The up-to-date molecular genetics of cystic fibrosis

The up-to-date molecular genetics of cystic fibrosis

455 Focus The up-to-date molecular G Novelli ‘, F Sangiuolo’, (Received Summary and Cystic the 6 April -The cloning and sequencing of the cy...

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455

Focus

The up-to-date

molecular

G Novelli ‘, F Sangiuolo’,

(Received

Summary and

Cystic

the

6 April

-The cloning and sequencing of the cystic biochemical defect of this disease, and allowed

fibrosis

/ CFTR

I CF

mutation

/ CF screening

genetics of cystic fibrosis

P Maceratesi2, B Dallapiccola2*3

1994:

accepted

fibrosis gene development

I gene

Introduction Cystic fibrosis (CF, MIM* 219700) is the most common lethal autosomal recessive disorder among Caucasians and a leading cause of childhood death [2,7, 761. Frequency estimates in different white populations vary from 1 in 500 to 1 in 3800. CF is rare among American blacks (1 in 17,000 births), and Asians (approximately 1 in 90,000). About 1 in 20 to 1 in 25 people are heterozygous for this mutation. The disease is variable with three major manifestations: extremely salty sweat, and serious digestive and respiratory problems [7, 72, 761. There are two main clinical CF subgroups: pancreatic sufficiency (15%) and pancreatic insufficiency (85%) patients. In the latter group, pancreatic enzymes fail to reach the intestine with a subsequent incomplete digestion and adsorption. Recurrent pulmonary infection is the main determinant of morbidity and duration of survival [33, 72, 501. The median age of survival is 24 years, with over 25% of patients living into their 30s. Male patients are sterile because of damaged testicular tubules. Fertility is also consistently reduced in females [33, 721. The electrolyte transport is defective. This results in viscous, dehydrated secretions from exocrine glands causing obstruction of the lumina of tubular structures such as airways, pancreatic ducts, and bowel [20, 371. Five to 15% of CF neonates

25 July

1994)

has provided insight into the clinical of mass screening and gene therapy

heterogeneity programs.

therapy

have meconium ileus and often the intestinal disease is manifested during fetal life with echogenic bowel, dilated intestinal loops, echogenic masses, and intraperitoneal calcifications [20]. CF is the most outstanding example of successful application of positional cloning to isolate human disease genes [13]. The cloning of the CF gene has provided new understanding of the basic defect of this disease, development of accurate DNA diagnostic testing, and new therapeutic strategies [12, 32, 54, 58, 63, 761. The CF gene and protein Attempts to localize the CF gene involved several strategies following its initial assignment to the long arm of chromosome 7 [76]. Closely linked RFLPs were found which delimited the CF gene in a genomic region of about 1 x lo6 base pairs (bp) [76]. More than 250 random clones from this region were isolated and individually mapped. Combining the saturation cloning with linkage analysis, a series of putative gene sequences were identified and characterized [76]. One of them (10-l) detected a 6.5 Kb RNA transcript in the chloride-transporting epithelia and, cross-hybridized with different vertebrate species [76]. This clone was used as a probe to obtain overlapping cDNA clones and, as a starting point to isolate the complete coding sequence of this gene.

456

This gene spans about 230 Kb of genomic DNA at chromosome 7q3 1, and was predicted to code for a 1480-amino acid protein, named cystic fibrosis transmembrane conductance regulator, or CFTR [12, 761. CFTR maps in a region abundant with genes involved in ion traffic, including the exchange protein EPB3, the muscle chloride channel gene CLCNI, the amiloride-binding protein ABPl, and the mucin 3 gene [ 1, 761. The identification of CFTR mutations in CF patients but not in normal chromosomes has proved that CFTR was the CF gene [76]. The CFTR gene shows features of house-keeping genes (S’GC-rich region, absence of TATA element, presence of GC boxes, and API sites). However, differently from them it has a high tissue specific expression [28, 74, 761. The CF gene comprises 27 exons coding for a symmetrical protein member of the “ABC” (ATP-binding cassette) transporter superfamily. CFTR contains two nucleotide-binding folds (NBF) and multiple transmembrane-spanning domains, connected by a highly charged intracellular domain with multiple potential CAMP-dependent protein kinase phosphorylation sites (R domain) [32, 54, 58, 761. This gene is a member of a superfamily of membrane transporters called “traffic” ATPases, which include the multidrug resistance (MDR) protein, and the prokaryotic permeases [17, 45, 841. Recent studies suggest that the normal CFTR molecule has two separate functions: to provide a chloride channel and to act as a regulator, opening and closing the pore, that is, the gate and the gatekeeper [66, 671. This is in agreement with the “flippase” (transport through the interaction with phospholipid bilayer) activity of the mdr2 protein in mouse [17]. CFTR normally operates as a multi-ion pore converted to a single-ion pore with reduced conductance by disease-causing mutations [67]. The reduced Cl- conductance mediated by CFTR is the primary physiological defect in CF. Cl- conductance is in fact essential for fluid secretion and salt absorption in the wet epithelia affected by the disease [33, 79, 871. Failure in secretion blocks the exocrine ducts and results in a cascade of destructive effects in different organs including the pancreas, epididymis, and lung [33, 871. However, the pump function of CFTR may contribute to the disease phenotype. In fact, it is expected that the newly recognized “alternate functions” of CFTR as plasma membrane recycling, and acidification of intracellular vescicles, could explain in part the pleiotropic nature of CF [5, 401. Expression studies have demonstrated a

specific apical membrane localization of the CFTR protein in pancreatic duct cells, reabsorptive ducts of sweat glands, and intestine crypts [74]. In normal adult respiratory epithelia, CFTR is localized in the serous tubules of the submucosal glands and in a subpopulation of gland cells’ duct, with little expression in other respiratory epithelia [74]. High levels of CFTR expression were found in reproductive tissues such as the cervix, fallopian tube and epididymis [74]. CFTR is activated by a multistep procedure, starting from the phosphorylation in SERVO by the CAMP-dependent protein kinase A (PKA). followed by ATP hydrolysis [54, 871. This event is necessary for channel opening and chloride movement by its electrochemical gradient [5, 541. Little is known about the mechanism of regulation and activation of the alternate functions of CFTR. In vitr-o studies using mutant CFTR proteins have provided insight into understanding the mechanism of the defective Cl- permeability. The most common mutation (AF 508 CFTR) leads to a reduced CAMP-sensitive Cl- conductance in epithelial cells as a consequence of cellular mislocalization and a more rapid turnover, due to changes in protein structure and stability [35, 711. CFTR

mutntiotls

Nowadays, more than 400 mutations have been detected in the CFTR gene based on a systematic search of 43,000 mutant chromosomes (The Cystic Fibrosis Genetic Analysis Consortium, February, 1994) (fig 1). A 3 bp deletion at codon 508

EXONS

Fig I. Locations and number of the Genetic Analysis Consortium,

CF mutations 1994).

(Data

from

(AF508) is the most frequent mutation, accounting for approximately 70% of worldwide CF chromosomes [75, 761. CF mutations include the following types: general (observed everywhere) local (observed in two or three closed populations), demic (observed twice or more in the same population), and private (observed only in the worldwide survey of the CF Consortium). The majority of mutations are rare, accounting for less than 1% of the CF alleles. However, a wide variation in mutation frequency does exist between different populations. For example AF508, reaches 70-90% in North Europe and North America, while it is less frequent in the Mediterranean area, which accounts for less than 50% of CF chromosomes (table I) [55, 62, 75, 761. Twenty-four general mutations, (AF508, G85E, R117H, 612+ 1G + T, 711 + lG+T, 1078delT, T334W, R347P, A455E, A1 507, S549N, R560T. 1898 + 1G + A, 2184delA, 2789 + 5G + A, R 1162X, 3659delC, 3849 + 10 KbC + T, G542X, G55 1D, R553X, W 1282X, N 1303K, and 1717-1 G -+ A), account for 77.4% of CF chromosomes in the world [69]. To-date, over 90-99% of CF chromosomes have been characterized in eight ethnically discrete populations, including Ashkenazi Jews, Bretons, Hutterites, Danish, French-Canadians, Welsh, Belgians and southern French [ 10, 11, 22, 15, 57, 60, 691. These mutations include missense, nonsense, frame-shift, splice, and small insertions or deletions. No major deletion of a large part of the gene has been described (a complex gene rearrangement has been found, X. Estivill, personal communication, 1994). About 65% of mutations are substitutions or deletions of a single amino acid and occur in the first half of the gene (fig 2). Almost 80% of changes in the second half of the gene are nonsense, frameshift, or splice mutations [75]. Primutations are randomly vate and demic distributed within the gene, whereas local, and general mutations are particularly converged within the first transmembrane (MSl) and NBFl domains [62]. This is in agreement with the hypothesis that selective factors favoured the successful spreading of these mutations [61, 621. This is confirmed by their association with specific RFLP or VNDR markers, suggesting a common origin and spreading out through population migrations [61, 621. It has been estimated that AF508 arose between 65,000 and 215,000 years ago in a population genetically different from the present day Europeans [43]. The high frequency of a specific haplotype’associated with

6 z 6

Fig 2. Single-base Genetic Analysis

substitutions Consortium,

in CFTR 1994).

gene

(Data

from

the

general CFTR mutations has been explained by meiotic drive of specific alleles at other closely linked loci in disequilibrium with the CFTR locus, as gene(s) for an increased resistance to Cl--secreting diarrhoea [56]. This latter gene (CLD, MIM* 214700) has been mapped close to CFTR, but haplotype analysis failed to demonstrate allelic association between the two genes [31]. Although AF508, as well as most of the CFTR mutations, appears to have originated from a common haplotype, being identical by descent, it is probable that mutations occurring in hypermutable CpG dinucleotides (for example R553X) have originated from recurrent mutations [62]. The majority of CF gene mutations have been detected in the coding region. Therefore, it is likely that the remaining mutations (about 26%) are located in the splice sites or in the CFTR promoter. Furthermore, techniques currently used for detecting mutations (denaturing gradient gel electrophoresis, single strand conformational polymorphism, chemical cleavage, or hydrolink analysis) do not uncover all mutations, some of them being missed during gene scanning [21, 23, 261. Other more accurate strategies for detecting nucleotide variations, such as direct sequencing, can be used to characterize the remaining CF alleles. The distribution of CFTR mutations has important implications for understanding the complexity of the CFTR functions and predicting the phenotypic effects of particular mutation [70731. Naturally occurring and in vitro generated CFTR mutations have provided information. on functions of single CFTR domains. Clusters of

458 CFTR mutations are housed within the two NBF domains which are the most highly conserved portions of the protein, supporting their critical role for the CFTR function. CF mutants include: i) Null phenotypes, which are due to a block of CFTR from its proper insertion into the apical membrane, or a CFTR abolition due to premature STOP or alteration in protein folding and maturation. A typical example is AF508, which results in a temperature-sensitive defect in protein processing, and fails to reach the plasma membrane at 37°C with a complete absence of Cl- channels phenotypes, or mutations [791; ii) modified where the protein is correctly processed but the channel fails to open or become unreactive to stimulations; iii) limited phenotypes, or mutations with reduced Cl- conductance per cell (about 530% of the wild-type CFTRs) but with a proper location of the protein. Representative examples are the missense mutations altering different arginine residues in the transmembrane domains (R334W, R347P, R117H), which transform CFTR from a multi-ion to a single-ion channel [40]. Nucleotide substitutions (FSOSC, R75Q, M470V) that do not cause disease have been detected in the coding region of the gene and are considered to be neutral polymorphisms because they are located on the presumed wild-type chromosomes in obligatory heterozygotes (fig 3). These variations are often found in combination with CF chromosomes. This suggests that a second mutation inactivating these variant alleles occurs on the same CF chromosome. Alternatively, other genetic factors, including the type of allelic CF mutation in a compound heterozygote, or other gene loci, influence the phenotype [16].

40%

Fig from

3. Neutral nucleotide the Genetic Analysis

changes in the CFTR Consortium, 1994)

gene

(Data

Genot?lpe-phenot?.l,e

correlntiorl

Although the wide variety of clinical symptoms observed in CF patients indicates that environmental variables and genetic factors other than the CFTR locus influence the phenotype, it has been established that clinical features are determined by the genotype itself [76]. Following the identification of preferential allele associations (linkage disequilibrium), it was suggested that different CF mutations were associated with (85%) or without (15%) pancreatic insufficiency (PI). This hypothesis has been confirmed by genotype-phenotype studies in patients carrying different CFTR mutations. It has been shown that about 90% of these mutations, including AF508, are associated with PI, and 10% with a nearly normal pancreatic function (pancreatic sufficiency or PS). Homozygotes or heterozygotes for missense alleles such as R117H, R334W, R347P, A455E, or P574H are PS, whereas those with two copies of nonsense, frameshift, splice-site or particular missense mutations are almost invariably PI. This is in agreement with the location of these mutations within the CFTR protein [68, 75, 761. Other phenotypic differences, including severity of lung disease, age at diagnosis, sweat chloride levels, weight percentile and weight height ratio have been observed in patients with different CF genotypes. However, most of these associations are little supported, since satisfactory criteria for classification of the different degrees of these manifestations are often lacking or the examined parameters reflect pancreatic function [68]. The compound heterozygotes R117H/AF508 are PS, are diagnosed at the age of 10.2 f 10.5 years versus 2.5 f 4.3 years, and show lower sweat chloride concentrations compared to AF508 homozygotes [68]. This demonstrates that R117H is associated with PS and has a dominant effect on the AF508 mutation. This information has important implications on the prenatal and prognostic counselling of patients with this genotype. Patients with homozygous nonsense mutations frequently show a mild course of the disease, but this is not universal [59]. Homozygotes and compound heterozygotes for nonsense mutations known to cause severe reduction of CFTR mRNA (G542X, R553X, W1282X) do not show consistent phenotypic differences compared to AF508/AF508 patients [77, 801. The abnormal high incidence of CFTR mutations detected in patients lacking the classical symptoms of CF (eg infertile men with congenital

459 bilateral absence of vas deferens [CAVD]; patients with borderline sweat chloride or low sodium concentrations; patients with chronic bronchitis or brochiectasis) has suggested that these diseases are the “mild” forms of CF, due to pleiotropic function of the CFTR protein. Screening programs have recently demonstrated that 57% of patients with CAVD have AF508 compared to a 4% carrier frequency in the general population [6, 821. Usually AF508 is associated in these patients with rare and unknown CFTR alleles. However, among these compound heterozygotes, the R117H mutation is highly represented [48, 491. CAVD patients could be defined as an incomplete or variant form of CF, inherited as an autosomal recessive condition in sons of carriers of different CFTR mutations [48, 491. This indicates that the number of CF alleles into the gene pool is even higher than previously expected, increasing the population carrier frequency to - l/15, based on an estimated figure of 35,000 - 40,000 men affected by CAVD [48, 491. CF genetic testing is recommended and counselling of each couple with CAVD sterility requiring microsurgical epididymal sperm aspiration combined with in vitro fertilisation and tubal embryo transfer is also recommended [48, 491. Detection

of CF homozygotes and carriers

Identification of the CF gene and its CFTR protein has greatly improved the detection of homozygotes (before or after birth) and heterozygotes (before or after producing an affected child). Different CF tests are now available for in vitro diagnostics based on polymerase chain reaction (PCR). Most of them detect multiplexing CF mutations, with a 100% sensitivity and specificity [24, 53, 65, 851. General strategies have been developed to improve prenatal diagnosis and genetic counselling of CF. based on these approaches (table II) [23]. Direct detection of mutations in conjunction with microsatellite haplotype analysis grants the monitoring of all pregnancies in CF families with a one in four risk [44]. Close relatives (ie, sibs, aunts, uncles) of high-risk families, can be easily genotyped and their reproductive risk, determined. In couples in which one partner is a known carrier, it is possible to reduce their risk to 1 in 500, upon exclusion of 80% of CF mutations, or to 1 in 2000, following a negative screening result of 90% of CF mutations. Of course, these figures are related to geogra-

Table

I. Distribution

Mutation

of CF mutations

in Central

Italy.

Relative frequerq

AF508 Nl303K 1717-IGA G542X G85E Wl282X S4X 621 + IGT LlO77P Hl39R

LlO65P M348K D614G R347P MIV Dl IOH RI l7L C276X Rl066C

Fl052V 40 I6insT 3667ins4 S549R(AC) S549N G55lD Total

48.5 9.58 5.5 3.28 2.5 2.5 1.6 0.92 0.51 0.51 0.51 0.51 0.51 0.51 0.5 I

0.51 0.51 0.51 0.51 0.51 0.51 0.35 0.25 0.25 0.26 81.61

Relative frequency of each mutation is expressed as a percentage of total chromosomes scored. Table II. Indications to CF testing.

Carrier testing of individuals with a family history of CF Prenatal diagnosis for carrier couples Prenatal diagnosis where ultrasound indicates fetal meconium ileus, echogenic bowel or obstructed bowel Diagnosis for individuals with clinical symptoms suggestive of CF when classical methods are inappropriate or inconclusive Diagnosis of individuals with chronic bronchitis or

bronchietcasis Diagnosis of men with congenital absence of the vas deferens and carrier testing for their spouses Carrier testing of human semen donors

phiclethnic origin of the examined population [29]. In families with known specific risks, different protocols have been proposed for prenatal monitoring of CF. They include direct CFTR mutations (if applicable) testing on trophoblast DNA or amniotic fluid cells, segregation analysis of at risk haplotypes (phase previously established in proband), microvillar enzymes (MVE) assay in

460 amniotic fluid in the second trimester of gestation. This latter method, individually or in conjunction with the DNA analysis, is at the present time a valid and rapid test for monitoring pregnancies in which fetal CF is suspected based on an abnormal intestinal echography [3, 461. Our experience with MVE in more than 250 antenatal diagnoses has indicated a cumulative 1.3% falsepositive and false-negative results (unpublished data). Single-cell assay has been successfully used to diagnose CF before embryo implantation, and to evaluate the frequency of the mutation within the germ cell population of patients [34, 811. The success rate of single cell amplification is around 9597% and the birth of normal children after preembryo biopsies and reimplantation has been reported [ 861. Mass screening of neonates is currently performed in many countries by monitoring the blood levels of the pancreatic enzyme trypsin, which tends to be higher in babies with CF. However, this test has a high rate of error producing false-positive and false-negative results [78]. Simple molecular tests for detecting heterozygotes in the general population based on a variety of non-invasive protocols (DNA preparation from buccal brushes/swabs, urinary cells, hair cells and Guthrie blood ,spots) are available and used in most pilot studtes [83]. However, the reliability of the mass molecular testing is directly related to the frequency of mutations identified in the ex-amined population. For example, in a screening program in which 75% of the CFTR mutations are detected, a Caucasian individual with a negative test result has his (her) risk decreased from 1 in 25 to 1 in 100. In most of the tested subjects, the risk of having a CF child will be reduced from l/2,500 to l/40,000. In a program in which multiple mutations are assayed and 85% of mutations are detected, the negative-by-negative couple’s risk is decreased to - l/100,000. Therefore, it is suggested that a population-based CF carrier screening is inappropriate until 90-95% of mutations are detected (American Society of Human Genetics, National Institutes of Health, USA). This result rate has been reached at present in individual populations, where it has provided the basis for implementing large-scale screening. However, further evaluations in terms of costs and benefits are required before extending these programs in the general population [9, 411. Garber and Fenerty [25] have compared antenatal screening in families affected by CF with

a screening programme for the general population. They concluded that testing of families with children affected by CF leads to substantial net savings in resources, while testing of the wider population was found to be less cost-effective. On the other hand, Mennie et cl1 1391, measured benefit in terms of averted costs and concluded that the benefits of screening exceeded the costs. Specific groups of people can be investigated for CF mutations. For example, CF mutation screening of human semen donors has been proposed to prevent known carriers from being used for artificial insemination by donor therapy. This reduces to 1 in 16,100 the risk of an offspring being born with CF. According to Cairns and Shackley [9], CF screening is at a developmental stage and further studies are required for a definitive evaluation of the costs and benefits.

Following cloning of the CF gene in 1989, and the demonstration that a functioning copy of the wild-type CFTR gene could correct the ion transport failure in CF, different strategies have been developed for inserting functional CFTR genes into airway cells to restore the cellular phenotype and improve the clinical status. Viral (adenovirus, adeno-associated virus, retrovirus) and non-viral (liposomes, immunoglobulins, naturally-occurring lung surfactant proteins) vectors are currently under study as potentially useful agents for this purpose [ 14, 511. Each vector type has its advantage and disadvantage. For example, adenovirus vectors show a specific tropism for the airway, because they are naturally occurring pathogens for the human airway and their molecular biology is well understood. However, the treatment efficacy is transitory and requires repeated dosing which stimulates specific immune response [ 18, 19, 381. Adeno-associated viruses are capable of a specific integration, but have a limited packaging capability [ 121. Adenoviruses are also found to be able to reconstitute CFTR expression in the biliary tract in rat cftr-/cftr-, after infusion through the common bile duct of recombinants carrying the human CFTR. These studies suggest the possibility of preventing CF liver disease by somatic gene transfer [87]. Recombinant retroviruses show an efficient host integration, but lack airway tropism and require cell division for integration. This restricts the treatment only to the undifferentiated

461 epithelia of the airway early in development [47]. Vector-free systems efficiently bind CFTR-DNA and can deliver this gene to the nuclei of airway cells. Preliminary studies in rats and non-human primates have demonstrated the efficiency and safety of CFTR gene-delivery in the airways, although a dose-dependent airway inflammation, as a result of exposure to the adenovirus/DNA complex, was observed [ 1, 8, 18, 52, 881. Clinical trials of gene-based therapy using adenovirus-mediated and liposome-mediated delivery of CFTR to human nasal and airway epithelia, are currently underway [ 11. These preliminary tests have substantially confirmed a vector-induced inflammation and a gradual loss of gene expression, for adenovirus system in the first treated patients [J Wilson, personal communication]. However, new genetically engineered adenoviruses with a reduced immunogenicity can be used in the near future. Liposome-mediated DNA delivery is at present underway (Brompton Hospital, London) but the effects observed in treated Edinburgh mice (cf/cf) suggest that this non-viral vector and the low immunogenic system, may constitute a therapeutic alternative to adenoviral therapies [ 11.

Conclusion Since identification of the CF gene, less than 5 years ago, the rate of CF research has been noteworthy. Many aspects of the disease at the molecular level have been addressed and, although the definitive pathogenesis is still not fully understood, the necessary tools to tackle the development of this disease are now available. CF genetic testing by carrier screening and prenatal diagnosis is expected to reduce in the near future the birth-rate of homozygous babies in many countries, where at least 90% of the CF molecular defects have been identified and preventive programs have been initiated. The gain of such a preventive program for the future will be the objective for those populations where a consistent number of CF alleles remain unidentified. The complete elucidation of the CFTR functions and the development of safe and efficient gene therapy strategies is the next goal of the CF research.

Acknowledgments We thank the patients and their families for their gracious co-operation. This work is supported by the CNR (PF FATMA) and ASM Milan.

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