Genotypes and Phenotypes in Cystic Fibrosis and Cystic Fibrosis Transmembrane Regulator Related Disorders

Transcription

1 180 Genotypes and Phenotypes in Cystic Fibrosis and Cystic Fibrosis Transmembrane Regulator Related Disorders Cristina Bombieri, PhD 1 Manuela Seia, BSc 2 Carlo Castellani, MD 3 1 Section of Biology and Genetics, Department of Life and Reproduction Sciences, University of Verona, Verona, Italy 2 Molecular Genetics Laboratory, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milan, Italy 3 Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata, Verona, Italy Address for correspondence Carlo Castellani, MD, Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata, Piazzale Stefani 1, Verona 37126, Italy ( carlo.castellani@ospedaleuniverona.it). Semin Respir Crit Care Med 2015;36: Abstract Keywords cystic fibrosis CFTR-related disorders CFTR mutations genotype phenotype correlations CBAVD pancreatitis bronchiectasis modifier genes Cystic fibrosis (CF) is characterized by remarkable variability in severity, rate of disease progression, and organ involvement. In spite of the considerable amount of data collected on the relationship between genotype and phenotype in CF, this is still a challenging matter of debate. Barriers to the interpretation of this connection are the large number of mutations in the CF transmembrane regulator (CFTR) gene, the difficulties in attributing several of them to a specific mode of dysfunction, and a limited number of the almost 2,000 mutations so far detected, which have been clinically annotated. In addition to that, the heterogeneity of clinical manifestations in individuals with the same CFTR genotypes indicates that disease severity is modulated by other genes and by environmental factors, of which the most relevant is possibly treatment in its aspects of appropriateness, early start in life, and adherence. The phenotype variability extends to conditions, named CFTR-related disorders, which are connected with CFTR dysfunction, but do not satisfy diagnostic criteria for CF. The current level of knowledge does not allow use of the CFTR genotype to predict individual outcome and cannot be used as an indicator of CF prognosis. This might change with the development of treatments targeting specific mutations and possibly capable of changing the natural history of the disease. Multifaceted Clinical Manifestations Associated with the CFTR Gene Cystic fibrosis (CF) is a chronic and life-threatening genetic disease, caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although its frequency may vary according to the country of origin, the average incidence of 1 in 2,500 births makes it the most frequent severe autosomal recessive disease among Caucasian populations. 1 CF is characterized by a wide heterogeneity of clinical expressions: patients are diagnosed following various modes of presentation and at different ages, from birth to adulthood, and there is considerable variability in the severity and rate of disease progression of the involved organs. 2 Typically, CF is distinguished by obstructive lung disease, chronic bacterial infections of lower airways and sinuses, bronchiectasis, fat malabsorption caused by pancreatic exocrine insufficiency, male infertility due to obstructive azoospermia, and elevated sweat chloride concentrations, 3 and is diagnosed at birth through neonatal screening programs or detected early in infancy. Issue Theme Cystic Fibrosis and Non- Cystic Fibrosis Bronchiectasis; Guest Editor:AndrewM.Jones,MD,FRCP Copyright 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) DOI /s ISSN

2 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. 181 Conversely, several patients come to clinical attention later in childhood or in adulthood with less severe presentation and lung disease. Clinical features may be even milder when single organ involvement is coupled with inconclusive diagnostic tests. Individuals with such characteristics are considered affected by a CFTR-related disorder (CFTR-RD), a condition defined as a clinical entity associated with CFTR dysfunction that does not fulfil the diagnostic criteria for CF. 4 The most representative phenotypes associated with this definition are congenital bilateral absence of the vas deferens (CBAVD), chronic pancreatitis, and disseminated bronchiectasis. Obviously, these three conditions can be labeled as CFTR-RD only if they are associated with evidence of CFTR dysfunction and if other etiologies have been excluded. A fourth phenotype, which at least at its onset concerns more biochemical than clinical manifestations, has been named CFTR-related metabolic syndrome 5 or as recently suggested by the Newborn Screening Working Group of the European CF Society, CF Screen Positive Inconclusive Diagnosis (CFSPID). It is a condition exclusively detected through newborn screening and characterized by neonatal hypertrypsinogenemia plus either borderline sweat chloride values and/or two CFTR mutations of which at least one is not considered CF causing. Long-term clinical evolution of CFSPID is still unclear and may probably range from no evidence of disease to CF, depending on the CFTR genotype, on individual non-cftr hereditary characteristics and on environmental factors. In clinical practice, the boundaries among all these phenotypes can be quite nuanced and change with aging, so that it may prove difficult to fit patients into distinct disease categories. CFTR Gene and Its Mutations The CFTR gene is located on chromosome 7q31.2, spans approximately 190 kb of genomic DNA, 6 and consists of 27 exons. It encodes a 1480 amino acid protein of around 168 kda, 7,8 which is expressed in the apical membrane of exocrine epithelial cells. CFTR functions principally as a cyclic adenosine monophosphate induced chloride channel and has been implicated in many processes such as modulation of ion channels, membrane trafficking, ph regulation, and apoptosis. 9 All types of mutations are represented in the gene: missense, frameshift, nonsense, splice, small and large rearrangements, in-frame deletions, and insertions. To date, almost 2,000 sequence alterations have been identified and their number is continuously updated within the Cystic Fibrosis Genetic Analysis Consortium (CFGAC) database. 10 In the CFGAC, missense mutations account for 40%, frameshift for 16%, splicing for 12%, nonsense for around 8%, in-frame insertions/deletions for 2%, large insertions/deletions for 2.6%, promoter mutations for 0.8%, and sequence variations, which are not predicted to be disease causing, for 14% of all alleles. De novo mutations and uniparental disomy of chromosome 7 bearing a mutated CFTR gene are exceptional events. 2 These mutations are distributed throughout the entire coding and promoter regions of the gene, and occasionally in deep intronic positions. The most frequent mutation, F508del, accounts for approximately 70% of the CF chromosomes worldwide. Only four mutations (G542X, N1303K, G551D, and W1282X) have an overall frequency above 1%, and fewer than 20 occur at a worldwide frequency of more than 0.1%. 10,11 The majority of the others are peculiar to specific ethnicities, often unique to a particular family or found in a handful of cases across the world. The vast majority of CFTR mutations are associated with Caucasian populations. 12 They have occasionally been detected in African and East Asian ethnic groups, where they may be more frequent than usually reported because of issues of underdiagnosis. 13 Mutations are distributed according to the geographical area. The common F508del mutation shows a northwest to southeast frequency gradient across Europe, with the highest frequency in Denmark, and the lowest in Turkey The remaining third of alleles show variable frequencies in different populations, with some mutations reaching a higher frequency in certain populations, likely due to a founder effect in religious, ethnic, or geographical isolates. 17,18 Overviews of the distribution of CFTR mutations causing CF have been produced by WHO 11 and Bobadilla et al. 16 However, these reports may over-represent mutations easier to screen for compared with those technically more difficult to detect, do not contain data on intra-cftr rearrangements, and have limited information on mutations occurring in non European-derived populations. Functional Effects of CFTR Mutations Understanding how a sequence variation affects the functioning of a gene is critical to understand pathogenesis and to design efficient therapeutic approaches. Mutations in the CFTR gene have initially been attributed by Tsui 19 to four groups according to their mechanisms of dysfunction and to their consequences at the protein level. The list has subsequently been expanded and refined to accommodate new data 20,21 and now includes six classes. Class I: Defective Synthesis Nonsense, frameshift, aberrant splicing mutations, and large deletions and insertions may create a premature termination codon in the mrna, resulting in the production of a truncated protein. This is usually unstable and rapidly degraded by nonsense-mediated RNA decay (NMRD), a mechanism by which the cell degrades transcripts harboring codons signaling the premature termination of translation. 22,23 The ultimate consequence of this process is the absence of functional protein in the apical cell membrane. The most common Class I mutations are G542X, R553X, and W1282X, all usually associated with complete and severe CF phenotypes. A notable exception occurs when the mutation is in the last exon, where NMRD does not recognize aberrant RNA transcript. In this case, the truncated protein is folded, reaches the cell membrane, and can be sufficiently functional to result in mild or non-cf phenotypes, sometimes with single-organ dysfunction. 24,25

3 182 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. Class II: Defective Protein Processing and Trafficking These mutations result in the synthesis of CFTR proteins that fail to be properly processed to a mature glycosylated form. Hence, only minimal quantities of a partially functioning protein are transported to the apical membrane, leading to a severe phenotype. The most common mutation, F508del, belongs to this group. Interestingly, F508del is a target for therapies aimed at correcting the protein misfolding and at increasing opening time and chloride conductance. 26 Class III: Defective Regulation or Gating Alleles carrying these mutations produce a normal amount of CFTR protein that is correctly folded and trafficked to the apical membrane, but the channel opening time is greatly reduced. These so-called gating mutations are usually located within the nucleotide binding domains and prevent ATP binding and hydrolysis, which is required for channel activation. The most frequent gating mutation is G551D. 27,28 These mutations are responsive to the new Ivacaftor drug, which has been proved to potentiate the channel activity, and significantly improve pulmonary function while decreasing sweat chloride values. 26,29,30 Class IV: Defective Chloride Conductance These mutations alter the conductivity of the CFTR channel. A normal amount of the protein is produced, but with only residual function. R117H, R334W, and R347P are examples of class IV mutations. In some cases, the mutations are located within the ion conduction pore of the CFTR channel, 31,32 whereas others appear to affect conductivity through allosteric mechanisms. 33 Mutations in this class are usually connected with milder phenotypes. Class V: Reduced Synthesis or Trafficking This class includes mutations that are associated with reduced biosynthesis of fully active CFTR, due to partially aberrant splicing ( kbC > T), 34 promoter mutations, or inefficient trafficking (A455E). 35,36 These mutations result in reduced expression of functional CFTR channels in the apical membrane. Class V mutations are also usually associated with milder manifestations of CF. Class VI: Decreased Stability This additional class includes mutations that result in instability of an otherwise fully processed and functional protein. 9 They are usually nonsense or frameshift mutations (Q1412X, 4326delTC, and 4279insA) causing a 70- to 100-bp truncation of the CFTR C-terminus and are mostly associated with a severe CF presentation. According to Haardt et al, 37 truncation of the C-terminus has no effect on CFTR channel biogenesis, function, or localization, but reduces from five to six times the half-life of the mature complex-glycosylated CFTR. Genotype/Phenotype Correlation Functional classes of CFTR mutations can only partially explain the relationship between genotype and phenotype. Patients carrying two class I III mutations tend to exhibit a phenotype associated with pancreatic insufficiency (PI), higher frequency of meconium ileus (MI), more severe deterioration of lung function, higher incidence of malnutrition and of severe liver disease, and earlier mortality. Class IV V mutations are usually associated with milder lung disease, longer survival, and pancreatic sufficiency (PS) and are phenotypically dominant when occurring in combination with class I III mutations. However, these general dispositions apply to patient populations more than to the single individual and should not be used for personal prognostic predictions. 2 As a matter of fact, the potential of a mutation to produce clinical disease and a more or less severe one depends on multiple factors: type of mutation, molecular mechanism at the cellular level, position in the gene, site of expression (organspecific pathophysiology), and other intragenic factors such as the presence of other sequence changes within the same gene (complex alleles) and the influence of the other allele mutation. 9 Besides, the wide range of clinical severity and the different rate of disease progression is connected with modifier genes and environmental factors, including the beneficial or harmful effects of treatment. 41 The impact of non-cftr factors on clinical phenotype is organ specific, with the vas deferens being the less affected and the lung widely influenced. 9 Mutations and Clinical Consequences In the 2008 study, Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice, CFTR mutations were clustered into four groups according to their predicted clinical consequences 2 : A. Mutations that cause CF disease B. Mutations that result in a CFTR-RD C. Mutations with no known clinical consequence D. Mutations of unproven or uncertain clinical relevance Groups A and B partially overlap, as some mutations may be detected either in association with pancreatic sufficient CF or with CFTR-RDs. The age-related progression of the disease plays an important role in the clinical heterogeneity of patients carrying these borderline mutations. Until recently, the great majority of mutations were categorized in group D, and only very few were universally acknowledged as part of group A. These were the 23 mutations included in the panel for CF carrier screening suggested by the American College of Medical Genetics (ACMG). 42,43 Although the 23 mutations represent altogether 85% of the mutated CF alleles worldwide, many other CF-causing mutations were left out, especially in areas where the panel did not represent appropriately the local mutation distribution. Hence, sequence variations not included in the ACMG panel but showing reasonable evidence of being CF causing such as insertions, deletions, and nonsense mutations introducing a premature termination signal were predicted to cause CF and often also tested. Further criteria, with a lower degree of certainty, were sometimes used to assess the pathogenic potential of missense mutations. The detection of the mutation in

4 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. 183 asymptomatic individuals already carrying a known CF-causing mutation suggested a minimal or nonexistent pathogenic potential, whereas its presence in several unrelated individuals with CF or the modification of a highly evolutionarily conserved amino acid residue was considered suggestive of a sequence variation potentially causing CF. Predicting the disease-causing potential of splicing mutations was also controversial. While splicing mutations that abolish exon recognition (such as 621 þ 1G > T, 711 þ 1 G > T, and G > A) lead to complete absence of correctly spliced transcripts, and thus belong to the CF-causing group, 44,45 those that retain a fraction of correctly spliced transcripts together with a majority of aberrantly spliced transcripts may be either CF causing or associated with CFTR- RDs (e.g., 3849 þ 10kbC > T, 2789 þ 5G > A, A > G, and IVS8-T5). 34,46,47 Patients carrying one of the latter mutations often show variable disease expression with a relatively mild phenotype, minimal lung disease, PS, and male fertility, but occasionally with wider and more severe disease expression. 48 The extent of disease expression is correlated with the quantity of correctly spliced transcripts, that is, lower levels of wild-type transcripts are associated with more severe disease, and higher levels with a milder phenotype. 34,49 53 Unfortunately, the difficulties in obtaining sufficient quantities of cells expressing CFTR and their laborious analysis make the individual determination of the extent of abnormal splicing impractical in routine diagnostics tests. Besides, the quantity of correctly spliced transcripts may differ among the various organs of the same patient, contributing to differential organ disease severity. 49,54 56 The IVS8- T5 allele is a typical example of this, 50,57 as it has been shown that in CBAVD males, the level of correctly spliced transcripts is lower in the epididymis than in the respiratory epithelium, whereas in an infertile male CF patient with severe lung disease the level of correctly spliced transcripts is low in both tissues. A similar correlation was also shown for the 3849 þ 10kbC > Tmutation. 58 Recently, a codification of CF disease clinical liability has been introduced by the Clinical and Functional Translation of CFTR (CFTR2) project. 59,60 The project collected clinical and genetic information on approximately 40,000 CF patients from registries and large clinics in Europe and North America. Patients mutations were available for approximately 70,000 alleles, while data on sweat chloride, lung function, and pancreatic status (pancreatic sufficiency or insufficiency) could be analyzed for one-third to one-fourth of the reported cases. The 159 mutations with an allele frequency of 0.01% went through a complex procedure which involved clinical, functional, and epidemiological steps. To be considered CF causing, a mutation had to meet the following three criteria: 1. At least three patients carrying that mutation in trans with a known CF-causing mutation had to have an average sweat chloride above 60 mmol/l. 2. The mutation introduced a premature termination codon or resulted after functional analysis in less than 10% of the level of wild-type CFTR mrna transcript, wild-type CFTR protein, or chloride current. 3. The mutation was not detected in trans with a CF-causing mutation in a sample of more than 2,000 fathers of children with CF. Mutations were considered non-cf causing when they complied with the following two criteria: 1. Not been labeled CF causing in the previously described assessment 2. Detected in trans with a CF-causing mutation in at least two fathers of children with CF. The results of this analysis can be consulted in a dedicated Web site, 61 where CFTR mutations are clustered into four groups according to their predicted clinical consequences: CF-causing mutation: the mutation, when in trans with another CF-causing mutation, would result in CF. Mutation of varying clinical consequence: the mutation has variable expression or penetrance and when in trans with another CF-causing mutation, can either result in CF or in a CFTR-RD, or in no disease. Non CF-causing mutation: themutationwhenin trans with another CF-causing mutation will not result in CF. This does not exclude the possibility that the mutation may contribute to CF-like clinical characteristics in certain individuals. At times, patients with the mutation (combined with a CF-causing mutation) may develop mild symptoms in some organ systems and/or be considered affected by a CFTR-RD. Mutation of unknown clinical significance: mutations not fully analyzed yet. CF-causing mutations, mutations of varying clinical consequence, and non CF-causing mutation as so far determined by CFTR2 are reported in Table 1. The clinical data included in the Web site are as follows: average age; average sweat chloride value at the time of diagnosis; a range of FEV 1 percent predicted values for three age groups (<10, 10 20, and >20 years); percentage of patients whose sputum culture was positive for Pseudomonas aeruginosa at the time the information was collected; and percentage of pancreatic-insufficient individuals. These data are relevant to genotype/phenotype correlation studies in large-scale assessments but, as repeatedly stated in the Web site, should not be used to predict the clinical course of individual patients. CFTR2 is an active project and a second round of data collection is presently ongoing, with an expected data output from more than 70,000 CF patients, also including extra European and extra North American countries. CFTR Genotype and Pulmonary Involvement is the major cause of morbidity and mortality in CF. Hence, a reliable prediction of the individual evolution of pulmonary involvement would be of the greatest importance and a useful support to modulate aggressiveness of treatment. Unfortunately, of all the organs involved in CF, the lung is the one showing the greatest variability of disease severity.

5 184 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. Table 1 CF-causing mutations, mutations of varying clinical consequences, and non CF-causing mutations so far determined by CFTR2 according to the July 22, 2013, version (legacy names in brackets) CF-causing mutations c.1521_1523delctt (F508del) c.2875delg (3007delG) c _273 þ 10250del21kb (CFTRdele2,3) c.3909c > G (N1303K) c.3752g > A(S1251N) c.1624g > T (G542X) c.2988 þ 1G > A (3120 þ 1G > A) c _4242 þ 577del (CFTRdele22,23) c.613c > T (P205S) c.1021t > C (S341P) c.948delt (1078delT) c.2988g > A (3120G > A) c.328g > C (D110H) c.200c > T (P67L) c.1397c > A (S466X(C > A)) c.1022_1023instc (1154insTC) c G > A (3121 1G > A) c.3310g > T (E1104X) c.3937c > T (Q1313X) c.1397c > G (S466X(C > G)) c.1081delt (1213delT) c A > G ( A > G) c.1753g > T (E585X) c.658c > T (Q220X) c.1466c > A (S489X) c.1116 þ 1G > A (1248 þ 1G > A) c.3528delc (3659delC) c.178g > T (E60X) c.115c > T (Q39X) c.1475c > T (S492F) c.1127_1128insa (1259insA) c.3659delc (3791delC) c.2464g > T (E822X) c.1477c > T (Q493X) c.1646g > A(S549N) c.1209 þ 1G > A (1341 þ 1G > A) c.3717 þ 12191C > T (3849 þ 10kbC > T) c.2491g > T (E831X) c.1573c > T (Q525X) c.1645a > C (S549R) c.1329_1330insagat (1461ins4) c.3744dela (3876delA) c.274g > A (E92K) c.1654c > T (Q552X) c.1647t > G (S549R) c G > A (1525 1G > A) c.3773_3774inst (3905insT) c.274g > T (E92X) c.2668c > T (Q890X) c.2834c > T (S945L) c.1418delg (1548delG) c.262_263deltt (394delTT) c.3731g > A (G1244E) c.292c > T (Q98X) c.1013c > T (T338I) c.1545_1546delta (1677delTA) c.3873 þ 1G > A (4005 þ 1G > A) c.532g > A (G178R) c.3196c > T (R1066C) c.1558g > T (V520F) c G > A (1717 1G > A) c.3884_3885inst (4016insT) c.988g > T (G330X) c.3197g > A (R1066H) c.3266g > A (W1089X) c G > A (1717 8G > A) c.273 þ 1G > A (405 þ 1G > A) c.1652g > A (G551D) c.3472c > T (R1158X) c.3611g > A (W1204X) c.1679 þ 1.6kbA > G (1811 þ 1.6kbA > G) c.274 1G > A (406 1G > A) c.254g > A (G85E) c.3484c > T (R1162X) c.3612g > A (W1204X) c G > A (1812 1G > A) c.4077_4080deltgttinsaa (4209TGTT > AA) c.2908g > C (G970R) c.349c > T (R117C) c.3846g > A (W1282X) c.1766 þ 1G > A (1898 þ 1G > A) c.4251dela (4382delA) c.595c > T (H199Y) c.1000c > T (R334W) c.1202g > A (W401X) c.1766 þ 3A > G (1898 þ 3A > G) c.325_327deltatinsg (457TAT > G) c.1007t > A (I336K) c.1040g > A (R347H) c.1203g > A (W401X) c.2012delt (2143delT) c.442dela (574delA) c.1519_1521delatc (I507del) c.1040g > C (R347P) c.2537g > A (W846X) c.2051_2052delaainsg (2183AA > G) c.489 þ 1G > T (621 þ 1G > T) c.2128a > T (K710X) c.1055g > A (R352Q) c.3276c > A (Y1092X (C > A)) c.2052dela (2184delA) c.531delt (663delT) c.3194t > C (L1065P) c.1657c > T (R553X) c.3276c > G (Y1092X (C > G)) c.2052_2053insa (2184insA) c.579 þ 1G > T (711 þ 1G > T) c.3230t > C (L1077P) c.1679g > A (R560K) c.366t > A (Y122X) c.2175_2176insa (2307insA) c.579 þ 3A > G (711 þ 3A > G) c.617t > G (L206W) c.1679g > C (R560T) c.2215delg (2347delG) c.579 þ 5G > A (711 þ 5G > A) c.1400t > C (L467P) c.2125c > T (R709X)

6 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. 185 Table 1 (Continued) c.2453delt (2585delT) c.580 1G > T (712 1G > T) c.2195t > G (L732X) c.223c > T (R75X) c.2780t > C (L927P) c.2290c > T (R764X) c.720_741delagggagaat GATGATGAAGTAC (852del22) c.2490 þ 1G > A (2622 þ 1G > A) c.2583delt (2711delT) c.1364c > A (A455E) c.3302t > A (M1101K) c.2551c > T (R851X) c.1675g > A (A559T) c.1a > G (M1V) c.3587c > G (S1196X) c.2657 þ 5G > A (2789 þ 5G > A) Mutations of varying clinical consequences c.3454g > C (D1152H) c.3154t > G (F1052V) c.3208c > T (R1070W) c.2930c > T (S977F) c.3808g > A (D1270N) c.3205g > A (G1069R) c.350g > A (R117H) PolyTG/PolyT c.1736a > G (D579G) c.3209g > A (R1070Q) c.220c > T (R74W) Non CF-causing mutations c.1727g > C (G576A) c.3485g > T (R1162L) c.224g > A (R75Q) c.3080t > C (I1027T) c.91c > T (R31C) c.3705t > G (S1235R) c.2991g > C (L997F) c.2002c > T (R668C) c.2260g > A (V754M) Non CFTR-related circumstances such as modifier genes, treatment, and environmental and social factors contribute to clinical heterogeneity Studies 66,67 of CF twin and siblings show that heritability (the proportion of CF variance that can be attributed to genetic factors) ranges from 0.6 to 0.8. Patients with Class I, II, and III mutations are inclined to have lower lung function with a faster decline in FEV 1 and higher risk of more severe lung disease than patients who carry one Class IV or V mutation However, there is considerable overlap within the two groups, 63 and homozygotes or compound heterozygotes for class I III CFTR mutations may present with relatively mild lung disease, while patients who carry at least one class IV V mutation may develop severe respiratory manifestations. Thus, for the individual, it is impossible to predict reliably the severity of pulmonary disease based only on CFTR genotype. 9,21,71,72 CFTR Genotype and Pancreatic Status Because of the large functional capacity of the exocrine pancreas, PI occurs when less than 5% of the exocrine pancreas is functioning. 73 When this happens, exogenous pancreatic enzyme replacement therapy is required to prevent severe fat malabsorption and subsequent malnutrition. The term pancreatic sufficiency (PS) is used instead when the residual exocrine pancreatic function is preserved enough to allow sufficient fat absorption. Class I III mutations on both alleles tend to be associated with PI, while at least one class IVor Vallele is usually enough to confer PS. PS mutations produce a PS phenotype with a dominant mechanism, that is, one class IV or V mutation in the genotype is enough to preserve sufficient nutrient assimilation. 74,75 However, these correlations are not absolute, and some mutations may be linked with either pancreatic sufficiency or insufficiency. A valid aid to assess the probability of a mutation to confer PS is the Pancreatic Insufficiency Prevalence (PIP) score. 76 The PIP score is the ratio between PI patients carrying a specific mutation and all PI and PS patients carrying the same mutation when in a homozygous state or in a heterozygous combination with F508del, G551D, or a class I mutations. A mutation with a high PIP score tends to be associated with a PI phenotype, whereas a mutation with a low PIP score is associated with a PS phenotype. PS is associated with better nutritional status, and also with a 15 to 20% risk for developing pancreatitis. 74,75 Ooi et al 76 suggested that the limited penetrance of pancreatitis in PS patients may be connected with the variable equilibrium between the degree of pancreatic acinar preservation and the extent of pancreatic acinar/ductal plugging due to inspissated secretions. The same authors 76 also showed that a genotype composed of two mutations with a PIP score 0.25 was associated with a significantly higher cumulative proportion and greater risk of pancreatitis than those with at least one allele carrying a mutation with a PIP score > CFTR Genotype and Male Infertility CFTR appears to be crucial in the development of the vas deferens. A relatively minor disruption of its function is

7 186 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. associated with CBAVD, 77,78 a condition characteristically found in the vast majority of male patients with CF. A small subset of patients retain some degree of vas deferens patency and thus fertility. This characteristic has been associated with specific CFTR mutations, and in particular with the mutation 3849 þ 10kbC > T. 2 CFTR Genotype and Other Disease Manifestations Liver disease, MI, and distal intestinal obstruction syndrome occur almost exclusively in patients carrying severe class I III mutations on both alleles. 79,80 Endocrine pancreas involvement expressing as CF-related diabetes is typical of patients with PI, which in turn is also strongly associated with Class I, II, and III mutations. 79 The high familiar recurrence risk of MI (29 39%) is suggestive of genetic predisposition patient or family cohorts to increase statistical power of association analyses for various CF clinical traits and specific covariates. Various degrees of evidence have been collected on the role of CF modifiers of a few genes. 72,84 86 More research is needed on these and other putative CF modifiers, and caution has to be exercised in interpreting the results so far available. Some of the genes found in association with disease variability in CF are listed in Table 2 and more exhaustive presentation of these modifier genes can be found in specific reviews. 72,87 90 Candidate genes are expected to modulate the severity of lung disease through several mechanisms, including the following: Modulation of Susceptibility to Infections Modifier Genes The wide range of clinical expression among patients carrying the same CFTR genotype suggests that genetic determinants different from the CFTR gene can modulate disease severity. These are named modifier genes. The earlier studies on CF modifier genes, mostly examined on small populations, have not been replicated and reached contradictory conclusions. This led to a different approach in study design, and nowadays the most relevant research on modifiers consider large CF Table 2 Candidategenemostlystudiedasmodifier of CF phenotype CF patients heterozygous for one of three variants in the mannose-binding lectin gene (MBL2) have been shown to have reduced levels of MBL, a protein playing a significant role in innate immune defense. The same patients had low lung function, earlier infection with P. aeruginosa, a higher prevalence of infection with Burkholderia cepacia, and poor survival. 91 Polymorphisms affecting the production and composition of surfactant by the surfactant proteins A and D (SP-A and Function/pathway Gene CF phenotype Reference Inflammatory response TGFB1 84, IL8 IL10 TNFA Innate immunity/infectious susceptibility MBL2 Lung and liver disease DEFB1 AGER Neutrophil effector function IFRD1 Protease/antiprotease imbalance SERPINA1 Liver disease Ion transport SLC4A4 MI SLC6A14 SLC26A9 SLC9A3 SLC8A3 SCNN1B MI and lung disease MI and diabetes MI and lung disease Disease severity Metabolism, electrolyte balance ACE Liver disease Tissue damage and repair GSTP1 Lung and liver disease GSTM1 NOS1/NOS2 trna processing CDKAL1 Diabetes Signaling by TGF-beta receptor complex CDKN2A/B Diabetes Insulin growth factor regulator IGF2BP2 Diabetes 99,102 84,100,103 84,97,103 84,91, ,96, ,112, ,116 84,115, ,100,119 84,97, ,120, Abbreviations: CF, cystic fibrosis; MI, meconium ileus.

8 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. 187 SP-D genes) may contribute to the heterogeneity of lung disease. Decreased levels of these proteins, which are also active in host defense mechanisms, have been shown in bronchoalveolar lavage fluid from CF patients. 92,93 The antimicrobial activity of β-defensin 1 (DEFbeta1) and 2 (DEFbeta2) is reduced when airway surface fluid is hypertonic, like in CF. 94 Polymorphisms in DEFbeta1 and DEFbeta2 genes might reduce their expression and further compromise the lung s antimicrobial defenses. 95,96 Modulation of Inflammatory Response Heterozygosity for a tumor necrosis factor-α (TNF-α) polymorphism (G-308A) that may influence serum levels of TNF-α has been associated with significantly worse pulmonary function and nutritional status in CF. 97 Tissue Damage and Repair Glutathione S-transferase (GSTM1) conjugates several organic compounds with glutathione and thus contributes to prevention of lung damage. There are two functionally active alleles and a common null allele, which produces no protein. CF patients homozygous for the null allele have been shown to be affected by more severe disease. 97 CFTR Mutations and CFTR-Related Disorders Soon after the CFTR gene was discovered, several individuals with CBAVD and idiopathic chronic and acute recurrent pancreatitis were found to carry one or two CFTR mutations with a frequency considerably higher than expected in the general population If two mutations were detected in the same individual, at least one was a non CF-causing mutation. These and other conditions were later called CFTR-RD. 4 Exploring the association between CFTR mutations and these disorders is often difficult. The prevalence of CFTR mutations depends on the number of mutations tested (e.g., a limited panel or full gene sequencing) and patients selection criteria often are not homogeneous. Besides, these clinical entities may have other, non CFTR-related etiologies. 4 Like in CF, CFTR-RDs do not show a clear genotype phenotype correlation and no specific mutation has been associated with any of these disorders. The best studied CFTR-related disorders are CBAVD, idiopathic recurrent acute or chronic pancreatitis, and diffuse bronchiectasis A prevalence of CFTR mutations higher than expected in the general population has been reported also in rhinosinusitis, allergic bronchopulmonary aspergillosis, and sclerosing cholangitis Congenital Bilateral Absence of the vas Deferens CBAVD accounts for 1 to 5% of cases of male infertility. 138 Approximately 80 to 90% of men with CBAVD carry at least 1 CFTR sequence variant and 50 to 60% 2 CFTR sequence variants. 139,140 In the latter case, only one mutation is CF causing, while the other (or the two if none of them is CF causing) belongs to group B or D in the ECFS categorization 4 or is labeled either mutation of varying clinical consequence or mutation of unknown clinical significance in the CFTR2 classification. 60, The most frequent mutation in CBAVD is F508del, which is found in approximately 40% of patients, followed by the IVS8- T5 variant in approximately 35%. 140,142 The IVS8-T5 allele is found in most CBAVD patients in cis with the IVS8-TG12 or TG13, a polymorphic dinucleotide repeat lying immediately upstream of the IVS8-T repeat. 144 R117H has also frequently been reported (30%), 132 usually in association with the IVS8-T7 variant. 145 CFTR gene defects in CBAVD are essentially point mutations. Large rearrangements, which are associated with severe disease, have been identified in less than 1% of CBAVD alleles, a proportion lower than in CF patients. 146,147 This possibly reflects the minor contribution of severe alleles to the pathogenesis of CFTR-related disorders. 4 Some complex alleles are also found in CBAVD patients, the most common being p.[g576a;r668c], p.[d443y;g576a;r668c], p.[r74w; V201M;D1270N], and [S1235R;IVS8 5T]. The most frequent compound heterozygous genotypes are F508del/IVS8-T5 and F508del/p.R117H (6%). 25,146,148,149 Several studies provide evidence for genetic heterogeneity in CBAVD. A quota of extensively studied men with CBAVD 146,147 does not display any abnormalities in the CFTR gene. Although it cannot be excluded that they carry unknown or undetected CFTR defects, it may well be that their infertility is not related to CFTR mutations. This speculation is supported by reports of discordant familial segregation analysis, 150,151 and in fact an altogether different pathogenesis has been proposed in a minor proportion of CBAVD males suffering from concomitant urogenital abnormalities, such as unilateral renal aplasia. CFTR mutations are observed in CBAVD males with normal renal systems and it has therefore been suggested that CFTR dysfunction alters the vas deferens after its embryological separation from the renal system and that most cases of CBAVD with associated renal abnormalities represent a distinct clinical entity, presumably due to organogenesis defects independent of CFTR. 4,152,153 Isolated CBAVD is diagnosed primarily in asymptomatic young adult males consulting for infertility. Some of them do not show any pulmonary or gastrointestinal manifestations consistent with CF; others have elevated sweat chloride concentrations, nasal polyps, and respiratory or pancreatic symptoms 154 compatible with a mild CF phenotype. 143,155 The clinical overlap between CBAVD and CF may become more evident later in life and occasionally the progressive worsening of symptoms has justified moving from an initial diagnosis of CFTR-RD to one of CF. A long-term follow-up targeting respiratory and gastrointestinal involvement is advisable for these individuals. 4 Idiopathic Chronic Pancreatitis Acute and chronic pancreatitis are complex inflammatory disorders with unpredictable severity, clinical courses, and complications. 156 In approximately 40% of cases, no clear etiology is detected, 157 and probably a combination of genetic, environmental, and metabolic factors contribute to the development of the disorder. 158 Three genes have been associated

9 188 Genotypes and Phenotypes in CF and CFTR-Related Disorders Bombieri et al. with susceptibility to acute and/or chronic pancreatitis: cationic trypsinogen (PRSS1), serine protease inhibitor Kazal 1 (SPINK1), and CFTR. Moreover, anionic trypsinogen (PRSS2), chymotrypsinogen C (CTRC), and calcium-sensing receptor (CASR) genes were also associated to pancreatitis, with smaller increases in risk Approximately 30% of individuals with idiopathic chronic pancreatitis (ICP) have at least one CFTR mutation, whereas approximately 10% are compound heterozygotes who carry a CF-causing mutation plus a non CF-causing sequence variation on the other allele. 159 The most common CFTR mutations found in ICP are F508del, the IVS8-T5 variant, R117H-T7, L206W, D1152H, R1070Q, R347H, R334W, and 2789 þ 5G > A The risk of developing chronic pancreatitis is increased by, respectively, 6.3-, 2.4-, and 37-fold in the presence of a single CF-causing mutation, the IVS8 5T allele, and a CFcausing mutation plus a milder allele in trans, respectively. 164,165 PRSS1 gene encodes cationic trypsinogen, the most abundant isoform of trypsinogen in human pancreatic juice. Biochemical studies demonstrated that most of the missense mutations considered disease causing in the PRSS1 gene (D19A, D22G, K23R, N29I, N29T, R122H, and R122C) led to enhanced trypsinogen autoactivation and/or increased trypsin stability. R122H and N29I are the most common mutations causing hereditary pancreatitis, with a penetrance of approximately 80%. 157 The SPINK1 gene encodes a serine peptidase, Kazal type 1, which is a trypsin inhibitor secreted by the pancreas. 157 Gene gene interactions have been documented in ICP individuals who inherit both low-penetrance SPINK1 variations and CFTR mutations. Coinheritance of the common SPINK1 N34S allele and at least one abnormal CFTR allele accounts for 1.5, 4, 5.1, and 7.7% of the total patients analyzed, respectively, in four studies 159,160,162,164 in which all the CFTR and SPINK1 exons were analyzed and the diagnosis of ICP was unambiguous; a total of approximately 4.1% ICP patients were transheterozygotes of SPINK1/CFTR variations. 164 Pancreatitis risk is increased approximately 40-fold by the association with two CFTR mutations, 20-fold with N34S, and 900-fold with both CFTR and SPINK1 mutations. 159 Disseminated Bronchiectasis Bronchiectasis are heterogeneous conditions sharing a large number of potential contributory factors and a poorly understood pathogenesis. 166,167 The genesis of approximately 50% of individual cases of bronchiectasis cannot be identified (idiopathic bronchiectasis). 168 Among them, an increased incidence of CFTR gene mutations has been found. At least one CFTR mutation has been reported in 10 to 50% of series of patients, two mutations in 5 to 20%. 83, A wide spectrum of CFTR mutations are involved: most are uncommon and likely to result in residual CFTR function, but no particular mutation can be directly associated with idiopathic bronchiectasis. A high incidence of the IVS8 5T allele has been reported in bronchiectasis, but not as high as in CBAVD , Nasal potential difference measurements in patients with diffuse bronchiectasis and a normal sweat test found intermediate values, neither typical of healthy subjects nor of CF patients. These values were distributed in a continuum strongly associated with the CFTR gene (i.e., bearing zero, one or two mutations) and with the clinical phenotype as expressed by bacterial colonization. 177 Weaknesses in the literature on CFTR analysis in bronchiectasis patients include the low numbers of patients enrolled in most studies and the different mutation scanning approaches used, not always considering the whole CFTR gene. Besides, when two mutations were detected, no information were provided explaining whether a segregation analysis had been performed to establish if the mutations were carried in cis or in trans. Finally, criteria for clinical selection of patients were not adequately explained and appropriate investigations not performed, so that known etiologic factors were not completely ruled out and in some cases the selected patients may actually have been affected by undiagnosed CF A thorough clinical examination at a specialized CF center and follow-up of the most suspicious cases should be recommended, as the symptoms often suggest undiagnosed CF. 166 Future Developments Several ongoing projects and initiatives have the potential to improve our understanding of CFTR genotype phenotype correlations and to provide insights into the molecular mechanisms of CFTR dysfunction in CF and CFTR-RDs. Next generation sequencing techniques will help identify still unknown CFTR mutations. Epidemiological and clinical data collection in worldwide public databases coupled with functional analyses, such as the above-mentioned CFTR2 project, is providing new information on the disease liability of CFTR mutations both in CF and CFTR-RDs. Heterologous cells expressing identical amounts of CFTR mutants, together with primary cultures of epithelial cells isolated from patients, will be helpful to better understand the molecular mechanism of CFTR dysfunction. Finally, high throughput genotyping analyses conducted by international consortia on vast numbers of patients will contribute to understand the role played by modifier genes in the disease process. Identification of genetic modifiers and the determination of their clinical effect could become an integral part of assessing disease prognosis. Conclusion Almost 2,000 mutations have been described in the CFTR gene. The vast majority of them are very rare. Mutations are located throughout the entire gene region, with all kinds of molecular lesion represented. A wide spectrum of clinical manifestations is associated with CFTR dysfunction, ranging from the classic multiorgan form of CF, to milder CF phenotypes with limited clinical features, to CFTR-RDs with single-organ involvement. No robust relationship of CFTR mutations with individual clinical phenotype has been found, except for the conditions of PS or insufficiency.

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