J. Mayo et al. (1980) showed that the ability to produce the factor co segregated with human chromosomes. The location of the gene responsible for the production of the factor was tentatively mapped to chromosome 4, since this was the only nondiscordant chromosome. However, the number of hybrids analyzed was not large and single discordances were also seen for chromosomes 2, 6, and 18; more importantly, chromosomes 3, 5, 7, 8, 13, 15, and 22 were not studied. Although inconclusive, this report later led to linkage studies on chromosome 4 (see below, p. 193). Bowman and co-workers used the oyster cilia assay to monitor partial purification of the factor [summarized in Bowman et al. (1980)]. Because of its association with IgG, it was thought that the dyskinesia activity was due to an immunological reaction. Results from a detailed examination of the interaction of the factor with the oyster gills were, however, not consistent with an immune reaction (Herzberg et al., 1973). Furthermore, the factor could be dissociated from IgG by treatment with guanidine, leading to a small polypeptide of 6-11 kD (Bowman et al., 1975). The properties of the factor isolated from heterozygote sources were similar to those of the factor from homozygotes. Blitzer and Shapira (1982) also purified the factor from serum using the rabbit tracheal assay. A glycopeptide fraction in the 5-kD range was found in both CF and control sera after chromatography in DEAE-cellulose, ultrafiltration, and Bio-Gel P-lO column chromatography. The CF samples were from both an individual sample and from pooled samples and had dyskinesia activity, while those from individual control sera did not. The glycopeptides appeared not to have aromatic amino acids. Only one band was seen in thin-layer chromatography and isoelectric focusing. The CF material caused ciliary dyskinesia within 30 sec and subsequently led to swelling and secretion of mucus like droplets. Control materials had no effect even at a 50-fold excess (Blitzer and Shapira, 1982). A subsequent publication showed that the dyskinesia activity was lost when an O-linked oligosaccharide was removed and also that the dyskinesia activity could be inhibited by the normal glycopeptide as well as by mannose, glucose, and N-acetylgalactosamine (Blitzer and Shapira, 1984).


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Cystic Fibrosis Protein

While none of the above studies led to purification of CFF, Wilson et al. (1975) showed that a specific protein could be identified using isoelectric focusing. Analysis of serum samples in a pH 5-10 gradient allowed the detection of a broad band with pI of 8.4-8.5 in sera from CF patients (homozygote) and carriers (heterozygote). In the initial report, this protein (named CFP) was detected in 63 of 65 CF homozygotes, 57 of 61 heterozygotes, and only 9 of 105 controls. The presence of CFP was then shown to correlate with the ability of the serum to cause ciliary dyskinesia in the rabbit tracheal assay in all 31 CF homozygote or heterozygote samples, whereas 13 of 14 control samples did not have CFP or cause dyskinesia (Wilson et al., 1977). In addition, 7 samples from patients with asthma had dyskinesia activity, but did not show the CF protein at pI 8.4-8.5. These investigators suggested that several substances in human serum could cause dyskinesia, but only one was a specific marker for CF. Attempts in other laboratories to use this assay as a diagnostic tool met with mixed results. Some of the difficulty was the result of unrecognized experimental variables or failure to follow the specified protocol (Wilson et al., 1984; Thomas et al., 1977). In most instances it was found that while the high-pI band was present more consistently in donors with at least one copy of the defective CF gene, the correlation was not sufficiently high to allow this method to be used in diagnosis (Hallinan et al., 1981; Nevin et al., 1981; Vickers et al., 1982; D. J. H. Brock et al., 1982). D. J. H. Brock et al. (1982) concluded, however, that these results were consistent with the existence of a protein associated with the mutant gene. Manson and Brock (1980) purified the pH 8.4-8.5 band and used it to raise antibodies in guinea pigs. This antibody yielded positive reactions in a rocket immunoelectrophoresis assay with 16117 CF homozygote serum samples, 8/9 heterozygote samples, and only 1115 control samples. In general the size of the homozygote rockets was higher than that of the heterozygotes, consistent with a gene dosage effect. A subsequent antibody with greater specificity allowed Bullock et al. (1982) to use quantitative immunoprecipitation and immunoradiometric assays to give greater than 94070 reliability in the assignment of the three genotypes (13/14 homozygotes, 28/29 heterozygotes, and 22/23 controls). Cystic Fibrosis Antigen

As search of CFP continued, the protein detected by the Brock antibodies was termed CF antigen (CFAg) to distinguish it from Wilson's

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protein. Van Heyningen et at. (1985) discovered that CFAg was synthesized by granulocytes and that it was also found in elevated amounts in the sera and cells of patients with chronic myelogenous leukemia (CML). Peripheral leukocytes from a patient with CML were used to construct a series of somatic cell hybrids with a mouse myeloid stem cell line. The segregation of CFAg was concordant with human chromosome 1 in 16 sub clones from four independent hybrids (van Heyningen et at., 1985), but it was clear that CFAg was not the CF gene, because the disease locus was mapped to chromosome 7 by linkage analysis (Tsui et at., 1985a; Knowlton et at., 1985). Monoclonal antibodies against CFAg were raised by immunization with CML lysates (Hayward et at., 1986). Analysis of the immunoprecipitated labeled antigen by SDS-polyacrylamide gel electrophoresis showed that all ten monoclonal antibodies bound to a 12.5-kD protein. Using an enzyme-linked immunoabsorbent sandwich (ELISA) assay, Hayward et at. (1987) measured quantitative differences in serum samples of the three genotypes and found that, while the means were statistically significant different (all comparisons P < 0.001), there was considerable overlap among the individual values. To provide a better comparison between CF patients and controls, the levels of the antigen were examined in sera of 50 CF patients, 34 heterozygotes, 25 "disease" controls, and 60 normals in relationship to those of another granulocyte-derived protein (lactoferrin, Lf) and also to the acute-phase reactant C-reactive protein (CRP), a marker of acute inflammation. Disease controls consisted mainly of individuals with respiratory disorders. The results of the study confirmed that the levels of CFAg were elevated in CF patients relative to normals, with the heterozygote yielding intermediate values; however, the same pattern was detected with Lf and CRP, such that differences in the ratios CF Ag/Lf or CFAg/CRP became nonsignificant for several of the comparisons. The latter observation raised the question of whether there was any connection between CF Ag levels and the CF gene or whether these represented the compound effect of granulocyte proliferation and active tissue damage. Since both CF patients and disease controls were clinically affected, it would be difficult to separate the effects of disease from those of the mutant gene. Hayward et at. (1987) therefore focused on the comparison between CF heterozygotes and normal subjects, since it was assumed that both of these groups would be clinically normal. In this comparison, the levels of CF Ag were raised in the heterozygotes relative to normals (P < 0.001), while those of Lf were not. The ratio CFAg/Lf was


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therefore significantly different (P < 0.001). This observation was interpreted as indicating some association between CFAg and the CF gene (Hayward et af., 1987). Calcium-Binding Proteins

The close relationship between CFAg and CF suggested that production of CF Ag could be a specific secondary consequence of the basic defect and that understanding its structure and/or function could shed light on the role of the mutant CF gene product. Dorin et af. (1987) used a partial amino acid sequence from a purified sample of CFAg to isolate cDNA clones from a library constructed with mRNA from CML cells and found that the complete sequence of this protein had significant homologies to that of calcium-binding proteins. Bruggen et af. (1988) claimed that the sequence of the CF antigen cDNA was identical to one of two leukocyte proteins (MRP-8 and MRP-14) that also mapped to chromosome 1. While the sequence of CFAg was almost identical to MRP-8, levels of MRP-8 were not elevated in CF samples. On the other hand, levels of MRP-14 were elevated. They suggested that Dorin et af. (1987) probably had isolated the CFAg as a complex of the two proteins and had obtained the sequence only of MRP-8 because that of MRP-14 was blocked at the N terminus. Andresson et af. (1988) have proposed that MRP-8 and MRP-14 proteins correspond to the light and heavy chains, respectively, of the major leukocyte protein, L1. Wilkinson et af. (1988) subsequently reported that monoclonal antibodies specifically recognizing CFAg allowed the immunopurification of two proteins, 11 and 14 kD in size, that they named calgranulin A and B. These are identical to the proteins described by Bruggen et af. (1988) and Andersson et af. (1988). Both of the genes coding for these proteins mapped to chromosome 1 (Wilkinson et af., 1988). Murao et af. (1989) attempted to define the biological role of CFAg by purifying a protein complex that contains MRP-14 and that is expressed during normal myeloid differentiation (Odink et af., 1987). The complex was isolated from human spleen using a monoclonal antibody that recognizes a myeloid cell nuclear antigen. The complex inhibits the activity of casein kinases I and II, but not of cAMP-dependent protein kinase, protein kinase C, v-abl, or insulin receptor tyrosine kinases. Addition of the purified complex to a nuclear extract prevented the protein kinase-mediated stimulation of RNA polymerase activity. Since defects in functions related to the activity of protein kinases have been observed in the study of ion

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transport in CF epithelial cells (see below, p. 188), it is still possible that the presence of elevated levels of CFAg is a consequence of the mutant CFTR. In summary, the long history of the CF dyskinesia factor/CF protein/ CF antigen/calgranulins is still a footnote to the main story. It illustrates vividly how little is known about the specific metabolic disturbances caused by the CF gene and therefore how difficult it has been to establish whether physiological or biochemical phenotypes seen in CF patients and heterozygotes represent a direct line to the mutant gene or only an adventitious finding.

Cellular Drug Resistance Ouabain Resistance

The elevated amounts of Na + and CI- in CF fluids and the observations that CF sweat and saliva could interfere with ion transport in salivary and sweat glands of normal individuals (Mangos et al., 1967; Taussig and Gardner, 1972) led Epstein and Breslow (1977) to examine aspects of ion transport in CF fibroblasts. They examined the survival of CF and control fibroblasts after exposure to the cardiac glycoside ouabain, known to inhibit ion transport through its effect on Na/K ATPase. They found that in medium deficient in K +, CF fibroblasts were resistant to the cytotoxic action of ouabain. For example, at 10- 6 M ouabain, survival of CF fibroblasts was 50%, while that of normal fibroblasts was 10070. A comparison of five CF strains and five control strains showed that the ratio of the relative survivals (CF/N) at 10- 7 M ouabain ranged from 2.7 to 4.8 in 12 separate determinations. It should be noted, however, that the survival curves reported by these investigators were quite different from those observed in other laboratories. While Epstein and Breslow (1977) found 10% survival at 10- 6 M ouabain, Mankovitz et al. (1974) found that exposure to that amount of ouabain reduced survival to less than 1 in 106 . The increased resistance of CF fibroblasts to ouabain was reported to be due to a decreased binding of ouabain (Breslow et al., 1977). Dexamethasone Resistance

This phenotypic difference between CF and control fibroblasts was then extended to include dexamethasone (Epstein et al., 1977), sex steroids


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(Breslow et al., 1978a), and dibutyryl cyclic AMP and drugs that raise intracellular levels of cAMP (Epstein et al., 1978). The increased resistance to dexamethasone, which can be determined with a technique easier to use than that for ouabain, was proposed as an assay to detect CF heterozygotes and for prenatal diagnosis. Breslow et al. (1978b) compared seven CF homozygote strains from five families, nine CF heterozygotes, and eight controls and found that all three genotypes could be distinguished. For example, at 10 - 8 M dexamethasone, the survival of the CF homozygote fibroblasts was 106 ± 70/0 (relative to drug-free plating), that of CF heterozygotes was 51 ± 5%, and that of controls was 37 ± 5%. While individual values sometimes overlapped, especially between CF heterozygotes and controls, use of the whole survival curve as the diagnostic tool allowed them to distinguish all three genotypes unambiguously. Similar results were obtained with amniotic fluid cells. An amniocyte culture and the corresponding fetal fibroblast strain from a fetus with a 114 risk of having CF gave curves indistinguishable from those observed with CF fibroblasts. However, it was not possible to confirm the diagnosis of CF in the fetus. Attempts by other laboratories to reproduce these results were not successful. Kurz et al. (1979) found no differences in the survival of CF or control fibroblasts after exposure to dexamethasone or dibutyryl cAMP. In fact, very little killing could be detected even at concentrations where Epstein et al. (1977) reported survivals of 10%. Similarly, Weichselbaum et al. (1980) found very little killing of human fibroblasts by a series of sex steroids and dexamethasone. Daniel et al. (1981) did find differences in the survival of fibroblasts of the three genotypes after exposure to dexamethasone. They studied 5 CF homozygotes, 10 CF heterozygotes, and 17 controls; at 10- 6 M dexamethsaone the survival of homozygote cells was 117 ± 60% (relative to no dexamethasone), that of heterozygote cells was 96 ± 61 %, and that of controls was 50 ± 52%. However, these differences were not statistically different. Sensitivity to membrane-active drugs could therefore not be used as an assay to distinguish control from CF fibroblasts.

Sodium Transport Abnormalities

Notwithstanding the contradictory reports mentioned above, Breslow et al. (1981) reported that the sodium transport properties of CF fibroblasts were distinguishable from controls and could be used for diagnosis. CF

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fibroblasts accumulated less 22Na than controls when incubated in the presence of ouabain. Eight CF strains, 8 heterozygotes, and 13 controls that had previously been analyzed for drug sensitivity and had been found to respond in the manner described above were compared. When expressing 22Na accumulation as the ratio ouabain-exposed/ -unexposed, controls gave values of 2.2 ± 0.2, and CF homozygotes, heterozygotes, and three sibs gave values of 1.3 ± 0.1. These differences were highly significant (P < 0.001). In response to questions on the validity of the assay to serve as a diagnostic tool (Erbe et al., 1981), a blinded study was performed. This revealed that a set of 28 strains showed either low or high sodium accumulation. However, there was no association between this phenotype and the presence of the CF gene. Thus, the assay could not be used for CF diagnosis (Breslow and McPherson, 1981). Interestingly, alterations in the transport properties of Na + and Cl- in CF fibroblasts have been demonstrated more recently (Mattes et al., 1987; Lin and Gruenstein, 1987), and it is still possible, therefore, that these early measurements lacked specificity due to some uncontrolled experimental variable. However, it is also possible that too few samples were analyzed and results were not the consequence of the CF gene, but of some other genetic or cell culture variable.

Protease Activity A series of interrelated but controversial observations were reported in the 1970s on the amount of proteolytic activity in CF fluids that led, at one point, to their use in prenatal diagnosis of CF. Kallikrein Deficiency

Rao and Nadler (1972) first reported that trypsinlike activity measured as the hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester (BABE) could be detected in human saliva. This activity could be inhibited by soybean trypsin inhibitor (STI). Comparison of saliva from CF patients, parents, and controls led to significant differences. The value for 19 controls was 1.65 ± 0.66 p.mole BABE hydrolyzed/hr per mg protein, for 20 patients was 0.22 ± 0.22, and for 20 parents was 1.05 ± 0.51 (P < 0.001 CF vs. controls or parents; P < 0.02 parents vs. controls). The inhibition of the


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activity of STI and the fact that other substrates such as alpha-N-benzoylL-arginine-amide or alpha-N-benzoyl-oL-arginine-nitroanilide were not hydrolyzed suggested that this activity was similar to that of a number of kallikreins. Furthermore, they postulated that the presence of CF factors in fluids of CF patients and heterozygotes was the consequence of the reduced kallikrein activity. Rao et al. (1972) then reported a deficiency of kallikrein activity in plasma from CF patients. The deficiency could be measured as both total and as STI-inhibited kallikrein activities. The values for 35 controls of all ages were 42 ± 13 p.mole substrate hydrolyzed/hr per ml plasma (total) and 26 ± 12 (STI-inhibited), and for 37 CF patients were 21 ± 11 and 11 ± 6, respectively. The comparisons between controls and CF patients were significant (P < 0.001 for total activity and P < 0.02 for STI-inhibited activity, respectively). A study of 27 CF heterozygotes gave values of 32 ± 10 and 18 ± 8 for the same two measurements. These initial results set the pattern for subsequent observations: a large overlap in individual values coupled with heterozygote values that fell between those of CF patients and controls. While the observation that heterozygotes had intermediate values would be consistent with the phenomenon being closely related to the basic defect, the large dispersion in individual data points could result from either uncontrolled experimental variables or from the fact that the phenomenon was distantly related to the basic defect. To resolve these internal inconsistencies in the data, refinements in the measurements were attempted. Rao and Nadler (1974) partially purified the arginine esterase activity by chromatography and resolved the STI-inhibitable from the STI-resistant activity. In CF plasma the STI -inhibitable activity was reduced by about 30070, while the STI-resistant activity was not changed. A band was also missing in CF samples after resolution of plasma on polyacrylamide isoelectric focusing. The deficient activity was shown to be proteolytic by assaying the hydrolysis of peptide bonds using an artificial fluorescent substrate (Rao and Nadler, 1975). The decrease in plasma arginine esterase activity in CF was confirmed by Chan et al. (1977), who compared CF children, CF adults, and parents with age-matched controls and found statistically significant differences in all cases. Though the differences in the means were meaningful, large overlaps in individual values were detected. For example, the values for CF children was 28 ± 8 (range 13-42) and for controls was 45 ± 18 (range 12-82) (P < 0.003). Other laboratories reported similar observations (Coburn et al., 1974; Seale and Rennert, 1977).

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MUGB Hydrolysis

In further attempts to overcome the diagnostic problems caused by the overlapping data points, an easier assay using a fluorescent active-site titrant of serine proteases (methylumbelliferyl guanidinobenzoate, MUGB) was developed and used to quantitate the levels of proteases in fluids. Rao et al. (1978) quantitated the amount of protease present in plasma by the amount of MU liberated. They found that the reaction between MUGB and proteases was complete in about 1 hr and that the liberation of MU was proportional to the amount of plasma. The reaction value (nmole MU released/ml plasma) for 18 controls was 225 ± 18, for 13 heterozygotes was 168 ± 15, and for 20 CF patients was 135 ± 18. All three comparisons were significant (P < 0.001). These observations were extended to skin fibroblasts. Activity measured in the particulate fraction distinguished the three genotypes: controls (N), 1.27 ± 0.11 nmole MU released/mg protein; heterozygotes (H), 0.82 ± 0.12; and CF patients, 0.66 ± 0.10; P < 0.01 for all comparisons. Activity could also be detected in amniotic fluid cells. The assay of MUGB hydrolysis in amniotic fluid was then used for the prenatal detection of CF. Measurement of MUGB-reactive proteases in over 1000 midtrimester human amniotic fluid samples yielded a mean value of 2.37 ± 0.41 nmole MU released/mg protein and appeared not to be different in samples from abnormal pregnancies such as biochemical or chromosomal abnormalities, neural tube defects, or abortions/stillbirths (Walsh and Nadler, 1980). Analysis of four fluids from pregnancies with a known outcome of a CF child showed that the values of MU-releasing activity were lower (by about 112) than controls. Nadler and Walsh (1980a,b) reported on the prospective analysis of 13 pregnancies of obligate heterozygotes; 3 pregnancies leading to patients with CF had abnormal MUGB reactivity; however, the absence of a method to confirm the presence of CF in a midtrimester fetus made evaluation of this prenatal diagnosis assay difficult without allowing the fetuses to go to term. A further prospective study of 39 pregnancies at a 114 risk was also reported (Nadler et al., 1981a). At the time of the report 24 had been delivered and 14 were still in progress. Of the 24 reported, 4 were correctly predicted to be affected, 1 had been aborted after a prediction of CF, and 18/19 predicted unaffected were correct. There had therefore been one false-negative report. The value for MUGB-specific activity was 2.35 ± 0.45 (range 1.50-3.8) for 2000 controls, and was 1.21 ± 0.08 (range 1.08-1.34) for the CF pregnancies. Two other predicted pregnancies had values of 1.34-1.40. A subsequent report gave 45/49 correct results (Nadler et al., 1981b).


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Attempts by other laboratories to use this method were not successful. Green et al. (1982) used the MUGB assay in a retrospective study of 84 second- and third-trimester pregnancies, one of which was known to be CF. Control values were 4.25 ± 1.36 and 6.12 ± 1.04 nmole/mg protein for second- and third-trimester fluids, respectively. The value for the CF sample was 5.64. Schwartz and Brandt (1982) were not able to confirm even the original findings and could not distinguish CF plasma from control plasma, nor amniotic fluid from CF pregnancies from that from control pregnancies. D. J. H. Brock and Hayward (1983) studied 19 pregnancies at a 114 risk of CF. MUGB-reactive protease values suggested that all would be unaffected; however, among the first ten to come to term, five were affected. They concluded that the method was not suitable for prenatal diagnosis in CF. The inability of the assay to be replicated outside of the original laboratory led others to investigate the biochemical basis of the MUGB assay. Tummler et al. (1982) found that they could not distinguish control from CF plasma. Furthermore, they reported that the reaction was not characteristic of an active-site titration and that instead, a significant portion of the MUGB hydrolysis was catalyzed by the imidazole ring of histidine. Schwart (1982) reexamined the plasma reactivity by using diisopropylfluorophosphate (DFP) and found that MUGB and DFP were reacting with the same molecule. Using crossed immunoelectrophoresis and SDS-polyacrylamide electrophoresis of 3H-DFP-Iabeled plasma and amniotic fluid, Schwart found that the major proportion of MUGB hydrolysis was catalyzed by serum albumin. Branchini et al. (1983) compared plasma samples with commercial albumin samples and found that albumin had weak esterase activity. Since hypoalbuminemia was detected in 42070 of CF patients, but only in 6% of heterozygotes and 4% of controls, they concluded that the decreased levels of MUGB hydrolysis in CF were a secondary consequence of the hypoalbuminemia. Since there was no connection between MUGB reactivity and the basic defect in CF, the assay was not suitable for the detection of CF. Thus, the biochemical basis of the assay was not substantiated and the method was abandoned as a reliable test for CF.

Alpha-2-Macroglobulin

Concurrently with the MUGB studies, a series of papers examined other aspects of protease activity in CF serum. Wilson and Fudenberg (1976) found that a protein with a pI of 5.48 was deficient in plasma from

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most CF patients and heterozygotes compared to controls. Purification of this protein revealed that it was a fragment of alpha-2-macroglobulin (cx2M) derived from intact cx2M by proteolytic cleavage. There were, however, no quantitative differences in the cx2M levels in plasma samples of the three genotypes. Wilson and Fudenberg (1976) studied arginine esterase activity in plasma and found, similar to the results described above, that such activity was deficient in CF samples. They speculated that the lower of amounts of the pI 5.48 band could be due to either a protease deficiency in CF or to an abnormal cx2M. The cx2M from CF plasma was found to have reduced protease-binding activity (Shapira et al., 1976a). The average amount of 125I_trypsin bound to 10 normal plasma samples was 8 x 105 cpm/t.tg cx2M [range (6.2-10) X 105], for 13 heterozygotes it was 6 x 105 [range (4.3-7.1)] and for 15 CF patients it was 4.8 x 105, [range (3.5-6.2) x 105]. The differences in the means were statistically significant for all three comparisons (P < 0.01). Since the aberrant cx2M activity could be detected with several proteases, they proposed that CF cx2M had an abnormal structure. Kinetic and immunological evidence in favor of this hypothesis was presented in several subsequent papers. Shapira et al. (1976b) raised antibodies against the pI 5.5 protein from controls and used it to analyze plasma from CF samples and controls. Upon immunoelectrophoresis, controls yielded four precipitin arcs, while only three could be seen in the CF samples. Testing with specific antisera revealed that the missing arc in the CF samples was cx2M. Comparing the amount of cx2M determined immunologically with the amount detected by trypsin binding gave a ratio of 0.9 for control samples and 0.65 for CF samples. Shapira et al. (1976b) suggested that the cx2M in CF samples had a defective capacity to bind proteases. A double-blind study of 15 control and 15 CF samples led to the correct identification of 13 controls and 14 CF samples. Kinetic comparisons between purified cx2M from three controls and three CF samples gave lower Ki (for the inhibition of binding by BAEE) and higher Km (for trypsin) for the CF enzyme (Shapira et al., 1977a). Gel chromatography of the normal enzyme preincubated with trypsin led to formation of cx2M fragments, but this was not observed with the CF enzyme. The complex formed between trypsin and cx2M purified from normal and CF individuals was subsequently studied. The complex with the CF enzyme had no activity toward high-molecular-weight substrates, but retained activity toward low-molecular-weight substrates (Shapira et al., 1977b). Ben-Yoseph et al. (1979) found normal amounts of hexose, but approximately 40070 less sialic


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acid in purified CF a2M compared to controls. Three CF samples had 24-28 residues per molecule of purified a2M, three heterozygotes had 34-37 residues, and three controls had 39-41 residues. As a consequence, the binding of CF a2M to immobilized concanavalin A and wheat-germ agglutinin was markedly reduced (CF: 16-20070, H: 44-50%, N: 60-68%). Surprisingly, Shapira and Menendez (1980) found the opposite; CF a2M gave increased binding to concanavalin A. The above set of results led to the suggestion that the absent arginine esterase activity mentioned above was due to a missing a2M-protease complex. Two sets of observations ultimately led to the rejection of the view that a2M was defective in CF. First, Romeo et at. (1978, 1979, 1980) and Denaro and Romeo (1980) showed that 80% of CF patients, 30% of their parents, but only 3% of controls had antibodies to the bovine or porcine trypsin that was part of the normal CF therapeutic regime. This anti-trypsin antibody competed with a2M in binding trypsin and, thus, individuals with elevated levels of anti-trypsin antibody would also show decreased levels of trypsin-a2M complexes (Romeo et at., 1979, 1980). Second, a2M isolated from CF and control plasma had similar trypsin-binding activity (Parsons and Romeo, 1980; Burdon, 1980; Schidlow and Kueppers, 1980; Bridges et at., 1982; Bury and Barrett, 1982; Beck and Alhadeff, 1983). Parsons and Romeo (1980) purified a2M from five CF and five control plasmas and could not detect any differences between them by isoelectric focusing, trypsin binding, and subunit molecular weight. Burdon (1980) also purified a2M from six CF patients and a similar number of controls and could find no differences in subunit cleavage. Schidlow and Kueppers (1980) reported a moderately increased molar binding ratio of trypsin to a2M in CF as well as in several other disorders. Bridges et at. (1982) studied the binding of proteases to purified control and CF a2M and found no differences in molar protease-binding ratio, in the interaction of the bound protease with small-molecular-weight substrates, in the stability of the complex, or in the subunit structure of a2M. Bury and Barrett (1982) found that the arginine esterase being studied was indistinguishable from plasma kallikrein, and that a2M -bound activity could not be distinguished between controls and CF samples. Beck and Alhadeff (1983) measured the enzymatic activities and isoelectric points of purified a2M and found no differences between CF and control molecules. Thus, all attempts to demonstrate a CF-specific difference in the activity or structure of proteases or protease-binding proteins were ultimately negative and these assays have not been used to detect CF genotypes.

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Trypsinogen Assay

In sharp contrast to the attempts to link the CF basic defect to protease activity, the use of a phenotypic assay involving trypsin has been successful for screening newborns. In 1979 two groups reported that elevated levels of immunoreactive trypsin (lRT - in reality, trypsinogen) were characteristic of CF patients in the first few months of their lives. Whereas elevated levels of IRT were consistently seen in children under the age of 1 year, older children with CF gave inconsistent results until about the age of 6 years, when values were near O. However, CF children with residual pancreatic function continued to have elevated IRT levels (Crossley et aI., 1979). King et at. (1979) were able to show that elevated levels of serum IRT could be detected in the dried blood spots collected for screening of other inherited diseases, even after storage for more than 3 years. A subsequent retrospective study of 24 cases confirmed that elevated blood IRT levels were characteristic of aIr newborn CF patients, regardless of their pancreatic function status (Crossley et at., 1981). Several more extensive studies were then reported. Crossley et at. (1981) studied 5040 newborns prospectively. Approximately 0.2070 of the samples had levels greater than 250 arbitrary units/liter and were therefore retested. Only one of the retested samples had higher than normal values. The child had no symptoms of CF, but yielded two abnormal sweat tests. No false negatives were known. Heeley et at. (1982) studied 14,000 newborns in East Anglia, United Kingdom, and also retested approximately 0.2070 of the samples. Five affected infants were identified. At the time that the report was published, no false-negative results had been discovered. Wilcken et at. (1983) reported the results of screening 75,000 newborn infants in New South Wales, Australia. A total of 433 samples were retested (0.6070), leading to 38 samples with persistent high values. Sweat testing confirmed the diagnosis of CF in 35 babies and was normal in 2 others. One was refused. Among the 35 positive cases, 13 had meconium ileus or an already diagnosed sibling. The other 22 cases represented unsuspected CF patients, although some suggestive symptoms were present in 18. Cassio et at. (1984) reported the use of the IRT assay to screen 47,127 newborn infants from the Emilia-Romagna region of Italy. They retested 0.9070 of samples and found 11 with repeated high values. Of these, 6 had positive sweat tests, while 5 had normal results with no evidence of CF. No false-negative results from the screen were known. Thus, this test, while


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clearly based on the phenotype of the patient, has a high degree of specificity and sensitivity and has become the test of choice in current screening protocols.

Lysosomal Hydrolases Because of the presence of abnormal amounts (and perhaps abnormal types) of mucus in CF secretions, it was hypothesized that CF could be due to an abnormality in glycoprotein metabolism. A large body of literature examining this area of biochemistry in CF exists, but no specific abnormality in glycoprotein metabolism has been convincingly demonstrated (see Boat et at., 1989). However, a series of puzzling and provocative findings involving enzymes of glycoprotein metabolism were published in the late 1970s by Hosli and co-workers that to this day remain unexplained. Lysosomal Enzyme Induction

Hosli et at. (1976) suggested that CF could be considered a lysosomal storage disease because fibroblasts showed metachromatic staining. In studying lysosomal storage disorders, Hosli (1976) had been able to show, through the use of micromethods that measured enzyme activities in single cells, that exposure of fibroblasts to nondigestible macromolecules led to the coordinate induction of intracellular digestive enzymes such as alkaline phosphatase or acid lysosomal hydrolases. Thus, in alpha-glucosidasedeficient Pompe cells, exposure to glycogen would lead to the coordinate induction of the digestive hydrolases. Working from the assumption that CF was a disorder of abnormal glycoprotein catabolism, Hosli et at. (1976) examined the response of control and CF fibroblasts to a challenge with the human Tamm-Horsfall urinary glycoprotein (THP). They found that alkaline phosphatase could be induced in CF, but not in control, fibroblasts. Induced alkaline phosphatase activity in ten CF strains was in the range (19-59) X lO-13 mole phosphate produced/hr per 20 cells and in three controls was (4-12) X lO-13. Pairwise comparisons gave a Student's t-test significance of P < 0.01. These differences represented a combination of higher basal activities in CF fibroblasts and a higher degree of induction, but the two contributions were not separated. Hosli et at. (1976) suggested that this test could be used for prenatal diagnosis of CF.

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Lysosomal Enzyme Leakage

Hosli and Vogt (1977) then showed that induced CF cells leaked lysosomal hydro lases into the culture medium. They found that the amounts of ten hydro lases in the culture medium ranged from 130070 to > 4000% of controls, while intracellular levels were usually less than 50070. The only exception was beta-glucuronidase, where levels were near normal. Data from parents were similar, but not as extreme as seen in the affected children. The procedure was tested by assaying 9 control and 17 CF strains and by assessing two further modifications of the induction protocol. With THP alone, the controls gave an induced value of (7-11) X 10- 13 mole MU released/hr per 20 cells and the CF strains (20-55) x 10- 13 • Values after induction with THP and isoproterenol and theophylline or with these two plus ascorbic acid were not much different (Hosli and Vogt, 1978). Analysis of 11 different amniotic fluid cell samples gave alkaline phosphatase activities similar to controls, consistent with the idea that this assay could be used for prenatal diagnosis. A double-blind study of 6 control, 12 heterozygote, and 6 CF fibroblast strains measured alkaline phosphatase activity before and after induction with THP in combination with isoproterenol and theophylline (Hosli et al., 1978). All 24 strains were correctly identified. For the 6 controls, the ratio of activity induced/noninduced ranged from - 3.0 to + 2.8 (mean - 0.2), for the 12 heterozygotes ranged from 0.8 to 6.8 (mean 3.6), and for the 6 CF strains ranged from 14.7 to 24.7 (mean 20.6). Using one-sided tolerance limits, the authors concluded that 99% of THP-induced CF fibroblasts have alkaline phosphatase activities higher than 19 x 10- 13 mole MU released/hr per 20 cells and that more than 95% of CF heterozygotes and more than 97.5% of controls have alkaline phosphatase activities lower than this value. While it is not possible to use the induced activities to distinguish individual CF homozygotes and heterozygotes, as a group the difference between them was statistically significant (P < 0.01). To explain the phenotypes of elevated amounts of alkaline phosphatase seen upon induction and of increased enzyme leakage it was hypothesized that CF was a disorder of abnormal enzyme recognition, similar to I-cell disease (Hosli et al., 1979). The mutation in CF affected a gene coding for a processing enzyme (e.g., a specific glycosyltransferase or phosphokinase) leading to abnormal enzyme recognition sites. The altered enzyme recognition sites interfered with the appropriate processing of hydrolases, causing their leakage into the extracellular space. Further aspects of this proposed


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biochemical cascade accounted for the presence of CF factors, impaired catabolism of macromolecules, impaired mucin metabolism, deranged cAMP/cGMP levels, and finally, abnormal function of exocrine glands. Hosli et al. (1979) reported that 131 cell lines had been studied with THP induction and all but 2 CF samples (belonging to sibs) were correctly identified. Using the enzyme leakage test, 57/60 were correctly identified; the 3 incorrect were the 2 sibs mentioned above and their mother. However, a second double-blind study with 27 other cell lines led to only 7 correct diagnoses. The discrepancy with the original double-blind study could not be explained. The presence of normal medium during induction of CF cells with THP led to inhibition of increase in alkaline phosphatase (Carey and Hosli, 1979). In contrast, CF medium did not stimulate control cells. This result was interpreted on the basis of the above hypothesis, namely that CF cells lacked a component (enzyme?) and that this absence led to the increased induction and leakage. This component was provided by the medium from normal cells and corrected the defective CF phenotype. Such "corrective factors" had been detected by in vitro complementation studies of 35S accumulation in MPS fibroblasts (Fratantoni et al., 1969). The MPS corrective factors were found to be enzymes responsible for glycosamino glycan and glycolipid degradation (Neufeld et al., 1975). These assays employed micro methods that were difficult to set up, since they required specialized materials, and had discouraged investigators' from exactly replicating the above observations. Using conventional assays, THP-stimulated induction of alkaline phosphatase (Wijcik et al., 1979; Aitken and Hoogeveen, 1980) or increased leakage of lysosomal enzymes into the medium of CF fibroblasts (Harris, 1981; Jessup and Dean, 1982) could not be reproduced. In addition, some work could not be reproduced outside of the Hosli laboratory (Carey and Pollard, 1979).

Temperature-Sensitive Lysosomal Hydrolases

Since the CF lysosomal hydrolases were predicted to have abnormal recognition markers, it was possible that other structural abnormalities were also present. Hosli and Vogt (1979a) tested this possibility by analyzing the thermo stability of lysosomal hydrolases. Incubation of a-mannosidase from the culture fluid of fibroblasts or from plasma of the three genotypes led to markedly different degrees of inactivation. After 4 hr

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Lap-Chee Tsui and Manuel Buchwald

at 41°C, the normal samples had not lost any activity, but that from a heterozygote had approximately 50070 residual activity and that from a CF homozygote had 5-20% activity_ A further publication (Hosli and Vogt, 1979b) reported that CF acid phosphatase was also more heat-labile (at 36.5°C) than those from heterozygotes and controls. Values for activities of a-mannosidase and acid phosphatase from 20 normal plasmas were 95 ± 5 % and 94 ± 4 %, respectively, of unheated values after 200 min of heating. Activities from 22 heterozygotes were 51 ± 3% and 45 ± 5%, respectively, and from 5 CF patients were 19 ± 6% and 10 ± 4070, respectively. Since thermo stability measurements were easy to perform, several laboratories immediately repeated the experiments with plasma (Hirani and Winchester, 1979; Patrick and Ellis, 1979; Harris et al., 1979; Hultberg et al., 1981; Kohlschutter and Jacobsen, 1983) and with culture fluid of fibroblasts (Butterworth, 1980), but all failed. Ceder and Kollberg (1983) measured the uptake of acid hydro lases from medium conditioned by CF fibroblasts into three cell lines with different enzyme deficiencies and found that CF hydrolases were taken up normally. This result was not consistent with the defective enzyme recognition hypothesis. On the other hand, it was possible for other investigators to reproduce the heat lability assay in Hosli's laboratory (Sack et al., 1980; Katznelson et al., 1981), including a doubleblind test in which 45 samples were correctly identified (Katznelson et al., 1983). Furthermore, Ceder et af. (1983) reported a three-generation study of four CF families where the genotypes could be clearly distinguished from each other by measuring heat lability, extracellular secretion of hydrolases, and intracellular induction of alkaline phosphatase. The conflicting results have never been resolved. The inability of the Hosli laboratory to determine the molecular basis of the increased lability of CF lysosomal enzymes and, perhaps, the existence of uncontrolled variables that made the measurements irreproducible in other laboratories discouraged the further exploration of the mechanism underlying the changes in plasma and fibroblasts described above. Interestingly, Morse (1986) has suggested that perhaps Hosli was using an incorrect substrate. He found that alpha-mannosidase was not heat-labile at 41°C, but that beta-galactosidase (29 observations) and beta-glucosidase (31 observations) gave heat lability curves that distinguished CF heterozygotes from controls. A limited study of CF homozygotes (three patients) suggested that the enzymes from two of them were more labile than those from heterozygotes. It will be particularly interesting to determine, once the function of the CF gene product is defined, whether these observations do bear a relationship to the CF basic defect.


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Microvillar Enzymes Gamma·Glutamyl Transpeptidase and Aminopeptidase M

Because CF is a disorder of abnormal fluid and solute transfer across exocrine epithelial membranes, Brock and co-workers investigated the activities of enzymes known to reside in the surface of intestinal microvilli. The activities of intestinal hydrolases, such as gamma-glutamyl transpeptidase (GGTP) and aminopeptidase M (APM), had been used for the diagnosis of pancreatic and hepatobiliary disease (Rutenberg et al., 1963; Gibinski et al., 1963) and it therefore seemed reasonable to measure these enzymes in amniotic fluids of pregnancies at risk for CF. Carbarns et al. (1983) reported on the levels of activity of GGTP and APM in 132 midtrimester amniotic fluids obtained from women at an elevated risk for a variety of genetic disorders. Pregnancies with a normal outcome accounted for 110 samples; the remaining 23 were from women who had a previous child with CF. Of these, 14 led to a normal outcome and 9 to a child with CF. Activities of both enzymes in normal pregnancies decreased with gestation age from 15 to 25 weeks. The levels of the two enzymes in fluids from pregnancies carrying a fetus affected with CF were significantly below the normal range (P < 0.0005 for both enzymes) and below the levels of at-risk samples that led to non-CF outcomes (P < 0.002 for both enzymes). These results indicated that pathological changes in affected fetuses, such as abnormal development or atrophy of microvilli in the small intestine, could be detected in midgestation and that this phenotype could be useful for prenatal diagnosis. This initial report was confirmed subsequently by Baker and Dann (1983), who also were able to detect decreased activity of GGTP and APM in samples from affected pregnancies. The separation between CF and control samples was not absolute, but was greatest in the early part of the second trimester. Disaccharidases

Subsequently, van Diggelen et al. (1983) reported that the activities of four intestinal disaccharidases were decreased in amniotic fluids from affected fetuses. They assayed sucrase, lactase, maltase, and trehalase and found that the activities of the four enzymes were highly correlated. Three affected CF fetuses yielded values of 57, 16, and 94 nmole sucrase/hr per mg protein in comparison to a range of 200-1920 in 70 controls. A fourth

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affected fetus with a borderline normal value for GGTP also gave normal values with the disaccharidases. However, Morin et al. (1983) did not obtain such clear-cut results with three affected fluids and suggested that caution should be used in interpreting this test since the relationship between levels of disaccharidases and the severity of the disease in the affected fetus was not clear. Alkaline Phosphatase

D. J. H. Brock (1983) extended these studies by analyzing the isozymes of another intestinal enzyme, alkaline phosphatase (ALP). He found that the proportions of phenylalanine- and homoarginine-inhibitable ALP were constant throughout the second trimester and that in CF pregnancies the amount of the phenylalanine-inhibitable ALP isozyme was profoundly reduced. When phenylalanine and homo arginine were used to define ALP isozymes in previously obtained amniotic fluids, the correct diagnosis was made in 9/10 affected samples (>800/0 of phenylalanine-resistant activity; < 65 % of homoarginine-resistant activity), while only 9 out of 831 controls gave false-positive results. The one exception among affected samples was one that previously had given false results with the GGTP, APM, and disaccharidase assays (Carbarns et al., 1983; Baker and Dann, 1983). Some samples (5/64) from fetuses with chromosome abnormalities also gave false-positive results. The ALP isozyme detected in amniotic fluid is an electrophoretically unique fetal entity that probably has its origin in desquamated fetal intestinal mucosa and enters the amniotic fluid by passage of meconium from the gut (Mullivor et aI., 1978, 1979). Deficiencies in intestinal enzymes in amniotic fluid from fetuses with CF probably arise because the presence of viscous meconium in CF leads to a retardation in the passage of the enzymes from intestine through meconium to the fluid. This view is supported by the observation of intestinal obstruction in utero and by the finding of meconium ileus equivalent at postmortem in CF fetuses (Muller et al., 1984a; Papp et al., 1985; Caspi et al., 1988). Because of the observed false-negative and -positive rates, D. J. H. Brock (1984) estimated that the sensitivity of the assay was 90% and that the odds of a fetus being affected upon a positive result were 28:1. These calculations were made on the assumption that the pregnancy had a prior risk of 114 and he recommended that this test be used only in such situations. In all other situations (i.e., mother had CF, one member of a couple had a CF sibling, or there was no history of CF in the family), the


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odds in favor of the fetus being affected upon a positive test ranged from 1.7:1 to 1:30 (D. J. H. Brock, 1984). The method was improved by technical modifications (Muller et al., 1984b), but some results were still difficult to interpret (e.g., intermediate, 60-70"70, residual activities in the presence of phenylalanine and homoarginine). On the basis of retrospective and prospective analyses, Muller et al. (1984b) recommended an initial assay at 17 weeks and, if intermediate or inconsistent results were obtained, a second amniocentesis performed 10-15 days later. Radioimunoassays with monoclonal antibodies to alkaline phosphatase were also developed (D. J. H. Brock et al., 1984; Mu1livor et al., 1985). Large·Scale Trials

The microvillar enzyme assay was used in three large series of prenatal diagnoses of pregnancies at a 114 risk of CF. Boue et al. (1986) studied 200 pregnancies prospectively with the GGTP, APM, and ALP assays. Normal values were derived from more than 2000 fluids. Mullivor et af. (1987) studied 111 pregnancies at a risk of 114 CF, assaying GGTP, ALP, and leucine-aminopeptidase activities and derived normal values from approximately 500 control samples. D. J. H. Brock et af. (1988) reported on the outcomes of 251 prospective pregnancies analyzed over a period of 45 months studied with the GGTP, APM, and ALP assays. In all three trials as many as possible of the aborted fetuses were examined for evidence of the disease. On the basis of these results and of the outcome of the pregnancies that went to term, the microvillar enzyme assay was estimated to yield the correct diagnosis 95-98% of the time. While it is probable that in the future microvillar enzyme assays will only be provided in specific instances, review of the extensive literature on the prenatal diagnosis of CF shows that this has been the only reliable method for providing this service before the development of diagnostic tests based on the location of the CF gene. These latter tests are discussed in a later section of this review.

Calcium and Mitochondria Calcium Homeostasis

The role of Ca2 + in influencing the physical properties of mucins and in the regulation of secretion has stimulated a large body of work on the

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possible involvement of Ca2+ homeostasis in the basic defect in CF [reviewed in McPherson (1988) and Shapiro (1989)]. However, no consistent evidence for elevated levels of total whole-cell Ca2+ has been found in tissues as diverse as fibroblasts, lymphocytes, parotid acinar cells, or nasal epithelium [see references in Murphy et at. (1988) and Shapiro (1989)]. Based on some of their own data of elevated amounts of 45Ca exchange into lanthanum-washed skin fibroblasts (Feigal and Shapiro, 1979a), Feigal and Shapiro (1979b) examined mitochondrial Ca2+ uptake and found that CF mitochondria took up about twice the amount of Ca2+ compared to controls. A similar result was obtained by von Ruecker et at. (1984), who found that the ratio of uptake CF / control fibroblasts was 2.4 (N = 11; P < 0.0005) with no overlap in values. Unlike Feigal and Shapiro (1979a), von Ruecker et at. (1984) could also distinguish heterozygotes from controls. Since mitochondrial calcium accumulation could be due to either A TP hydrolysis or respiration, Feigal et at. (1982) studied calcium uptake in the presence of inhibitors and found that the excess accumulation by CF fibroblasts was due to altered respiration (oligomycin-inhibited). Oxygen Consumption

Statistically significant differences in oxygen consumption among the three genotypes were detected (Feigal and Shapiro, 1979b). CF cells consumed approximately twice the amount of O 2 than did controls, with heterozygotes consuming approximately 1.5 times the amount. Addition of the electron transport inhibitor rotenone inhibited O 2 consumption in the order CF > H > N (P < 0.005), suggesting that the excess O 2 consumption involved the activity of NADH dehydrogenase, the enzyme of energy conserving site I (complex I) of the mitochondrial electron transport system (Shapiro et at., 1979). Stutts et at. (1986) also observed increased oxygen consumption in CF nasal epithelium when compared to controls. CF nasal polyps consumed 6.2 ± 0.6 Itl 02/mg dry wt per hr (N = 10) vs. 2.4 ± 0.2 (control turbinates, N = 17) and 3.0 ± 0.3 (atopic nasal polyps, N = 10) (CF vs. control or atopic, P < 0.001). Because a higher portion of the oxygen consumption was ouabain-insensitive, the data were consistent with the existence of a higher metabolic rate in the CF tissue. NADH Dehydrogenase

The kinetics of NADH dehydrogenase was analyzed in detail by Shapiro et at. (1979), leading to the observations that the CF enzyme had a


183

pH optimum at pH 8.6 (N = 5), the heterozygote enzyme at pH 8.3 (N = 5), and the control enzyme at pH 8.0 (N = 10). Similarly, when Km values for NADH were measured, the most dramatic differences were observed in the CF strains. The Km value was approximately 10 JlM at pH 8.6 and 30 JlM at pH 8.0. In contrast, the value for control strains was about 22 JlM at pH 8.6 and 19 JlM at pH 8.0. The Km value at pH 7.9 for 7 CF strains ranged from 11 to 16 JlM, for 5 heterozygote strains from 21 to 26, and for 9/12 controls from 32 to 43. The other 3 control strains (including one set of identical twins) yielded values in the heterozygote range (Shapiro et at., 1982). One attempt to reproduce the differences in pH optimum using white blood cells was unsuccessful (Sanguinetti-Briceno and Brock, 1982), but the differences in Km were replicated by Dechecchi et at. (1988). These authors reported that the rotenone-sensitive NADH cytochrome c reductase activity of 16 white blood cell preparations from patients ranging in age from 1 month to 24 years had Km (NADH) values from 4.7 to 10 JlM (mean 7 ± 1), while those of 13 controls (2 months to 45 years) were from 10 to 16 JlM (mean 13 ± 2) (P < 0.001). Except for cells from one CF patient of 4 months of age, all other values were below 8.3 JlM and did not overlap controls. Corresponding values from nine heterozygotes were 11 ± 2 (H vs. CF, P < 0.001; H vs. N, nonsignificant). Studies of two CF and three control fibroblast strains also yielded lower Km values for the CF strains (4.6,5.3 vs. 8.0, 9.7, 9.4 JlM). Shapiro (1989) has suggested that the most parsimonious explanation for the altered kinetics of NADH dehydrogenase is to postulate that one of the components of complex I is different in CF. Since CF is not inherited as a mitochondrial disorder, this would require that the CF nuclear gene product either be a component of or interact with mitochondrial complex I. An alternative proposed role for Ca2+ in CF pathology is an involvement in the derangement of protein secretion and electrolyte transport. The evidence in favor of this hypothesis will be discussed below.

Beta-Adrenergic Responses Autonomic Nervous Dysfunction

The secretory response of exocrine glands is controlled by the autonomic nervous system. The possibility that the defect in CF is caused by a malfunction of this system has been investigated both in vivo and in vitro

184


[reviewed in Davis and Kaliner (1983)]. It must be recognized that the autonomic system is complex and that a derangement in any part of the pathway could lead to the conclusion that the system is abnormal. For example, abnormalities in the regulation of secretion could occur at the level of innervation, at the level of cellular receptors, or in an intracellular second messenger. CF patients have a reduced pupillary response to stimulation and the blood pressure of young adults is lower than that of controls (Rubin et al., 1966; Lake et al., 1979). In addition, animal models that mimic CF have been developed by chronic overstimulation of the autonomic nervous system (Martinez et al., 1975). However, although the endpoint in the animal models might be similar to the disease in patients, such a finding would not prove that the underlying mechanisms were the same. Cyclic AMP levels

CF lymphocytes and granulocytes have a decreased responsiveness to stimulation by beta-adrenergic agents as measured by cyclic AMP levels. Davis et al. (1978) showed that the mean level of intracellular cAMP following stimulation of leukocytes with 10- 5 M isoproterenol was 3.5 (±0.5)-fold for 21 normal subjects compared to 2.2 (±0.3)-fold for 20 CF patients. Heterozygotes yielded intermediate levels, but there was considerable overlap among all three groups. Subsequent studies showed that the defect in CF cells could be observed when measuring the activity of isoproterenol-stimulated adenylate cyclase. Membrane preparations from CF leukocytes or granulocytes yielded 50010 of control adenylate cyclase activity when the enzyme was stimulated with isoproterenol (P < 0.005), but normal activity when stimulated with prostaglandin E1 (Davis and Laundon, 1980). However, measurement of beta-adrenergic receptors led to conflicting reports. Galant et al. (1981) compared the binding of dihydroalprenolol to neutrophils and found that CF cells had significantly fewer receptors than did controls (400 ± 50 vs. 820 ± 70, P < 0.005). CF heterozygotes produced 50% less cAMP than did controls, but had the same number of receptors (910 ± 90). Davis et al. (1983) used the same method to study lymphocyte and granulocyte membrane preparations and found no difference in the number of receptors in CF and controls. They postulated that the defect in CF cells was due to defective receptor-cyclase coupling. Since Lemanske et al. (1981) had shown that the more severely affected patients have a lower beta-adrenergic granulocyte responsiveness,


185

the difference between the results of Davis et al. (1983) and those of Galant et al. (1981) were attributed to the inclusion of patients with more severe disease in the Galant series. Studies of beta-adrenergic responsiveness in fibroblasts have been inconclusive. The disparate results could reflect the use of samples from different patients or the effects of uncontrolled cell culture variables (Buchwald, 1976; Davis et al., 1980; Kurz and Perkins, 1981; Markovac et al., 1981). More recently, several studies with epithelial cells have confirmed that CF cells may be underresponsive to betaadrenergic agents. Most of these studies have involved measurements of ion transport or of a specific CI- channel and will be discussed in more detail in the section on epithelial ion transport. Secretion Abnormalities

Bloomfield et al. (1976) showed that CF parotid glands hyper secreted zymogen granules with a concomitant increase in salivary Ca2 +. Elevated levels of Ca2+ have also been observed in submandibular saliva (Chernick et al., 1961; Bloomfield et al., 1973). To determine if CF glands had defective regulation of secretion, McPherson et al. (1985, 1986a) studied the adrenergic secretory response of acinar cells as measured by amylase and mucin release following treatment of the isolated cells with beta-adrenergic agonists. A comparison of cells from three controls and three patients showed that treatment of cells with isoproterenol stimulated the amylase release from controls by 4.3 (±0.6)-fold relative to unstimulated cells, but increased CF cells by only 1.6 (±0.3)-fold (P < 0.01). Similar results were also seen for mucin release. The defect was partially corrected when cells were incubated with a phosphodiesterase inhibitor, leading to the suggestion that the defect was distal to the production of the intracellular messenger (McPherson et al., 1986a). McPherson et al. (1986b) proposed that the defect might involve a calmodulin function that modified both Ca2 + and cAMP pathways, though calmodulin itself was excluded on biochemical and genetic bases (Scambler et al., 1987a). This hypothesis has been tested by the analysis of calmodulinbinding proteins in submandibular tissues. Cytosolic tissues from three controls had 3.1 ± 0.1 {tg calmodulin/mg protein, while four CF samples gave a value of 4.8 ± 0.2 when the activity was measured by activation of cyclic AMP phosphodiesterase (P < 0.001). There was no significant difference in calmodulin when measured by radioimmunoassay (Shori et al., 1988). These results led the authors to suggest that submandibular

186


glands had modulators of calmodulin activity and that this was the site of the CF defect. Western blot analysis of binding of 125I-Iabeled calmodulin to boiled homogenates showed a 61-kD band in 5/6 CF samples and 0/3 controls. The binding required the presence of Ca2+ . Subsequently Shori et al. (1989) showed that the 61-kD band is underphosphorylated in CF. Quantitative densitometry of the peak area of the 61-kD band gave 80 ± 11 mm 2 for five control glands and 9.4 ± 1.1 for seven CF glands (P < 0.001). No significant differences were seen for the phosphorylation of the two other major bands. Use of inhibitors gave results consistent with the presence of both protein kinase A and C in the extracts catalyzing the phosphorylation of the 61-kD band. In addition, this band cross-reacts with an antiserum against calcineurin, also a substrate for protein kinase A and C. Since defects in cellular CI- -channel responsiveness to these two protein kinases have also been observed (Hwang et al., 1989; Li et al., 1989), it is possible that the observations on the 61-kD band represent a secondary consequence of the defective gene product.

EPITH ELiAL ION TRANSPORT The various studies discussed above have been brought together by a series of observations that are consistent with the hypothesis that CF epithelia have a derangement in ion transport. In particular, it has been shown that in CF there is aberrant regulation by intracellular second messengers of CI movement across the apical membrane. Initial observations on tissues have been followed by detailed studies of cultured epithelial cells, both from the sweat gland and the respiratory tract. These results have shown that the defect resides in the cell membrane and is not caused by a humoral circulating factor or by the pathological sequelae of the disease. In this view, the increased mucus secretion in CF is a secondary consequence of the primary abnormality in fluid and ion transport. Thus, the basic defect in CF must involve a membrane function involved in the transport of ions [reviewed in Welsh and Fick (1987), Quinton (1990), and Welsh (1990)].

Studies on Sweat Glands Quinton (1983) measured the permeability of isolated sweat ducts and found that those from CF patients had more negative potentials (relative to


187

the serosal bath) than those of controls. The average luminal potential differences (PD) in 150 mM NaCI for 7 controls subjects was - 6.8 ± 2.5 mV and for five CF subjects was -76.9 ± 13.2 mV (P < 0.001). If SO/was substituted for Cl-, the potential of the control ducts was -75.5 ± 11.1 m V, consistent with the view that the differences between control and CF ducts were accounted for by a decreased permeability of the latter to CI-. This hypothesis was confirmed by direct measurement of ductal transport rates. In 34 glands from six normal subjects the Na + and CIreabsorption rates were 252 ± 64 and 208 ± 47 pmole/min per gland, respectively, and were 101 ± 18 and 41 ± 15 in a similar number of glands from CF patients. The sodium difference was significant to P < 0.025 and the chloride difference to P < 0.005 (Quinton and Bijman, 1983). Subsequent studies showed that in this tissue CI- impermeability is due to the almost complete absence of an electrodiffusive shunt for CI- across the epithelium (Bijman and Quinton, 1984; Bijman and Fromter, 1986; Quinton, 1986).

Studies with Respiratory Epithelia Similar results had been reported by Knowles et al. (1981) in respiratory epithelia. They found little overlap in values between CF subjects and controls, both with and without disease. They compared 23 CF patients ranging in age from 3 months to 32 years of age, 24 parents, and 14 sibs to 32 "disease" and 54 healthy controls. Average PD across nasal epithelia was -53.0 ± 1.8 mV in the older patients (>3 years), -24.7 ± 0.9 mV in healthy controls, and - 20.5 ± 1.3 m V in disease controls. All patient values were above three standard deviations from the mean of the healthy controls. No differences compared to normal values were observed with sibs or parents. Similarly, more negative potential differences were measured in trachea and bronchi of three CF subjects in comparison to seven controls. Amiloride caused a decrease in the PD for both sets of subjects and abolished any differences between CF and controls, the implication being that the lack of CI- permeability in CF epithelium results in no shunting of the voltage generated by amiloride-sensitive sodium absorption. Hay and Geddes (1985) also detected a more negative PD in the distal inferior turbinate of CF patients in comparison to controls ( - 25 ± 13 mV vs. - 13 ± 5 mY; P < 0.02), but found overlap between the two groups. Nonetheless, measurement of trans epithelial potential difference remains a useful diagnostic tool.

188


Subsequent studies have also found defective chloride transport in pancreatic (Kopelman et al., 1988), intestinal (Berschneider et al., 1988; Taylor et al., 1988; Orlando et al., 1988; Baxter et al., 1989), and rectal (Goldstein et al., 1988) tissues of CF patients, confirming that most, if not all, CF epithelia have similar abnormalities.

Studies on Cultured Sweat Gland Epithelial Cells Sato and Sato (1984) first showed that the secretory coil in CF sweat glands had an abnormal response to the stimulation of secretion by beta-adrenergic agents. All 14 isolated CF sweat glands failed to secrete fluid when stimulated with 5 x 10- 5 M isoproterenol (with or without theopylline) in comparison to 17/18 control glands that secreted a mean rate of 1.1 nllmin. Stimulation of glands with metacholine led to near-normal responses from the CF glands. After in vivo stimulation of sweat glands by injection of isoproterenol and theophylline, 26/28 CF patients failed to show any sweating, while the other 2 had a small response the first time and failed the second time. All 35 age- and sex-matched controls responded to the stimulus by secreting a mean of 0.72 nllmin. This failure to stimulate secretion was not due to a defect in cAMP generation, since both control and CF glands had similar dose-response curves and produced the same amounts of cAMP after treatment with isoproterenol. Cholinergic and alpha-adrenergic responsiveness of CF subjects was not significantly different from controls. Sato (1984) studied the effect of isoproterenol on luminal potentials in isolated segments of the secretory coil of the sweat gland and found that, unlike in controls, where treatment with the drug generated a mean negative potential of 1.0 m V, no changes could be observed in CF. Hyposecretion of beta-adrenergically induced sweating has also been observed in CF heterozygotes. Behm et al. (1987) found that the mean sweat response of 20 heterozygotes was about 50070 that of 19 controls (P = 0.0013). However, there was significant overlap in individual values. There was no difference in the cholinergic sweat response. As observed earlier by Sato and Sato (1984), five CF homozygotes did not respond at all to the beta-adrenergic stimulation. In conjunction, these various studies are consistent with the hypothesis that the defect in CF epithelia is due to a failure to respond to beta-adrenergic stimuli and that the defect is distal to the generation of cyclic AMP, probably at the level of the intracellular response to the elevated cAMP.


189

Studies on Cultured Respiratory Epithelial Cells Observations of faulty beta-adrenergic responsiveness have also been made in respiratory cells and tissues. Widdicombe (1986) reported that in cultured CF tracheal cells, the short-circuit current (IsJ response following treatment with 10 - 5 M isoproterenol was 1110 that seen in normal cells. In six control subjects the mean rise in Ise was 2.8 ± 0.3 p,A/cm 2 (range 1.3-4.4), while in four CF subjects the increase was 0.21 ± 0.09 p,A/cm2 (range 0.0-0.4). The resistances of the two groups of cells were comparable. Similar results were seen with CF and control nasal polyp cells. Measurement of the basal and isoproterenol-induced cAMP levels in CF and control tracheal and nasal polyp cells showed no differences, again consistent with the view that the defect was in a step beyond the synthesis of the second messenger. Studies on cultured human tracheal cells localized the ion permeability defect to the apical membrane (see Fig. 1). Widdicombe et al. (1985) showed that treatment with isoproterenol decreased the fractional resistance and depolarized the apical membrane of normal cells, but not of CF cells. Cotton et al. (1987) measured membrane PD in six controls and six CF patients using intracellular microelectrodes. The PD across the apical membrane in CF cells was smaller than in controls ( - 11 ± 3 m V vs. - 29 ± 4 mV), whereas it was similar across the basolateral barrier. Reduction of luminal CI- led to a significant depolarization of the control apical PD (-29 ± 4 mV to -1 ± 7 mY), but did not change the CF PD. Furthermore, in contrast to normal membranes, the CF membrane did not respond to isoproterenol. The Na + channel blocker arniloride induced a larger apical membrane hyperpolarization (CF: - 9 ± 4 m V to - 32 ± 3 mV; N: - 29 ± 3 m V to - 38 ± 3 mV) and a greater increase in the voltage divider ratio in CF epithelia (CF: 0.51 ± 0.02 to 0.69 ± 0.04; N: 0.52 ± 0.06 to 0.57 ± 0.07). Therefore, both reduced apical CI- and increased Na + conductance contributed to the abnormal function of respiratory epithelial cells in CF patients.

Single-Channel Recordings These demonstrations of defective apical permeability in CF epithelia led to direct measurements of chloride-channel activity through the use of single-channel recordings. Welsh and Liedke (1986) found that excised,


190

",.",-

8 . .0111_

ApI ..1 _m~

H2 O I" CI '

I N T

E A S

CONTROL CELL

Na+

K'

L U M

T1ghl Junction

T I T I U M

E

N

,cr

CYSTIC FIBROSIS CELL

7

Na

+

K' H2 O

r

Fig. 1. Model to explain differences in fluid and electrolyte absorption between normal (control) and CF airways. In CF cells the impermeability of the apical membrane to Cl- ions is compensated by an increased Na + ion permeability. Since Cl- can transverse the cellular layer from the lumen to the interstitium via a paracellular pathway, NaCI is absorbed by the epithelium at a greater rate than in normal cells. In sweat glands there is no increased Na + permeability or paracellular movement of Cl- ions, resulting in decreased absorption. The permeability of the basalateral membrane to Cl- ions has not been completely defined. [Adapted from Quinton (1990).]

cell-free patches of membranes from CF airway cells contain Cl- channels with the same conductive properties as from normal cells. However, in CF the channels could not be detected in cell-attached recordings. Frizzell et at. (1986) observed CI- channels in control cells upon stimulation by epinephrine, but could not evoke a response in CF cells with epinephrine, forskolin, or cAMP. The CI- channel activity could be observed if patches were excised from cells. In both of these publications, the reported CI- channels were outwardly rectifying (greater conductivity at depolarizing voltages), were anion-selective, and had conductances of 26 pS (Welsh and Liedke, 1986) and 50 pS (Frizzell et al., 1986). An outwardly rectifying anion channel has also been detected in control and CF sweat glands, though its


191

regulation has not been investigated (Krouse et al., 1989). Schoumacher et al. (1987) and Li et al. (1988) subsequently found that phosphorylation by the catalytic subunit of cAMP-dependent protein kinase A and ATP opened chloride channels in control membrane patches, but failed to do so in CF patches. The presence of the channels could be detected in the CF patches by depolarization. Schoumacher et al. (1987) studied cells from seven controls and three CF patients and Li et al. (1988) analyzed five controls and four CF patients. Similar results were obtained in all instances. These authors postulated that the defect resided in the channel itself or, perhaps, in a molecule that regulated the channel. Similar observations, not yet extensively replicated, have been reported in lymphoblasts (Chen et al., 1989). In addition, two groups have reported abnormal regulation of the CI- channel in CF by protein kinase C. Hwang et af. (1989) found that activation of the channel by protein kinase A could be detected in 6/7 control fetal tracheal epithelia, but not in 9/9 adult CF airway epithelia. Activation by protein kinase C was seen in 7/8 control fetal tracheal epithelia and 3/3 adult airway epithelia, but not in 7 adult CF airway epithelia. Li et af. (1989) found that at a high calcium concentration, protein kinase C inactivated CI- channels, while at a low calcium concentration, it activated the channels (five normal subjects). In CF epithelia (four subjects) the inactivation at high Ca2+ was not affected, but the activation at low Ca2 + could not be elicited. The precise molecular basis of the defective CI- permeability in CF is uncertain. More recent reports have presented evidence that the apical membrane of CI- -secreting cells (e.g., cells from fetal pancreatic duct or T84) have, in addition to the outwardly rectifying channel mentioned above, a small linear anion channel with a conductivity in the range of 4-8 pS (Gray et al., 1989; Cliff and Frizzell, 1990; Tabcharani et al., 1990). The probability of observing the activity of the small channel was increased by raising intracellular cAMP levels, whereas the outwardly rectifying channel was stimulated by mechanisms involving Ca2+. These results are therefore consistent with the view that the linear, rather than the outwardly rectifying, channel is defective in CF (see below, p. 227).

CF GENE MAPPING The major difficulty in mapping the CF locus (CF, the presumed CF gene) was the lack of obvious chromosomal rearrangements, such as

192


deletions or translocations, associated with the disease. Several cases of CF children with a chromosome abnormality were reported in the early literature (Lindenbaum et al., 1972; Edwards et al., 1984), but they all turned out to be false leads. The alternative method to map CF was then through linkage analysis. The objective was to identify a set of genetic markers closely linked to the disease locus by screening markers distributed over different regions of the genome in families with CF individuals. Although the approach was by no means an easy task, the goal was certainly achievable, with the requirement of only a finite number of genetic markers to be tested in order to saturate the entire genome (Bostein et af., 1980; Kidd and Ott, 1984; Edwards, 1987). Because of the autosomal recessive mode of inheritance of the disease, most of the families available for the linkage analysis were nuclear (two-generation) families each with a small number of CF children [see examples given in Tsui et af. (1986)}; there were only a few large pedigrees of Amish and Mennonite origin (Klinger, 1983; Ober et al., 1987). Fortunately, although the amount of linkage information per family was limited, this deficiency was compensated by the large number of families available for study due to the high prevalence of the disease. Linkage studies would be ineffective if a significant portion of the CF cases were caused by mutations in a second locus. In fact, genetic heterogeneity was once considered to be one of the possible explanations for the high frequency of the disease (Thompson 1980); however, two population studies provided no evidence suggestive of the existence of more than one CF gene (Danks et af., 1984; Romeo et al., 1985), although the data were not entirely conclusive (Lander and Botstein, 1986). Nevertheless, these preliminary analyses pointed to the feasibility of mapping the disease locus by linkage analysis.

Negative Results As might have been predicted, early linkage studies only yielded negative data, but these, in a positive sense, excluded several locations from being the site for the CF locus. The chromosomal regions that could be examined were limited by the small number of conventional, polymorphic protein markers available. For example, Steinberg et al. (1956; Steinberg and Morton, 1956) obtained results for eight different blood group markers


193

and Goodchild et al. (1976) extended the study to include several additional enzyme markers. The linkage approach received greater attention after the introduction of DNA markers defined by restriction fragment length polymorphisms (RFLPs) (Kan and Dozy, 1978; Botstein et al., 1980), since the number of polymorphic DNA sequences is essentially unlimited and their use should allow coverage of the entire genome. The number of protein markers suitable for linkage studies also increased substantially during this time (see reports of Human Gene Mapping Workshops). The use of RFLP analysis had provided an easy method to study gene loci (or chromosome regions) suggested by previous biochemical or cytological observations. For example, Davies et al. (1983) performed family analysis with the gene coding for complement C3, which was thought to be involved in ciliary dyskinesis (see above, p. 162), and showed that C3 was not linked to CF. Scambler et al. (1985a) tested and failed to demonstrate linkage for a number of markers on chromosome 4, which had been inferred to contain the gene for the CF ciliary dyskinesia factor (B. J. Mayo et al., 1980). Scambler et al., (1986a) also examined for linkage with the coagulation factor X located on chromosome 13(q34) to test whether the reported chromosome translocation involving this region (Edwards et al., 1984) was etiologically related to CF, but obtained negative data with eight families. Other chromosomes were implicated in CF patients with trisomies, but none were conclusive (Syman et al., 1984). Although the search for the CF locus was tedious, there was considerable cooperation among the different research groups. For example, Tsui et al. (1985b,c) and Scambler et al. (1986b) reported the negative linkage data for a large number of protein and DNA markers covering a substantial portion of the human genome, so that other investigators could choose to avoid the so-called excluded regions. Similarly, Eiberg et al. (1985b) reported preliminary linkage data between CF and coagulation factor F13B, so that other investigators could confirm their observation. Wainwright et al. (1986a) also demonstrated the utility of multiple markers in excluding an entire chromosome.

Localization to Chromosome 7 While most investigators had directed their search based on DNA markers, the first linkage to CF was discovered by Eiberg and his co-workers, using a series of polymorphic protein markers (Eiberg et al.,

194


1985a). One of the markers, a genetic determinant for the varied levels of paraoxonase activity in serum (locus symbol PON), was found to be approximately 10 centimorgans (cM) from CF (Eiberg et al., 1985a); Schmiegelow et al., 1986). This finding confirmed the existence of a CF locus and demonstrated the feasibility of the linkage approach toward identification of the gene. The linkage did not provide adequate information for further targeting of the CF locus, however, because the chromosome location of PON had yet to be determined and it could not be done readily on the basis of enzyme expression in somatic cell hybrids. Linkage with 07815

A turning point was the mapping of the CF gene by the demonstration of linkage between CF and D7S15 (formerly DOCRI-917) (Tsui et al., 1985a). The most likely distance between the two was estimated to be 15 cM based on linkage analysis with 39 informative families. The results of two-point and three-point linkage analysis with D7S15, PON, and CF confirmed the linkage and suggested the order D7S15-PON-CF, with estimated intervals of 5 and 10 cM, respectively (Tsui et al., 1985a). A brief description of this marker is included in Table III. The chromosome location for D7S15 was initially unknown, but mapping of the DNA marker was relatively straightforward. The chromosomal location for D7S15 and by inference CF was determined by DNA hybridization analysis, using a panel of human-rodent somatic cell hybrid lines containing different human chromosome complements (Knowlton et al., 1985; Tsui et al., 1986b). The results suggested that the most probable location for D7S15 was on the long arm of chromosome 7, between the centromere and band q31. Linkage with MET and 0788

Further information on the localization of CF was derived from the almost simultaneous discovery of close linkage between CF and two other markers, MET (R. White et aI., 1985) and D7S8 (Wainwright et al., 1985) (Table III). Both of these markers were estimated to be about 1 cM from CF and were known to map on chromosome 7 (Dean et al., 1985, Bartels et al., 1986). To confirm these observations and to determine the relative order of the three closely linked loci, a joint study involving seven different research


195

groups was conducted and data from 211 CF families, representing over 1200 genotyped individuals, were compiled (Beaudet et al., 1986). Unfortunately, while the result of this collaborative analysis provided overwhelming support for the close linkage, as initially reported, the order of MET, D7S8, and CFwas equivocal (Beaudet et al., 1986; R. White, 1986). The most likely order, MET-CF-D7S8, was only ten times more favored than the next most likely order, CF-MET-D7S8. Better supporting data for the order MET-CF-D7S8 were derived from a subsequent study using additional markers and reference pedigrees (Lanthrop et al., 1988). In addition to their utility in serving as two closely linked reference points for the isolation of additional DNA segments near the CF locus, MET and D7S8 have also been remarkably useful as markers for genetic diagnosis (see below, p. 236). Further, the linkage data provided a strong argument that the majority of the CF mutations in the population would map within the same locus, although the possibility of a small proportion (5070) of CF families segregating mutations at another unlinked locus could not be totally excluded by the linkage analysis (Beaudet et al., 1986). There has been no direct evidence in support of a second locus causing CF, however. One observation (Tsui et al., 1986a) in which genetic heterogeneity in CF was suggested was subsequently discovered to be due to a diagnostic error (Tsui and Buchwald, 1988).

Linkage with Other Chromosome 7 Markers A number of other genetic markers known to be on chromosome 7 were also examined for possible linkage to CF. These markers included COLIA2 (Scambler et al., 1985b; Buchwald et al., 1986), TCRB (Wainwright et al., 1985, 1986b), D7S13 (Estivill et al., 1986; Wainwright et al., 1987b; Bohm et al., 1988), PAIl (Klinger et al., 1987), EPO (Watkins et al., 1986), and NPY(Meisler et al., 1987). Although none ofthese markers were found to be closer to CF than was MET or D7S8, they were useful in confirming the chromosome location of CF. Some of them were also useful for genetic diagnosis based on linkage (see below, p. 236). In addition, many RFLP markers were identified by the various molecular cloning experiments described in the next section. A representative list of these markers is shown in Table III and the current understanding of the genetic map of the CF region is shown in Fig. 2. Some of the marker-marker linkage relationships shown were derived from study of reference pedigrees (Barker et al., 1987; Lathrop et al., 1988, 1989).


196

TABLE III. List of Polymorphic DNA Markers Spanning the CF LocusQ Probe name

Enzyme

metD

Ban I

met D

Taq I

metH

Taq I

E6

Taq I

E7

Taq I

pH131

Hin fl

W3Dl.4

Hin dIll

XV2C or H2.3A

Taq I

EG1.4

Hin clI

EG1.4

Bgf II

KM19 or JG2El

Pst I

E2.6 or E.9

Msp I

H2.8A

Nco I

Mp6d9 or E4.1

Msp I

J44

Xba I

1O·IX.6

Ace I

1O·1X.6

Hae III

T6/20

Msp I

H1.3

Nco I

CE1.0

Ndel

132

Sac I

Fragment (kb)

Allelic association [AJ

[.::lJ

Reference

0.60

0.10

0.66

0.06

0.35

0.05

0.45

0.06

J .E. Spence et al. (1986), Kerem et al. (1989b) R. White et al. (1986), Kerem et al. (1989b) White et al. (1985), Kerem et al. (1989b) Kerem et al. (1989a,b)

0.47

0.07

Kerem et al. (1989b)

0.73

0.15

0.68

0.13

0.64

0.09

Rommens et al. (1988), Kerem et al. (1989b) Rommens et al. (1988), Kerem et af. (1989b) Estivill et af. (1987a,b), Kerem et af. (1989b)

0.89

0.17

Kerem et af. (1989b)

0.89

0.18


0.88

0.18

Estivill et af. (1987a,b), Kerem et al. (1989a,b)

13 8.5 25 8 12 8.5 + 3.5

0.85

0.14


0.87

0.18


0.77

0.11

Estivill et af. (1989a), Kerem et af. (1989b)

15.3 15 + 0.3 6.5 3.5 + 3 1.2 0.6 8 4.3 2.4 +1.4 5.5 4.7 + 0.8 15 6

0.86

0.13


0.90

0.24


0.91

0.25


0.51

0.54


0.87

0.15


0.41

0.03


0.17

0.02

Iannuzzi et af. (1989), Kerem et af. (1989b)

7.6 6.8 6 4.8 7.5 4.0 4.4 3.6 3.9 3 + 0.9 0.4 0.3 20 10 2.1 1.4 3.8 2.8 20 15 7.8 6.6


197 TABLE III. (Continued)

Probe name

Enzyme

13.11

MspI

Fragment (kb) 4.2

Allelic association [A]

[.6.]

0.29

0.04

0.36

0.06

1.8

129

Pvu II

9 6

Reference Wainwright et Kerem et 01. Iannuzzi et 01. Kerem et 01.

01. (1985), (1989b) (1989), (1989b)

"Table adapted from Kerem et 01. (1989b). Yule's association coefficient A = (ab - be)/(ad + be), where a, b, e, and d are the numbers of normal choromosomes with DNA marker allele 1, CF chromosomes with 1, normal chromosomes with 2, and CF chromosomes with 2, respectively. Relative risk (RR) can be calculated using the relationship RR = (1 + A)/(1 A) or its reverse. The correct .6. association values are calculated according to Kerem et 01. (1989b) with consideration of the frequency of CF chromosomes (0.02) in the population.

Chromosomal Localization

The knowledge on the chromosome localization of CF was crucial in the subsequent identification of the CF gene itself. As there were no functional tests available for the gene, chromosome mapping became a rapid means of testing candidate genes or other DNA segments for their

7q lU~

11.23

21.1 21.2 21.3 22

31.1 31.2 31.3 32 33 34 35 36

MET C0L1A2 ] - - 07515 EPO ]-PON 07513 07818

] - - TCRB

500 kb

078340 078122 07823 078399 ---078411

0788

Fig. 2. Genetic map of the CF gene region. The chromosome localization and relative positions of the genetic markers surrounding the CF gene are shown. The transcription direction of the CF gene (CF) is indicated by an arrow.

198

Lap-Chee Tsui and Manuel Buchwald

possible involvement in CF _ Since MET and D7S8 were determined to be about 1 cM from CF, the map position for CF could be inferred from mapping of these closely linked markers_ MET was assigned to a region between 7q21 and 7q31 in the initial mapping study by in situ hybridization to metaphase chromosomes (Dean et al., 1985). D7S8 was placed in the 7q22 region in an early study using a panel of somatic cell hybrids (Bartels et al., 1986). On the basis of these observations, CF was assigned to band q22. Subsequent studies, however, showed that these two markers were more likely to be within band q31 (Zengerling et al., 1987; Wainwright et al., 1987a; van der Hout et al., 1988). Consistent with this assignment, a more centromeric marker, D7S18, was found to map between 7q31.1 and 31.2 (Buckle et al., 1987). These data therefore suggested that the CF gene was located below 7q31.1 or q31.2. Moreover, a recent study with two more closely linked DNA markers indicated that the gene might be within band q32 (Duncan et al., 1988). Mapping of the CF gene has so far not been formally performed with the isolated gene itself.

IDENTIFICATION OF THE CF GENE The knowledge about the precise location of the CF locus permitted the use of a variety of molecular cloning techniques to attempt to isolate the gene itself. The general idea was to narrow the region of interest with additional DNA markers, clone the DNA segments from the region, identify candidate genes, and study these for their possible involvement in the disease.

Cloning Strategies Isolation of DNA Segments from Flow·Sorted Libraries

Scambler et al. (1986c) isolated a random DNA segment from a flow-sorted chromosome 7-specific library (Deaven et al., 1986) and showed that the clone, 7C22 (D7S18 or formerly D7S16) (Table III), was close to the CF gene (Farrall et al., 1987b; Wainwright et aI., 1988). A subsequent long-range physical mapping study showed that this marker was approximately 100 kilobase pairs (kb) from MET, but on the opposite side of CF


199

(Poutska et al., 1988). Jobs et al. (1990) studied 41 DNA segments from chromosome 7, but none of them appeared to be closely linked to the CF locus. Melmer et al. (1989) and Burns et al. (1990) detected clones that were most likely associated with genes, but the clones had not been tested for linkage to CP. In a more systematic approach, Rommens and co-workers (Rommens et al., 1988) isolated over 250 DNA segments from a chromosome 7-specific library (Deaven et al., 1986). Of these, 58 were found to map within band 7q31. This number correlated well with the estimated size of chromosome 7 (150,000 kb) and that of band q31 (Y5 of the chromosome). This so-called saturation mapping approach allowed these investigators to isolate two additional DNA segments (D7S122 and D7S340) located between MET and D7S8 (Rommens et al., 1988) (Fig. 3) and closer to the CF locus. As described below (p. 207), molecular cloning studies originated from these two clones eventually led to the isolation of the CF gene (Rommens et al., 1989a; Riordan et al., 1989; Kerem et al., 1989b). Chromosome· Mediated Gene Transfer Scambler et al. (1986d) introduced a small region of human chromosome 7 presumed to contain the CF locus into a mouse cell line. These investigators took advantage of an activated copy of the met prot 0 oncogene (MET) from a chemically transformed human osteosarcoma cell line (MNNG-HOS) (Cooper et al., 1985). With the chromosome-mediated gene transfer (CMGT) technique, cell lines with various amounts of human DNA sequences were isolated. Although the DNA sequences of the met gene appeared to have undergone complex rearrangements and translocations in the original MNNG-HOS (Park et al., 1988; Scambler et al., 1990), the region around the CF gene was thought to be intact in at least one of the CMGT-derived cell lines (Scambler et al., 1986d). These cells were useful for isolation of additional DNA sequences near MET because the human DNA could be easily distinguished from that of the mouse on the basis of species-specific repetitive DNA sequences. One of the above described CMGT-derived cell lines, clI, appeared to be particularly useful. Taking advantage of the knowledge that undermethylated CpG-rich sequences were frequently found to be associated with genes, Estivill et al. (1987a) isolated a DNA segment (D7S23) from this cell line using a cloning vector designed to isolate CpG-rich sequences. This DNA segment was found to contain a gene now known as IRP (or INTlLI)

3.3

matH

TeJ'

melD

MET XV2C

075399

CF

-soo 5

-10 ..10

::~)IT?W4~")&e("'~

JG2E1 E2.6 H2.8 E4.1 J44 10.1X.6 6120 H1.3 CE1 .0 CS.7 KM19 (E.9) ~.9

07523

E6 E7 pH131 W301.4 H2.3A EG1.4

075122

J29

075426

iJr.,oo T

07S8 J32 p3H-3 pJ3.11

075424

Fig. 3. Physical map of the CF gene region. The genetic markers are identified by their locus names, laboratory probe names, and the corresponding RFLPs (see Table III). The distances (as indicated in kilobase pairs) between markers are determined by pulsed-field gel electrophoresis and molecular cloning. The extent of the CF gene locus (CF) is represented by the thickened line. The inverted triangle indicates the position of the major CF mutation (.6.F508). [Adapted from Kerem et 01. (J989b).J

kb

RFLP

Probe

Locus


201

(Wainwright et al., 1988) (Fig. 3). Although IRP was not found to be the long sought-for CF gene, the RFLPs associated with this gene (KM.19 and XV2C) became extremely useful markers for genetic diagnosis (see below, p. 235). The location of IRP was also an important landmark for physical mapping of the CF region (see below, p. 202). A number of other DNA segments were also isolated from the cII cell line. EstiviIl et al. (1989a) reported the isolation of Mp6d.9 (D7S399) in the interval between D7S23 and D7S8, further narrowing the critical region for the CF gene. Ramsey et al. (1990) combined the use of the cII cell line with pulsed-field gel electrophoresis and isolated a DNA segment (pG2; D7S411) from a restriction fragment near IRP (560 kb Sac II). A cosmid clone was isolated by chromosome walking from pG2 and it was subsequently determined to contain exons 20-23 of the CF gene (Ramsay et al., 1990). Similar experiments were performed by Dorin et al. (1989) and Porteous et al. (1990). Both of these attempts were based on the simian virus 40 (SV40) sequence found near the CF region. Dorin et al. (1989) succeeded in inserting a biochemically selectable marker into chromsome 7, in band 7q31-q35, by homologous recombination with an array of integrated SV40 sequences in this region. Upon a second round of transfection via CMGT, a 2- to 3-mb region of chromosome 7 was retained in a mouse recipient cell line, but the segment did not appear to contain genetic markers closely linked to the CF gene. Long-Range Restriction Map

In designing further strategies to localize and identify the CF gene, the long-range physical map of the CF region was a crucial piece of information. The first complete map of the region was generated by Poustka et al. (1988), who produced a map spanning almost 5 mb in size using DNA probes from the D7S23 locus to bridge the gap between MET and D7S8, which were estimated to be 1250-1500 kb apart. Using another source of DNA as starting material and partial digestion, Drumm et al. (1988) derived a map for the MET-D7S8 region; these investigators estimated the two flanking markers were 1300-1800 kb apart. Although a slightly different map was generated by Fulton et al. (1989) with additional DNA probes, the distance between MET and D7S8 was also estimated to be 1400-1900 kb. Rommens et al. (1989b) performed a detailed mapping for the two DNA markers (D7S122 and D7S340) known to be closely linked to the CF locus with respect to MET and D7S8 (Fig. 3). The study showed that the

202


distance between D7S122 and D7S340 was only about 10 kb and that they were about 500 kb from MET and 950 kb from D7S8. Further studies showed that D7S23 was between D7S340 and D7S8, about 80 kb from D7S340 (Rommens et al., 1989a). In addition, family studies showed that the CF locus was between D7S23 and D7S8 (Rommens et al., 1989b). A polymorphic Not I site noted by Julier and White (1988) with a met gene probe and pulsed-field gel electrophoresis (see Fig. 3). This site could have been used for family mapping studies to refine the location of the CF gene, but it turned out to be at the center of the CpG island isolated by Estivill et al. (1987a; Stanier et al., 1988), and the gene was placed distal to this site (Berger et al., 1987).

Chromosome Jumping, Microdissection, and Cloning from Pulsed· Field Gels

Collins et al. (1987) were the first to use the chromosome jumping technique involving circularization of large DNA molecules to isolate additional DNA segments from the CF region. They isolated one clone (CF63) from a "jump" estimated to be 100 kb from MET; however, the clone was subsequently mapped by long-range physical mapping studies to a region about 40 kb toward CF (Rommens et al., 1989b). These investigators (Iannuzzi et al., 1989) also initiated jumps from the other flanking marker D7S8; it was estimated that a total of over 300 kb was covered by two successive steps in one direction and one to the other. Three new RFLPs were detected along the way and use of these markers in family studies showed that they did not cross one of the recombination breakpoints between CF and D7S8, thus narrowing down the region to be studied to less than 500 kb. The chromosome jumping technique was used in combination with chromosome walking to cover about 280 kb of DNA and resulting in the cloning of the CF gene (see below, p. 207). Isolation of a specific region by direct dissection of chromosome regions from metaphase spreads is a powerful way to approach a disease gene locus. Kaiser et al. (1987) and Weber et al. (1990) reported the isolation of a large number of DNA segments from the 7q22-q32 region. Unfortunately, localization of these clones with respect to the CF locus was a tedious exercise; each clone had to be physically or genetically mapped. Physical mapping study based on long-range restriction mapping analysis


203

showed that one of the microdissected clones was about 50 kb from MET, distal to CF (S. Zengerling et at., unpublished data). Based on a long-range restriction map of the CF region, experiments were also attempted to clone specific chromosome regions from highmolecular-weight DNA fractionated by pulsed-field gel electrophoresis. Michiels et at. (1987) succeeded in isolating one of the ends of a 450-kb Not I restriction DNA fragment containing the met gene. Physical mapping studies showed that this end (Lcn2 or D7S24) was about 300 kb 5' of the met gene, thus further away from CF. Based on the physical map of the region known to contain the CF gene, Ramsay et at. (1990) isolated a DNA segment (p02; D7S41l) from a 560-kb Sac II fragment from a CMOTderived cell line containing approximately 4.2 mb of human DNA in a mouse background. Pulsed-field mapping data showed that this clone was 300-350 kb from IRP. Its relative position to CF could not be determined by family studies because of the lack of informative meioses, but the cosmid clone H34 isolated with p02 was subsequently determined to contain a portion of the CF gene (Ramsay et at., 1990).

Chromosome Walking

Fragments adjacent to a number of DNA markers closely linked to the CF locus were isolated sequentially from various genomic libraries to cover a larger portion of the genome. The purpose of this laborious exercise was initially to increase the informativeness of the region for genetic analysis and, when markers were sufficiently close to the CF gene, the overlapping segments were used to detect candidate gene sequences. Therefore, chromosome walking was generally used in combination with other cloning strategies. The common techniques used to detect genes included crossspecies hybridization, RNA blot hybridization, cDNA library screening, identification of undermethylated CpO regions, and direct DNA sequencing. Using a cDNA which spans 75 kb of genomic DNA, Dean et at. (1987) identified two additional RFLP markers toward the ,5' end of the met gene; a genomic DNA fragment which could be used to detect these polymorphisms was also isolated, but subsequent studies showed that the CF gene was located 3' to MET. Scambler et at. (1987b) also isolated a total of 160 kb of DNA containing the MET locus and 90 kb around D7S8; these efforts were

204


not continued, as the two markers were found to be too far from CF. From the cII CMGT-derived cell line (see above, p. 199), Estivill et al. (1989b) isolated a cluster of three cosmids spanning a total of 75 kb. One of the DNA framents (D7S399) was found to detect a DNA sequence polymorphism and it was placed 160 kb from D7S23 in the direction of D7S8 (see Fig. 3). Family studies showed that D7S23 and D7S399 were on the same side of CF. From the same cell line, Ramsay et al. (1990) isolated another DNA fragment (D7S411) and performed a chromosome walk experiment covering part of the CF gene (see below, p. 201). The isolation of the CF gene was the result of a continous walk and jump from two DNA markers (7S122ID7S340) 500 kb from MET, toward D7S8, by Rommens et al. (1989a). The effort, which was guided by a careful restriction map (Rommens et al., 1989a,b) and a large number of RFLP sites (Kerem et al., 1989b), spanned a total of 280 kb, covering both D7S23 and D7S399 (Fig. 3). While traditional chromosome walking experiments (as described above) were conducted with the use of phage and cosmid vectors, the development of yeast artificial chromosome vectors capable of accepting large (;::: 1 mb) fragments of mammalian DNA clearly provided the method of choice for future disease gene cloning studies based on chromosome localization. Green and Olson (1990) and Anand et al. (1991) showed that it was possible to clone the entire, contiguous CF gene locus in overlapping YAC clones.

Recombinant Families

While various DNA segments or genes could be isolated from the region of interest with molecular cloning techniques, testing for their involvement in CF could only be done by genetic analysis with families that showed recombination near the CF locus. Because of the close distance between MET and D7S8, only a small number of recombination events were noted within this interval in a collaborative study (Beaudet et al., 1986). Two families were shown to contain recombination between CF and MET (Tsui et al., 1986a; FarraH et al., 1986c) and one between CF and D7S8 (R. White et al., 1986). The recombination breakpoints in these families were confirmed by testing with flanking DNA markers. In two German CF families, one showed a recombination between XV2C and CF, and the other between MET and XV2C (Berger et al., 1987).


205

Four additional ones were reported for the XV2C and CP interval (FarraH et af., 1988), but flanking DNA markers were not informative in these families. Subsequent studies showed that one of these recombinants was located between KM19 and E2.6 (Kerem et af., 1989b) (see Fig. 3) and that another one thought to be between KM19 and E4.1 (Kerem et af., 1989b) was actually an error in diagnosis (Devoto et af., 1990). Taken together, all the available family data narrowed the CF gene to a region of at least 850 kb in size (see Fig. 3). Uniparental disomy was documented in two patients (J. E. Spence et af., 1988; Voss et af., 1989), but this information was not useful in pinpointing the CP locus. Attempts were also made to screen for deletions located in the CF region with the use of pulsed-field gel electrophoresis, but the results were negative (Morreau et af., 1988; L.-C. Tsui and D. Markiewicz, unpublished data).

Allelic and Haplotype Association

Allelic association, more commonly interpreted as linkage disequilibrium, has been detected for many of the DNA markers closely linked to CP. In those analyses, information about the market>alleles associated with the normal (N) and CF chromosomes was obtained from the parental chromosomes in each affected family by using the affected children to determine the phase. An association was first detected for MET and, to a much lesser degree, D7S8 (Beaudet et af., 1986; Tsui et af., 1986a). Haplotypes for these DNA markers could also be derived and an association was detected (Beaudet et af., 1986). This observation was confirmed by many other investigators (Mathy et af., 1987; Schmidtke et af., 1987). The ability to detect allelic and halotype association for these markers is consistent with their close linkage to the disease locus. Allelic association with a distantly linked marker, TCRB, was observed in one study (McMillan et af., 1989). As DNA markers closer to the CF gene were identified, the degree of allelic association became even higher. The most notable ones are the RFLPs associated with D7S23, as detected by the probes XV2C and KM.19. In an early study with the XV2C marker, the common allele (the 2.1-kb Taq I fragment) was found to represent almost 92070 (195/213) of the CF chromosomes, but only 44% (93/212) of the N chromosomes (Estivill et af., 1987a). The association found for KM.19 was even more striking, with 88%

206


(157/178) of the CF chromosomes carrying the common allele, which was present in only 29070 (50/175) of the N chromosomes (Estivill et al., 1987b). Further, it was shown that the common haplotype ("B") for these two markers accounted for 85070 of the CF chromosomes, but only 16070 of the N chromosomes. Similar observations were made for these and other DNA markers by many other investigators (Beaudet et al., 1988; Weber et al., 1988; Maciejko et al., 1989; Krawczak et al., 1988; Hill et al., 1989; Lucotte et al., 1989; Estivill et al., 1989a,b; Vidaud et al., 1989; Fernandez et al., 1990). The unusually high degree of allelic and haplotype association was interpreted to mean that a single mutation accounted for a great majority of CF cases in some populations, particularly the Danish population (Estivill et al., 1987b). In another study by Estivill et al. (1988) with Italian families, however, evidence was presented to suggest that two mutations were responsible for most CF cases in southern European CF families. DNA marker haplotype studies in other populations also revealed additional, possible mutations. Ober et al. (1987) and Fujiwara et al. (1989) raised the possibility that there were as many as three different CF mutations in the Hutterites. On the basis of DNA marker haplotype data, Cutting et al. (1989) predicted that there were at least three novel mutations in the American Black population. In another study (Martin et al., 1988), a group of American Black CF patients were found to have similar haplotypes as those found in the Caucasian population, indicating racial admixture. Another potential application of the observed allelic association was its use in estimating the distance between the test markers and the CF gene (Estivill et al., 1987a; Weir, 1989). There was, however, no good method available for the calculation. In an early report, the CF gene was placed within 10 kb from the two test markers (Estivill et al., 1987a), which turned out to be at least 150 kb away from the gene (Rommens et al., 1989a). Nevertheless, the strongest CF association wa subsequently found with two markers within the gene itself (Kerem et al., 1989b). The difficulty was partially overcome by a modified method of haplotype analysis (Cox et al., 1989). Based on the strong haplotype association between the closely linked DNA markers and CF, it was also possible to modify risk calculations in genetic counseling for families where no DNA or mutation information was available from the affected family member (see below, p. 236).


207

Cloning of the CF Gene In 1989, Rommens and colleagues (Rommens et at., 1989a) reported the isolation of approximately 280 kb of a contiguous DNA segment, by chromosome walking and jumping from two of the DNA markers (D7S122 and D7S340) that were found to be closely linked to the CF gene (Rommens et at., 1988). Small DNA segments from this region were purified and tested for the presence of genes by their ability to detect possible conserved sequences in other animal species by DNA hybridization, by hybridization to messenger RNA (mRNA) products, or to cDNA clones from affected tissues, and by identifying protein-coding regions by direct sequence analysis. These investigations led to the identification of several gene sequences (Rommens et at., 1989a); on the basis of predicted biochemical properties and genetic analysis one of them was found to be the gene responsible for CF (Riordan et at., 1989; Kerem et at., 1989b). Structure of the Gene

The CF gene spans approximately 250 kb of DNA and contains at least 27 exons (Rommens et at., 1989a; Zielenski et at., 1991a) (Fig. 4). The full-length cDNA sequence has been described in the report by Riordan et at. (1989) and the genomic DNA sequences flanking individual exons reported by Zielenski et at. (1991a); the sequence data may be obtained from Genbank with accession numbers M55106-M55131. The most characteristic feature of the predicted protein product for this gene is the presence of two repeated motifs, each of which consists of a domain capable of spanning the membrane six times and another containing the consensus sequence of nucleotide (ATP)-binding domains (NBFs). Another striking feature of this predicted protein is a region containing a rather high proportion of charged amino acid residues linking the two halves of the protein (Fig. 4). This domain is thought to have a regulatory function and was thus named the R-domain (Riordan et at., 1989). The overall structure of this protein resembles many other prokaryotic and eukaryotic transport proteins, most notably the mammalian P-glycoprotein. Further discussion on the predicted properties of this protein is presented below (p. 222). RNA blot hybridization revealed a transcript about 6500 nucleotides in length; it could be detected in a variety of tissues, including lung, pancreas, sweat gland, liver, nasal polyps, salivary gland, and colon, all of which are

I

b

i

i

50

4

i i1 "

1 2 3

CFTA protein

2 3

CFTRGENE

IV Zi

5 Sa 6b

i

». p).

7 i

8 i

9

100

I

10

FS08

t::.

ATPbinding

10 11 12

4 5 7 9 Sa8 6b

A-domaJn

13

11 12

19

in

out

14a 14b15 16 17a 17b 18 19

150

I

13 14b16 18 14a 1517a 17b

Membrane

ATPbinding

200

I

20

21

1480

a.a.

22 23 24 I

250 (kb)

Fig. 4. The CFTR gene and its protein product. (a) The exoniintron structure of the CFTR gene, with open boxes denoting the 27 exons. The introns between exons 6a and 6b, 14a and 14b, and 17a and 17b were identified after the initial publication (Rommens et at. 1989a; Zielenski et at., 1990a). (b) The predicted domain structure of CFTR, with the exon junctions included . (c) A schematic model of CFTR, according to the proposed domain structure. There are two N-linked glycosylation sites on the fourth exterior loop of the predicted protein.

o

a

III

a:

III

:IE

::r

()

c:

aI

c: !!.

::J

III

;:

a.

::J

III

5:.

~

::

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(:)

"g

r-

~ co


209

tissues affected in patients with CF (Riordan et al., 1989). While all these predicted biochemical properties were consistent with the CF phenotype, the absence of a functional assay for the CF gene product meant that the only way to ensure that the gene was the CF gene was to identify a mutation in it. Upon sequence comparison of the cDNA clones from normal and CF individuals, a 3-bp deletion (..:l508) was discovered among the CF cDNA clones (Riordan et al., 1989) (see below). In the original report, this deletion was present in 68070 of the 218 CF chromosomes examined, but none of the 198 normal chromosomes (Kerem et al., 1989b). This strict correlation between the deletion and the disease thus provided an overwhelming statistical argument for the gene being responsible for CF. The result of DNA marker haplotype analysis also argued that the 3-bp deletion was unlikely to be a sequence polymorphism (Kerem et al., 1989b). Since the original reports, many other CF mutations have been described (see below, p. 210). A functional assay for the CF gene product has also been developed to show that this product could correct the defective chloride channel regulation in CF epithelium (Rich et al., 1990; Drumm et al., 1990) (see below, p. 226). These results further confirm the identity of the CF gene. To avoid confusion with some of the previously named CF-related proteins (see above, pp. 159-163) and to reflect its most probable function, this CF gene product has been referred to as the CF transmembrane conductance regulator (CFTR) (Rommens et al., 1989a).

The Major CF Mutation

The most frequent mutant allele of the CF gene is a 3-bp deletion which results in the deletion of a single amino acid residue (phenylalanine) at position 508 of the predicted polypeptide (Riordan et al., 1989). While the original study showed that this mutation (..:lF508) accounted for about 68070 of the CF chromosomes in Canada (Kerem et al., 1989b), a subsequent study showed that the frequency was higher (75%) in an American Caucasian population (Lemna et al., 1990). Since then, there have been a large number of studies to examine the population frequency of the ..:lF508 mutation in various geographical locations. In order to facilitate identification of the remaining CF mutations and to study the frequency of each of the mutations among different populations around the world, a consortium

210


consisting of more than 80 research groups has also been formed. The results of 35 studies have been published in a single issue of Human Genetics (Volume 85, September 1990) and summarized in the first report from The Cystic Fibrosis Genetic Analysis Consortium (1990). While the overall proportion of .:lF508 remains at 68070 for the total of 17,000 CF chromosomes analyzed, there are remarkable differences in its distribution among different populations (Fig. 5). A generally higher frequency is observed for the northern European countries in comparison to southern European populations (Estivill et al., 1989c; McIntosh et al., 1989; European Working Group on CF Genetics, 1990), ranging from 30-35% in the Ashkenazic Jewish population (Lemna et al., 1990; Lerer et al., 1990) to 87% in Demark (Schwartz et al., 1990). Haplotype analysis of DNA markers closely linked to the CF gene has shown that there is a strict association of one haplotype group (Ia) with the .:lF508-bearing chromosomes, suggesting a single origin for this mutant allele (Kerem et al., 1989b). Similar analyses have also been performed for additional .:lF508-bearing chromosomes with DNA markers more distantly linked to the mutation (European Working Group on CF Genetics, 1990). The results of these studies show that, while there is a strong association between .:lF508 and the B haplotype, the mutation is also found on non-B chromosomes. The latter observation may reflect presumptive recombination events between the mutation and these DNA markers (KM19 and XV2c) (Serre et al., 1990). The observed southeast to northwest gradient of the relative frequency of .:lF508 has, however, allowed the European Working Group to hypothesize that there was a diffusion of this mutation during the Neolithic Age, at a time when immigration of early farmers started from the Middle East and slowly progressed toward the northwest of Europe (Ammerman and Cavalli-Sforza, 1984). A selective advantage of this mutation or an associated gene located in the same region on the chromosome, or both, may have contributed to the spreading of this mutation. Further discussion on this subject is presented below (p. 233).

Non·.:lF508 Mutations

For the remaining CF alleles, more than 60 different mutations have thus far been reported to the consortium. A representative list of these mutations is shown in Table IV and their corresponding positions in the CFTR coding region are shown in Fig. 6. The most intriguing mutation is

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