ABCMdb

PMID: 20706124

Lucarelli M, Narzi L, Pierandrei S, Bruno SM, Stamato A, d'Avanzo M, Strom R, Quattrucci S
A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation.
Genet Med. 2010 Sep;12(9):548-55., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation Marco Lucarelli, BS, PhD1 , Lorena Narzi, BS, PhD2 , Silvia Pierandrei, BS, PhD1 , Sabina Maria Bruno, BS, PhD1 , Antonella Stamato, BS2 , Miriam d`Avanzo, MD, PhD3 , Roberto Strom, MD, PhD1 , and Serena Quattrucci, MD, PhD2 Purpose: To evaluate the role of complex alleles, with two or more mutations in cis position, of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the definition of the genotype-phenotype relationship in cystic fibrosis (CF), and to evaluate the functional significance of the highly controversial L997F CFTR mutation. Login to comment 2 ABCC7 p.Leu997Phe According to a first CFTR mutational analysis, subjects appeared to be compound heterozygotes for a classic mutation and the L997F mutation. Login to comment 4 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe Results: We detected a new [R117L; L997F] CFTR complex allele in the four subjects with the highest sweat test values and CF. Login to comment 6 ABCC7 p.Leu997Phe Conclusions: The new complex allele partially explains the variable phenotype in CF subjects with the L997F mutation. Login to comment 18 ABCC7 p.Leu997Phe Although the CFTR mutation L997F has been known since 1992, it is still highly controversial from the functional point of view, and its pathogenetic role has not yet been fully elucidated. Login to comment 19 ABCC7 p.Leu997Phe L997F was initially believed to be a polymorphism6 then it was been reported to cause both CFTR-RD and no disease. Login to comment 20 ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe The presence of L997F has been linked to pulmonary diseases,7,8 disseminated bronchiectasis,9,10 neonatal hypertrypsinaemia with normal sweat test,11-14 idiopathic pancreatitis,12,15-17 and congenital absence of vas deferens.18 It has also been suggested that L997F negatively influences the clinical course of primary sclerosis cholangitis.19 In subjects with idiopathic pancreatitis, the L997F CFTR mutation has also been found to be associated with mutations of the serine protease inhibitor Kazal type 1/pancreatic secretory trypsin inhibitor (SPINK1) gene, namely the N34S mutation20 and a deletion encompassing the entire gene.21 However, there is also evidence suggesting that homozygosity for L997F does not cause disease.22,23 The frequency of L997F in the general population has been reported to be ϳ0.5%,14,24 which is neither high enough to clearly exclude its pathogenetic role nor so low as to strongly support the pathogenetic hypothesis. Login to comment 21 ABCC7 p.Leu997Phe To shed light on the general issue of the role of complex alleles in the genotype-phenotype relationship in CF, we studied the functional and clinical significance of the L997F CFTR mutation. Login to comment 22 ABCC7 p.Leu997Phe For this purpose, we studied 12 unrelated compound heterozygotes for a classic CFTR mutation on one allele and, according to a first CFTR mutational analysis, apparently only L997F on the other allele. Login to comment 33 ABCC7 p.Leu997Phe DOI: 10.1097/GIM.0b013e3181ead634 ARTICLE 548 Genetics IN Medicine • Volume 12, Number 9, September 2010 exons, adjacent intronic zones, and 5Ј-flanking regions to verify whether their phenotypic variability was linked to any additional CFTR sequence variations in cis with L997F. Login to comment 36 ABCC7 p.Leu997Phe We selected, from a larger group of subjects with a diagnosis or presumptive diagnosis of CF, 12 unrelated subjects who had a classic mutation and the L997F mutation in different alleles of the CFTR gene. Login to comment 55 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe RESULTS Genetic analysis, biochemical features of the mutations found, and state of conservation of L997F and R117L residues Five different CFTR mutations were found (those included in the CF-OLA panel were confirmed by sequencing) on one allele of the subjects analyzed (Table 1). Login to comment 57 ABCC7 p.Trp1282* W1282X (c.3846GϾA) is a nonsense mutation that produces a truncated CFTR form. Login to comment 58 ABCC7 p.Gly85Glu ABCC7 p.Leu320Val G85E (c.254GϾA), S549R(AϾC) (c.1645AϾC), and L320V (c.958TϾ G) are missense mutations located in the first membrane spanning domain-first transmembrane segment, in the first nucleotide-binding domain, and in the first membrane spanning domain- fifth transmembrane segment, respectively. Login to comment 59 ABCC7 p.Trp1282* ABCC7 p.Gly85Glu F508del, W1282X, G85E,35 and S549R(AϾC)36 are considered classic CFTR mutations that cause a severe CF form when in homozygosity or compound heterozygosity with another classic mutation. Login to comment 60 ABCC7 p.Leu320Val The L320V mutation has previously been found in two subjects with congenital bilateral absence of the vas deferens, with no other symptoms37; to our knowledge, no other phenotypical description of this mutation has been published. Login to comment 61 ABCC7 p.Leu997Phe On the other allele (allelic segregation was confirmed by analysis of the parents), L997F (c.2991GϾC) is a conservative aminoacidic substitution: both amino acids are nonpolar. Login to comment 62 ABCC7 p.Leu997Phe Because the position of L997F is in the second membrane spanning domain-ninth transmembrane segment, both amino acids fit the hydrophobic transmembrane environment. Login to comment 63 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe The R117L (c.350GϾT) was found in four subjects on the same allele as L997F (as assessed by parents` analysis); it is a nonconservative Table1CFTRgenotypes,sweattestvalues,andclinicalassessmentofthesubjectsstudied SubjectGenotypeSex Averagesweat testvaluea (mEq/L) Semen analysis Uponenrollment AgeCause Clinicalsymptoms (withouttherapy) Respiratory manifestations Pulmonarybacterial isolatesByFEV1Byrx 1F508del/͓R117L;L997F͔M90Ϯ9OA5yrSymptomsDehydrationevents, bronchopneumonia, rhinosinusitis ModerateModerateAbsent 2c F508del/L997FF56Ϯ8ND2moNeonatalscreeningAbsentTooyoungMildAbsent 3F508del/L997FM42Ϯ5Tooyoung5moNeonatalscreeningAbsentTooyoungAbsentAbsent 4c F508del/L997FF35Ϯ4ND1moNeonatalscreeningAbsentTooyoungMildAbsent 5c F508del/L997FM32Ϯ1Tooyoung2moNeonatalscreeningAbsentTooyoungAbsentAbsent 6F508del/L997FM22Ϯ3Tooyoung8yrSymptomsBronchopneumonia,bronchitis, rhinosinusitis AbsentMildAbsent 7G85E/͓R117L;L997F͔M102Ϯ10OA7yrSymptomsProductivecoughTooyoungMildS.aureus 8G85E/L997FM21Ϯ4Tooyoung11moNeonatalscreeningProductivecoughTooyoungMildS.aureus(sporadic) 9W1282X/͓R117L;L997F͔M96Ϯ4OA33yrSymptomsCholelithiasis,productive cough,bronchopneumonia MildModerateS.aureus,P.aeruginosa 10W1282X/͓R117L;L997F͔F80Ϯ5ND36yrSymptomsBronchopneumoniaModerateModerateP.aeruginosa 11L320V/L997FM77Ϯ5Tooyoung3yrSymptomsRhinosinusitisTooyoungAbsentAbsent 12c S549R(AϾC)/L997FM39Ϯ6Tooyoung2moNeonatalscreeningAbsentTooyoungAbsentAbsent Nosubjecthadeitherpancreatitisorliverdisease.ClassificationofpulmonarysymptomsbyFEV1isasfollows:absent,Ͼ90%;mild,from70%to90%;moderate,from40%to70%;severe,Ͻ40%.Classificationofpulmonary symptomsbychestx-rayisasfollows:absent,noradiologicalsigns;mild,limitedairtrappingorperibronchialinfiltration;moderate,denseareasorbronchiectasisrestrictedtoonelobe;severe,denseareasorbronchiectasisin bothhemithoraxes.Theseverityofcysticfibrosiswasclassifiedasreportedin"MaterialsandMethods-Biochemical,microbiologic,andclinicalcharacterization"section. a Eachsweattestvalueisthemeanofrepeatedsweattestmeasurements(from2to4)onenrollmentandduringfollow-up. b TheBransfieldscorerangesfrom25,nodisease,to0,highlyseveredisease(forreferencesee"MaterialsandMethods"section).27 c Thesefoursubjectshavealreadybeenpartiallydescribed14andareincludedheremerelyforcomparisonpurposes. d AllthesubjectshadpancreaticsufficiencywiththeexceptionofSubject1whohadinitialPS,whichgraduallyevolvedintopancreaticinsufficiencyfrom12yearsofage. OA,obstructiveazoospermia;ND,notdetermined;PI,pancreaticinsufficiency;PS,pancreaticsufficiency. Login to comment 66 ABCC7 p.Arg117Leu Because the position of R117L is in the first extracellular loop exposed to the aqueous extracellular environment, the substituting amino acid will have a lower fit than the original one. Login to comment 70 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe Both the isolated L997F and the [R117L; L997F] complex allele were associated with the 470V allele in all subjects. Login to comment 77 ABCC7 p.Gly85Glu ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe If one considers only the first two mutations found, the six subjects with apparently the same F508del/L997F genotype (Table 1, Subjects 1-6) showed highly varying sweat test values, ranging from 22 to 90 mEq/L, as did the two subjects (Subjects 7 and 8) with apparently the same G85E/L997F genotype, who had sweat test values of 102 mEq/L and 21 mEq/L, respectively. Login to comment 78 ABCC7 p.Trp1282* ABCC7 p.Leu997Phe By contrast, the sweat test values in the two subjects with apparently the same W1282X/L997F genotype (Subjects 9 and 10) were more similar (96 mEq/L and 80 mEq/L, respectively). Login to comment 79 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe However, the extended genetic analysis detected the R117L mutation on the same allele as the L997F mutation in Subjects 1, 7, 9, and 10, thereby revealing a new complex allele of the CFTR gene (an example of the sequencing analysis is shown in Fig. 1). Login to comment 81 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe The four subjects with the [R117L; L997F] complex Fig. 1. Login to comment 89 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe Subject 1, with a F508del/[R117L; L997F] genotype and the highest sweat test value, displayed clinically severe CF with late pancreatic insufficiency, whereas the five subjects with the F508del/L997F genotype and lower sweat test values displayed either a milder form of CF (Subject 6) or CFTR-RD (Subjects 2, 3, and 4) or no disease at all (Subject 5). Login to comment 90 ABCC7 p.Gly85Glu By contrast, the agreement between the presence of the complex allele and clinical outcome was not as good when we compared the two subjects with the same G85E mutation on one allele. Login to comment 92 ABCC7 p.Trp1282* Because the other allele in the two subjects with the W1282X mutation (Subjects 9 and 10) was characterized by the presence of the complex allele, no evaluation of the effect of simple compound heterozygosity was possible. Login to comment 97 ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe ABCC7 p.Leu320Val The five subjects with the F508del/L997F genotype, which were expected to be more severe than the L320V/L997F genotype, represent a paradigm for variability. Login to comment 103 ABCC7 p.Arg117His ABCC7 p.Arg347His ABCC7 p.Val754Met ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Ser1251Asn ABCC7 p.Asp979Ala ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg ABCC7 p.Ser1235Arg ABCC7 p.Phe508Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Arg117Cys ABCC7 p.Ile1027Thr ABCC7 p.Pro750Leu ABCC7 p.Ala1006Glu ABCC7 p.Gly628Arg ABCC7 p.Val562Ile ABCC7 p.Ser912Leu ABCC7 p.Arg352Trp ABCC7 p.Gly1244Val In vivo findings and, in some cases, in vitro functional characterizations have been reported for [F508C; S1251N],38 [R347H; D979A],39,40 [R74W; D1270N],41 [G628R; S1235R],42,43 [M470V; S1235R],42 [S912L; G1244V],44 [R117H; (TG)mTn],45-47 [R117C; (TG)mTn],46 [S1235R; (TG)mT5],48 [G576A; R668C],10,49 [V562I; A1006E],49 [R352W; P750L],49 [1198_1203del TGGGCT; 1204GϾA],49 [V754M; CFTRdele3_10,14b_16],50 and [F508del; I1027T].51 These complex alleles have been found in patients with either CF or CFTR-RD, although more often in the former. Login to comment 105 ABCC7 p.Arg334Trp ABCC7 p.Arg74Trp ABCC7 p.Arg1158* ABCC7 p.Arg1158* ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Phe508Cys ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Ser912Leu Both in vivo and in vitro studies have also highlighted cases in which there is one main mutation with the phenotypical effect that is worsened by a second mutation, which may even be a neutral variant when isolated, as occurs for F508C,38 R74W,41 S912L,44 and M470V.42 However, different effects have also been described, as in the case of the two M470 and R1235 variants, which give rise to a hyperactive CFTR when present on different alleles but have a suppressive effect when combined on the same allele.42 In addition, the finding of complex alleles in CFTR-RD seems to suggest that a second CFTR mutation may even lead to a partial reversion of the phenotype.43 Indeed, in a reduced number of complex alleles, the effect of the second mutation may partially correct the functional defect, thereby lessening the phenotypical effect, as has been demonstrated for the R553Q mutation in the [F508del; R553Q] complex allele by in vivo52 and in vitro53 studies and for the R553M mutation in the [F508del; R553M] complex allele by an in vitro study.53 A milder phenotypical effect has also been demonstrated for the [R334W; R1158X]54 and [-102T; S549R(TϾG)]55 complex alleles if compared with alleles carrying, respectively, isolated R1158X or S549R(TϾG). Login to comment 107 ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe L997F is the only known mutation of the 997 CFTR amino acid.37 As reported earlier, when isolated, L997F can cause either CFTR-RD (although not CF) or no disease at all.6-19,22,23 The conservative nature of the L997F substitution (both residues are hydrophobic) in the CFTR second membrane spanning domain-ninth transmembrane segment may constitute the molecular basis for the limited effect of such an isolated L997F. Login to comment 108 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg117Leu ABCC7 p.Arg117Cys ABCC7 p.Arg117Cys ABCC7 p.Arg117Pro ABCC7 p.Arg117Gly Five different CFTR mutations of the 117 CFTR amino acid are known: R117C, R117G, R117H, R117L, and R117P.37 All these mutations have previously been reported to be more likely to cause CFTR-RD than CF.13,37,46,56 However, R117H and R117C have been shown to yield high sweat test values and CF, even severe, if cis-acting with the T5 variant tract in CFTR intron 8.45,46 If we bear in mind that the pH range of airway surface fluid is pH 6.7-7.0,57,58 these mutations of the R117 CFTR residue represent both conservative and nonconservative substitutions. Login to comment 109 ABCC7 p.Arg117Leu In particular, the R117L is a nonconservative substitution that changes the basic residue to a hydrophobic residue. Login to comment 111 ABCC7 p.Arg117Leu A possible molecular mechanism for the reduced effect of the isolated R117L mutation may be that only 11 of the 15 amino acids that constitute the first extracellular loop domain in which the R117 residue is located have a charged or a polar side chain, whereas the other four are hydrophobic. Login to comment 112 ABCC7 p.Leu997Phe L997F was found in compound heterozygosity with another CFTR mutation in six subjects (four of whom had the complex allele) with CF (mild or severe), in five subjects with CFTR-RD, and in one asymptomatic subject. Login to comment 114 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe In these four cases, the mild effects of the isolated L997F and R117L mutations cumulate in the complex allele with a cis-acting effect, thereby inducing a well-defined, strong effect on both the Cl-transport (producing the highest sweat test values in the entire case series) and clinical outcome, resulting in CF (from mild to severe). Login to comment 123 ABCC7 p.Leu997Phe In the absence of complex alleles, both levels of variability may in general depend on the limited adverse effect of the L997F mutation, which can easily be modified (worsened or improved) by environmental factors and/or modifier genes.5 A particular class of modifier genes that specifically may influence Cl- levels are the alternative Cl-channels. Login to comment 127 ABCC7 p.Leu997Phe Although the L997F mutation seems to exert a limited influence on Cl-transport (possibly owing to the limited impact of this substitution on the pore structure), it may exert greater influence on other CFTR functions, giving rise to the disease despite having a reduced or even no effect on Cl-transport. Login to comment 139 ABCC7 p.Leu997Phe The late presentation of severe clinical symptoms in subjects with genotypes involving L997F mutation thus seems to be possible. Login to comment 141 ABCC7 p.Leu997Phe These findings shed light not only on the phenotypical variability in CF subjects with the L997F mutation but also, more generally, on the issue of the genotype-phenotype relationship in this disease. Login to comment 145 ABCC7 p.Arg117Leu ABCC7 p.Leu997Phe Whenever a L997F mutation is found, the search for the R117L mutation must be undertaken (and vice versa); if the complex allele is found, the onset of CF (in a mild or severe form) with high sweat test value is likely. Login to comment 146 ABCC7 p.Leu997Phe If the L997F mutation is the only mutation of the allele, the phenotypical results vary to a greater extent: neither mild CF nor CFTR-RD can be ruled out, although the disease may even be absent, irrespective of the sweat test value. Login to comment 147 ABCC7 p.Leu997Phe Because the late-onset of CF is possible even in the presence of the complex allele, particularly when L997F is found alone, a prolonged follow-up is recommended. Login to comment 151 ABCC7 p.Leu997Phe ABCC7 p.Leu320Val The initial genetic characterization of the subject with the L320V/L997F genotype was performed by the Regional Reference Center for Rare Diseases, Department of Pediatrics, University Hospital of Padova (Italy). Login to comment