Invited Review 1609776

European Journal Eur J Hum Genet 1996;4:65-73 of Human Genetics

Batsheva Kerema Eitan Keremb The Molecular Basis for Disease a Department of Genetics, Life Sciences Variability in Cystic Fibrosis Institute, Hebrew University and b Department of Pediatrics, Pulmonary and Cystic Fibrosis Clinic, Shaare Zedek Medical Center, Jerusalem, Israel

Key Words Abstract Cystic fibrosis Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in Variable phenotype the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The Male infertility disease is characterized by a wide variability of clinical expression. The clon­ Variable normal RNA ing of the CFTR gene and the identification of its mutations has promoted extensive research into the association between genotype and phenotype. Sev­ eral studies showed that there are mutations, like the AF508 (the most com­ mon mutation worldwide), which are associated with a severe phenotype and there are mutations associated with a milder phenotype. However, there is a substantial variability in disease expression among patients carrying the same mutation. This variability involves also the severity of lung disease. Further­ more, increased frequencies of mutations are found among patients with incomplete CF expression which includes male infertility due to congenital bilateral absence of the vas deferens. In vitro studies of the CFTR function suggested that different mutations cause different defects in protein produc­ tion and function. The mechanisms by which mutations disrupt CFTR func­ tion are defective protein production, processing, channel regulation, and con­ ductance. In addition, reduced levels of the normal CFTR mRNA are associ­ ated with the CF disease. These mutations are associated with a highly vari­ able phenotype from healthy individuals or infertile males to a typical CF disease. This variability in disease expression is associated with different lev­ els of normally spliced transcripts. Further understanding the mechanisms of CFTR dysfunction may suggest different therapeutic strategies for each class of mutations.

Cystic fibrosis (CF) is a common, lethal, autosomal by progressive lung disease, pancreatic dysfunction, ele­ recessive disorder among Caucasians. The frequency of vated sweat electrolytes, and male infertility [1], How­ the disease in the general population is 1:2,500 live birth, ever, there is a substantial variability in disease expres­ which calculates to a carrier frequency of 1:25. However, sion in all clinical parameters. In 1989 the gene causing the frequency varies between different ethnic and distinct CF was cloned and the protein designated cystic fibrosis geographic populations [1]. The disease is characterized transmembrane conductance regulator (CFTR) was iden-

© 1996 S Karger AG, Basel Batsheva Kerem, PhD Received: October 19,1995 KARGER 1018-4813/96/0042-0065$ 10.00/0 Department of Genetics Revision received: E-Mail [email protected] The Life Sciences Institute January 16, 1996 Fax+ 41 61 306 12 34 The Hebrew University Accepted: Jerusalem (Israel) January 22, 1996 tified [2-4], The most common CFTR mutation was one family. Only 10 mutations were found in more than found to be a 3 bp deletion, which leads to the removal of 100 patients, but in specific ethnic groups mutations oth­ a phenylalanine residue at amino acid 508 of the protein, er than the AF508 were found in relatively high frequen­ designated A 508. This mutation is carried by 70% of the cies [6]. For example, the stop codon mutation, W1282X, CF chromosomes worldwide but its frequency varies which is carried in the Ashkenazi Jewish population by greatly among different populations [5]. In Europe, the 50% of the chromosomes, appears worldwide in only 2% frequency of the A 508 mutation decreases from north­ of the CF chromosomes [6, 7], Among the Finnish, Ital­ west to southeast, ranging from 90% in Denmark to about ian, Amish, Hutterite, and other non-Ashkenazi Jews, rel­ 30% in Turkey. In many European populations, like those atively higher frequencies of specific mutations were in Belgium, Germany, The Netherlands, England and found [6,14]. This high frequency is probably the result of France, the frequency of the AF508 mutation is over 70% a founder effect or a genetic drift. The identification of the [5,6]. Among Ashkenazi Jews the frequency of the AF508 majority of mutations causing disease enables carrier test­ mutation is less than 30% [7]. ing of the general population for this mutation but the A recent extended haplotype analysis using highly abundance of the additional CFTR mutations hampers polymorphic microsatellite markers has been used to the availability and accuracy of such a test. It is generally study the origin and evolution of the AF508 mutation in accepted that as of today only 85-90% of the mutant Europe [8]. The results indicated that the AF508 muta­ alleles, in most populations, might be detected by muta­ tion occurred more than 52,000 years age, in a population tion screening. genetically distinct from the present European group. The mutation spread throughout Europe in chronologically distinct expansions, which might be responsible for the Spectrum of Mutations different frequencies of the AF508 in Europe. In addition to genetic analysis and prenatal counsell­ ing, the information on mutations causing disease may CFTR Gene Structure and Mutation Distribution provide an important insight into the CFTR function. Spectrum analysis of the mutations revealed that about The CFTR gene consists of 27 exons and spans over 50% of the mutations are missense mutations, 20% are 250 kb in chromosome 7q31. The CFTR protein consists frameshift mutations caused by small insertions or dele­ of 1,480 amino acids arranged in repeated motif structure tions and 15% are nonsense mutations. The rest are muta­ and of 12 membrane spanning regions (TM1 to TM12), tions affecting splicing and other variations [CF Genetic two ATP-binding domains (NBF1 and NBF2) and a high­ Analysis Consortium, pers. commun.]. The mutations are ly polarized domain named the R domain which is located along the entire CFTR gene, but there are several thought to have a regulatory function [3]. The CFTR pro­ mutations hotspots. For example, in exons 4, 7 and 17b, tein is allocated in the apical membrane of normal epithe­ which are parts of the two transmembrane domains, over lial cells by its two hydrophobic membrane-spanning 30 mutations in each exon were identified. A missense regions and the two nucleotide binding domains and the mutational hotspot in NBF1 is also apparent. In exon 11, R domain are oriented in the cytoplasm. It forms a chlo­ within a small region of 15 bp 11 different mutations were ride channel regulated by cyclic AMP [9-13]. In addition, identified. This pattern suggests that ATP binding or the CFTR appears to have other functions, including the hydrolysis are critical for normal CFTR function. In addi­ regulation of other channel proteins [13]. tion, there is a nonsense mutational hotspot in exon 13. The focus of this review is the genetic basis for disease Within 100 codons, 10 nonsense mutations were identi­ expression and phenotypic variability among different fied. This might indicate that the first half of the protein is patients. In addressing the question of how the different insufficient to confer the normal CFTR activity. mutations disrupt the normal functions of the CFTR pro­ Insight into the functions of the individual domains of tein and how these defects affect the disease, it is impor­ the CFTR protein has come from studies of the outcome tant initially to consider the nature and the frequency of of specific mutations. The TM1-TM12 appear to contrib­ the mutations causing the disease. ute to the formation of the Cl- channel pore, since muta­ So far over 500 different CFTR mutations were identi­ tions of residues within these regions alter the anion selec­ fied [The CF Genetic Analysis Consortium, pers. com­ tivity of the channel. The NBF1 and NBF2 control chan­ mun.]. Most of these mutations are rare, found in only nel activity through an interaction with cytosolic nucleo-

66 Eur J Hum Genet 1996;4:65-73 Kerem/Kerem tides. The regulatory domain also controls channel activi­ Table 1. Classification of mutations according to severity of phe- ty: phosphorylation of the regulatory domain, usually by notype cAMP-dependent protein kinase, is required for the chan­ Severe Mild Substantial variable nel to open [reviewed in detail in 1], 1078delT [33] RI 17H [31] G85E [55-57] AF508 [31] A455E [35] R334W [37] Association between Genotype and 1717-1G—> A [31] 3849+lOkb C—>T 5T [43-45,62] G542X [31] [15, 16,34] Phenotype in CF G551D [27] R553X[30, 31] CF is characterized by a wide variability of clinical 621 + 1G->A [31] expression: patients are diagnosed with various modes of W1282X [31] presentation at different ages, from birth to adulthood, N1303K [31] with considerable variability in the severity and rate of 1811+1.6 kb A->G [36] 1677delTA [32] disease progression of the involved organs. Although most CF patients are diagnosed in the first year of life This list includes mutations that had been studied in at least 10 with typical lung disease and/or pancreatic insufficiency patients. (PI), an increasing number of patients have recent years been diagnosed with atypical disease only in adulthood. Furthermore, although progressive lung disease is the most common cause of mortality in CF, there is a great variability in the age of onset and the severity of lung mutations, and was associated with earlier age of onset, disease in different age groups. Variability is also found higher sweat Cl- levels, younger age and PI [22], Despite in male infertility. Almost all male CF patients are infer­ the severe nature of this mutation, the severity of pulmo­ tile due to congenital bilateral absence of the vas defer­ nary disease varied considerably among the patients. This ens (CBAVD); however, recently fertile CF male patients association of homozygosity for the AF508 mutation with have been reported [15, 16]. The extent of the pancreatic a generally more severe disease presentation was subse­ disease also varies. Most affected individuals suffer from quently confirmed by other investigators [23-26], Several PI; however, approximately 15% of the patients possess subsequent studies analyzed the genotype-phenotype cor­ sufficient exocrine pancreatic function to permit normal relation in several mutations for which a large enough digestion (PS) [17-19]. A remarkable concordance of the number of patients (>10 patients) was available [15, 16, pancreatic function status was found within affected 27-37], These studies have shown that there are muta­ family members [20], suggesting that genetic factors tions other than the AF508 mutation that are associated could influence the severity of pancreatic disease and with severe disease presentation. The clinical presenta­ possibly its rate of progression. Evidence for a genetic tion of patients carrying two of these severe alleles is simi­ basis for the severity of the pancreatic disease came from lar to that of the AF508. studies of the distribution of haplotypes linked to the Only few mutations were found to be associated with CFTR locus and from the distribution of the AF508 the milder phenotype [15, 16, 29]. Patients carrying at mutation among PS and PI patients. More than 50% of least one mild mutation were diagnosed at a later age, the CF-PI patients were homozygous for the AF508 mu­ were older at the time of the study, had lower sweat chlo­ tation but none of the CF-PS patients suggesting that the ride levels, some with normal or borderline values, had a AF508 mutation is associated with PI, and that muta­ better nutritional status and most of them had PS. Inter­ tions associated with PS would be dominant over those estingly, some of these patients had normal or interme­ with PI [4,21], diate sweat chloride levels (40-60 mEq/1). In these pa­ The cloning of the CFTR gene and the identification of tients, the CF diagnosis could be made only after genotype its mutations have promoted extensive research into the analysis was available. Table 1 lists the CFTR mutations association between genotype and phenotype, which has which were shown to be associated with either the severe contributed to our understanding the molecular mecha­ or the mild phenotype. nisms of the remarkable clinical heterogeneity of CF. The Higher frequencies of CFTR mutations were found clinical presentation of patients homozygous to the among patients with incomplete CF expression. For many AF508 mutation was more severe compared to milder years, elevated sweat electrolyte levels were the gold stan-

Molecular Basis for Disease Eur J Hum Genet 1996;4:65-73 67 Variability in CF Table 2. Patients with increased CFTR mutation frequencies these mutations were found only in infertile males with CBAYD. This significantly higher frequency of CFTR Chronic bronchitis [64-66] mutations among men with CBAYD indicates that in Chronic bronchiectasis [64, 66-68] Pseudomonas bronchitis [68] many cases CBAVD is caused by defective CFTR alleles Chronic sinusitis with nasal polyposis [69] and might be considered an atypical CF disease. How­ Allergic bronchopulmonary aspergillosis [68] ever, the genetic basis of CBAVD in the other males with Congenital bilateral absence of the vas deferens [39-48] CBAVD, and its association with CF remained unclear. Extended haplotype analysis of polymorphic sites in the CFTR locus in familial CBAVD supported the hypothesis that in many families CBAVD is associated with two dard for CF diagnosis. Although patients with atypical CF CFTR mutations; however, in others it might be caused presentation and intermediate sweat electrolyte levels by other mechanisms such as homozygosity or heterozy­ were reported, it remained unknown whether they had CF gosity for partially penetrant CFTR mutations [48], More or some other similar diseases. The cloning of the CFTR recently it has been shown that 30-40% of the chromo­ gene and the identification of CFTR mutations enabled, somes of males with CBAVD carry a splice variant, 5T in many cases, a definite diagnosis of CF. Patients sus­ allele, in the acceptor/branch site of exon 9 [43-45]. This pected of having atypical CF presentation were tested for variant was found to cause high levels of exon 9 skipping the presence of CFTR mutations and certain mutations, leading to a nonfunctional protein. The high frequency of usually those associated with the milder phenotype were the 5T allele among males with CBAVD is significantly found. The clinical characteristics of atypical CF patients different from its frequency in the general population (3- are diagnosis above 10 years of age, survival into adult­ 5%) suggesting that the 5T allele is associated with hood, chronic sinopulmonary disease, pancreatic suffi­ CBAVD [43-45], ciency, and sweat chloride <60 mEq/1. It is recommended Males with CBAVD are currently participating in pro­ to refer such patients for CFTR genotyping. Table 2 lists grams of sperm aspiration and subsequent in vitro fertili­ clinical conditions with increased frequencies of CFTR zation. It is therefore important to perform CFTR muta­ mutations; however, their direct association with CF is tion analysis of these men and their female partners prior still unclear. Absence of a known common mutation does to their participation in this program. not rule out CFTR-associated disease, since in all popula­ tions there are still unidentified mutations. In summary, the results of these genotype-phenotype Molecular Mechanisms Underlying Disease studies indicate that the variability in disease presenta­ Variability among Patients Carrying Different tion is the result of the type of the CFTR mutation. Fur­ CFTR Mutations thermore, these studies confirmed the previous hypothe­ sis that the milder mutations are dominant over the A different approach to understanding the genotype- severe and therefore will determine the phenotype. phenotype correlation involved in vitro studies of the CFTR function. Sheppard et al. [49] studied the expres­ sion of normal and AF508 CFTR cDNA in epithelial Congenital Bilateral Absence of the Vas deferens cells. They found that the wild-type CFTR transfected with the recombinant vaccinia virus generated a mature Almost all men with CF are infertile, presenting with fully glycosylated form of the protein consistent with azoospermia but normal spermatogenesis, as a result of delivery to the plasma membrane. The mutant AF508 CBAVD [1], CBAVD is also found in otherwise healthy produced only the immature core-glycosylated form of the infertile men, although detailed clinical evaluation of protein, indicative of defective processing. The partially these men was not reported. Holsclaw et al. [38] hypothe­ glycosylated protein is degraded instead of trafficking to sized that CBAVD is a mild form of CF. Mutations in the the apical cell membrane. As a result, the AF508 CFTR CFTR gene were analyzed in men with CBAVD [39-48], cannot be detected at the cell surface, and thus, leads to a and 10-20% were found to carry two mutated CFTR loss of the cAMP-mediated chloride conductance. In alleles (with at least one of the mutations being mild), 40- another mutation, G551D, the protein can be processed 60% were found to carry one known CFTR mutation and and correctly targeted to the plasma membrane but lacks in 30-50% no CFTR mutations were found. Several of full responsiveness to stimulation by cAMP [50], Recom-

68 EurJ Hum Genet 1996;4:65-73 Kerem/Kerem Table 3. Classes of CFTR mutations Class I Class II Class III Class IV Class Y (protein (processing) (regulation) (conduction) (reduced level of normal production) RNA or protein)

G542X AF508 G551D R117H 3849+lOkb C—>T 621 + 1 G-+T N1303K R334W 5T 3905 insT S549R R347P 181 l+1.6kb A—>G AI507 A455E

The examples are of relatively common mutations. Modified version of the classification suggested by Welsh and Smith [52].

binant adenoviruses were used to transduce normal and Another class of mutations, class V mutations, was variant forms of CFTR into surface epithelial cells of recently introduced [53, 54] which includes mutations human bronchial xenografts grown in mice. Using immu­ affecting the level of normal mRNA transcript and pro­ noperoxidase electron microscopy analysis it was shown tein required for normal function. This class might in­ that the mutation G551D is predominantly localized to clude mutations affecting correct splicing of pre-mRNA the apical plasma membrane of the cells. In cells express­ transcripts by either exon skipping, inclusion of extra ing AF508 CFTR the peroxidase staining was localized to crypt exon(s) and by affecting mRNA and/or protein sta­ the nuclear envelope and the endoplasmic reticulum. bility (table 3). These studies suggested that different mutations cause different defects of protein production and function. This led Tsui [51] and subsequently Welsh and Smith [52] to Genotype-Phenotype Variations among Patients suggest four mechanisms by which mutations disrupt Carrying the Same CFTR Mutations CFTR function. Class I mutations cause defective protein production. As has previously been shown, CFTR mutations can be In this group there are mutations containing premature classified as associated with either severe or mild disease; termination signals and mutations causing truncated or however, there is a substantial variability in disease unstable protein. These mutations are expected to pro­ expression among patients carrying the same mutation, duce little or no CFTR chloride channels. Class II muta­ mainly in the extent of pulmonary involvement. This tions are associated with defective protein processing, as variability was found both among patients carrying the shown in the AF508 mutation. The abnormally processed same severe mutations and among patients carrying the protein which fails to progress through the biosynthetic same mild mutations. Several mutations show an extreme pathway is degraded. As a result it cannot be detected at variability among patients. One such mutation is the mis- the cell surface. However, the mutant AF508 protein was sense mutation, G85E [55-57]. All the tested clinical found to have near normal activity when it reaches the parameters were significantly more variable compared to cell surface [50], Class III mutations are associated with those found among patients with severe or mild muta­ defective regulation. Alanysis of some mutant proteins tions [57]. It is important to note that the variability in the that do reach the apical membrane shows that several G85E mutation was also found among siblings and have mutations in the nucleotide binding domains. Some among patients from the same extended family. For mutations cause total loss of ability to be stimulated by example, in one family, 2 children were diagnosed before ATP, and in others this ability is reduced. Class IV muta­ 1 year of age with typical CF presentation, had a severe tions are associated with defective conductance. Patch course and died at a relatively early age. Their subsequent clumping studies have shown that the CFTR chloride brother developed liver disease at the age of 12 with no channels are capable of generating cAMP-regulated chlo­ other signs of CF, including <60 mEq/1 sweat Cl-. His ride currents, though the amount of current is reduced. diagnosis was made by genotyping only. Thus, this muta­ Table 3 lists mutations according to these classes. tion cannot be classified as severe or mild mutation. The missense mutation R334W was also associated with an

Molecular Basis for Disease EurJ Hum Genet 1996;4:65-73 69 Variability in CF extremely variable expression [37], Discordance between The variability found among patients carrying class V sibs was found in pancreatic and lung disease. Another mutations might shed new light on the molecular mecha­ genetic variation associated with a high variability of phe­ nisms underlying the disease severity. In cases where the notypic expression is the 5T allele. This variant was found mutation affects the level of normal splicing and leads to to cause high levels of exon 9 skipping leading to a non­ transcription of normally and aberrantly spliced trans­ functional protein [58], As discussed above a substantial cripts, the disease might be caused by insufficient levels of portion of infertile males with CBAVD carry this allele. the normally spliced CFTR transcripts. Several mutations However, fertile males with the same genotypes were also were shown to lead to the transcription of both aberrantly identified [43-45]. In addition the 5T allele was shown to and normally spliced transcripts [15, 36, 58], The muta­ affect the disease severity of patients carrying the muta­ tion 3849+10kb C->T creates a partially active splice site tion RI 17H [59]. In patients carrying the RI 17H muta­ in intron 19 which can lead to the insertion of a new 84-bp tion on the 5T allele the phenotype was mild CF and in ‘exon’, containing an inframe stop codon. Patients carry­ patients carrying the mutation on a 7T allele the pheno­ ing the 3849+lOkb C->T mutation have milder clinical type was CBAVD only. More recently, the 5T allele was presentation with variable disease severity [15, 16]. Vari­ found among individuals with atypical and typical CF ability in the level of the aberrantly spliced transcripts was with no other identified CFTR mutation [44, 60], Analy­ found in respiratory epithelial cells from CF patients sis of their clinical presentation indicated that most pa­ carrying this mutation. The level of the aberrantly spliced tients suffered from respiratory disease presenting as asth- transcripts correlated with severity of pulmonary func­ ma-like symptoms, nasal polyposis, chronic sinusitis, tion. Patients producing undetectable levels of aberrantly chronic bronchitis or bronchiectasis. Several patients had spliced transcripts had normal or minimal lung disease, pancreatic insufficiency, 2 with meconium ileus. Sweat whereas patients with high levels of aberrantly spliced Cl- levels ranged from normal to elevated levels. Of the transcripts had severe lung disease. No correlation was males with respiratory disease who were old enough to be found with sweat chloride levels, pancreatic status or age evaluated for fertility status, several were fertile, one of [61], consistent with the hypothesis that tissue-specific them had pancreatic insufficiency [60]. Thus the 5T allele splicing factors affect the level of alternative splicing dif­ might be considered a mutation causing disease associat­ ferently in different tissues. The 5T allele should also be ed with an extreme variability of clinical presentation: included in this group. As mentioned above, the frequen­ from normal healthy fertile individuals or males with cy of this allele in the normal population is nearly 5%; CBAVD to the atypical or typical clinical phenotype of however, several studies found that among males with CF. Again, this extreme variability was found among CBAVD the incidence of the 5T allele was nearly 30% members of the same family. [43-45]. These results implicate that insufficient levels of normally spliced CFTR transcripts might be associated with infertility. More recently, the level of the aberrantly Molecular Mechanisms Underlying Disease spliced transcripts lacking exon 9 was studied in respira­ Variability among Patients Carrying the Same tory epithelial cells of different patients. As for the CFTR Mutations 3849+lOkb C->T mutation the level of the aberrantly spliced transcripts correlated with the severity of pulmo­ The molecular mechanisms underlying the variability nary function [62]. Futhermore, the level of the aberrantly found among patients carrying the same CFTR mutations spliced transcripts in epididymal epithelial cells of males are not well understood. The results of expression studies with CBAVD was consistently low. A significant differ­ of the AF508 mutation might suggest that among patients ence was found between the level of the aberrantly spliced homozygous for the AF508 mutation the variable lung transcripts in respiratory and epididymal epithelial cells disease might result from allelic differences in genes asso­ of the same individual. Allelic differences in splicing fac­ ciated with trafficking the mutant protein to the cell sur­ tors might contribute to the variability in disease expres­ face. Patients with a more severe lung disease might have sion in different tissues of the same individual and in the low levels of protein successfully transported to the cell same tissue of different patients. membrane while patients with a milder lung disease Understanding the mechanisms of CFTR dysfunction might have higher levels, which confers partial function­ may suggest therapeutic strategies. For class I and II ing of epithelial respiratory cells. mutations, pharmacological agents designed to enhance opening of mutant channels may not be very effective,

70 Eur J Hum Genet 1996;4:65-73 Kerem/Kerem since protein is not produced or little is present in the cor­ delivered to the cell membrane. Presumably the folding rect cellular location. Strategies to relocate class II muta­ process is able to occur, at least partially, at reduced tem­ tions including the most common AF508 mutation could perature. For patients with class IV mutations stimulation be beneficial. This has been shown to be possible by in of the opening of the mutant CFTR channels is required. vitro studies. Denning et al. [63] showed that reducing the Further understanding of the mechanisms regulating al­ incubation temperature of epithelial cells containing ternative splicing will contribute to potential therapy for AF508 CFTR from 37 to 23-30 °C caused some of the patients carrying class V mutations. Thus, if gene therapy mutant proteins to escape from the endoplasmic reticu­ may not be available each mechanism of disease causing lum, being fully glycosylated in the Golgi complex and mutations may require a different therapeutic approach.

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Molecular Basis for Disease Eur J Hum Genet 1996;4:65-73 73 Variability in CF