Spectrum of CFTR mutations in cystic fibrosis ... - Wiley Online Library

HUMAN MUTATION 16:143156 (2000)

RESEARCH ARTICLE

Spectrum of CFTR Mutations in Cystic Fibrosis and in Congenital Absence of the Vas Deferens in France Mireille Claustres,1* Caroline Guittard,1 Dominique Bozon,2 Françoise Chevalier,2 Claudine Verlingue,3 Claude Ferec,3 Emanuelle Girodon,4 Cécile Cazeneuve,4 Thierry Bienvenu,5 Guy Lalau,6 Viviane Dumur,6 Delphine Feldmann,7 Eric Bieth,8 Martine Blayau,9 Christine Clavel,10 Isabelle Creveaux,11 Marie-Claire Malinge,12 Nicole Monnier,13 Perrine Malzac,14 Hervé Mittre,15 Jean-Claude Chomel,16 Jean-Paul Bonnefont,17 Albert Iron,18 Michèle Chery,19 and Marie Des Georges1 1

Laboratoire de Génétique Moléculaire, CHU, CNRS UPR 1142, Institut de Biologie, Montpellier, France Laboratoire de Biochimie Pédiatrique, Hôpital Debrousse, Lyon, France 3 Centre de Biogénétique, Brest, France 4 Laboratoire de Biochimie Hôpital Henri Mondor, Creteil, France 5 Laboratoire de Biochimie et Génétique Moléculaire, Pavillon Cassini, Groupe Hospitalier Cochin, Paris, France 6 Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital Calmette, Lille, France 7 Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital Trousseau, Paris, France 8 Laboratoire de Génétique Médicale Hôpital Purpan, Pavillon Lefebvre, Toulouse, France 9 Laboratoire de Génétique Moléculaire, CHU Pontchaillou, Rennes, France 10 Conformations Cellulaires et Moléculaires, INSERM U314, CHR Maison Blanche, Reims, France 11 Laboratoire de Biochimie, Biologie Moléculaire et Enzymologie, Faculté de Médecine, Clermont-Ferrand, France 12 Service de Génétique, CHU d’Angers, Angers, France 13 Biochimie de l’ADN, Hôpital de la Tronche, Grenoble, France 14 Biologie Moléculaire Appliquée, Centre de Diagnostic Prénatal, Hôpital de la Timone, Marseille, France 15 Laboratoire de Biochimie B, CHU Georges Clémenceau, Caen, France 16 Laboratoire de Génétique Cellulaire et Moléculaire, CHU de Poitiers, Poitiers, France 17 Biochimie B, Tour Lavoisier, Hôpital Necker-Enfants Malades, Paris, France 18 Biochimie et Biologie Moléculaire, Groupe Hospitalier Pellegrin, Bordeaux, France 19 Laboratoire de Génétique, CHU Brabois, Vandoeuvre les Nancy, France 2

Communicated by Lap-Chee Tsui

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7,420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61–80), G542X (2.86%; range 1–6.7%), N1303K (2.10%; range 0.75–4.6%), and 1717-1G>A (1.31%; range 0–2.8%). Only 11 mutations had relative frequencies >0.4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of

Received 29 December 1999; accepted revised manuscript 7 April 2000.

Biologie, Boulevard Henry IV, 34060 Montpellier Cedex, France. E-mail: [email protected]

*Correspondence to: Mireille Claustres, Laboratoire de Génétique Moléculaire, CHU, CNRS UPR 1142, Institut de

Contract grant sponsors: Hospital of Montpellier; AFLM (Association Française de Lutte contre la Mucoviscidose).

© 2000 WILEY-LISS, INC.

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coding/flanking sequences, 516 of 654 (78.90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations. Hum Mutat 16:143–156, 2000. © 2000 Wiley-Liss, Inc. KEY WORDS:

congenital absence of the vas deferens; CBAVD; cystic fibrosis; CF; CFTR; ABCC7; France

DATABASES:

CFTR – OMIM:602421, 219700; (CF), 277180 (CBAVD); GDB:120584; HGMD:CFTR

INTRODUCTION Cystic fibrosis (CF; MIM# 219700) is the most common severe recessive disorder in populations of European descent, with an incidence of approximately one in 2,500 live births (carrier frequency 1 in 25), although this figure varies from 1/600 to 1/ 9000 depending on geographical locations and populations. The disease has also been reported in native Africans, Indians, Asians, and Arabians. CF is characterized by one or more of several features that vary in severity, including a progressive decline of pulmonary function secondary to chronic lung infections, exocrine pancreatic insufficiency, and elevated chloride concentrations in sweat [for a recent review, see Cutting, 1997; Stern, 1997; Rosenstein and Cutting, 1998], due to dysfunction or absence of the cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7). In addition, more than 95% of males with CF are also infertile as a result of obstructive azoospermia caused by maldevelopment of the mesonephric ducts leading to atresia of the epididymis, vas deferens, or seminal vesicles. Congenital bilateral absence of the vas deferens (CBAVD; MIM# 277180) in otherwise healthy males, due to bilateral regression of the mesonephric duct, occurs in 6% of azoospermic men [Oates and Amos, 1994]. The prevalence of CBAVD among infertile male patients is about 1– 2% in populations from Europe, the United States, or Japan [Okada et al., 1999]. Clinical symptoms of CBAVD are azoospermia with low semen plasma volume (T and 1811+1.6kbA>G) by specific PCR-restriction. The locus IVS8(T)n was analyzed by nested PCR according to Chillon et al. [1995] or by specific assays designed in order to readily distinguish the 5T, 7T, and 9T alleles (such as the use of alternative restriction enzyme, DGGE assays, or specific allele amplifications). Mutation Nomenclature

We followed recommendations from The Cystic Fibrosis Genetic Analysis Consortium [1999] and from the Nomenclature Working Group [Antonarakis et al., 1998]. RESULTS

Mutational genotype data were collected for 3,710 patients with CF and for 800 patients with CBAVD. Mutations Responsible for CF in France

A total of 310 different CF mutations were so far reported, accounting for 93.56% of CF genes (6,942/ 7,420) (Fig. 1). As the genetic centres screened their chromosome panels to different extents for diseasecausing alterations, the rate of undetected mutant genes (6.44%) varied according to laboratories from 2.06% (scanning of all 27 exons by DGGE) to 17.74% (screening for 12 known mutations). Types of mutations.

In addition to F508del, 309 different molecular defects were identified in this study, including missense mutations (41.61%), small insertions or deletions causing frameshifts (22.90%), nonsense mutations (19.03%), mutations that affect splic-

ing (13.23%), a small number of in-frame deletions (2.26%), and three large deletions (0.97%) (Fig. 1, Table 1). Five sequence changes (R31C, R75Q, F508C, G576A, R1162L) were reported as ‘‘mutations’’ in the forms; however, they are listed as ‘‘polymorphisms’’ in the CFGAC (designed respectively as 223C/T, 356G/A, 1655T/G, 1859G/ C, and 3617G>T). Ten mutations were reported as ‘‘complex alleles’’ (two sequence alterations associated in cis on the same gene): W57X+F87L, R117H+5T, R117H+I1027T, F508del+L467F, I507del+F508C, 1898+3A>G+186-13C>G, Y1092X+S1235R, 3732delA+K1200E, S1235R +5T, and D1270N+R74W. However, at least three of these changes are listed as neutral polymorphisms in the CFGAC: L467F (1531C/T), F508C (1655T/G), and I1027T (3212T/C). CF mutations were widely scattered throughout the gene, with at least one mutation identified in each of 27 exons/flanking intronic sequences. We noticed that 25/30 (83.33%) mutations in R domain should result in a premature termination of CFTR translation. Distribution of mutations according to frequencies.

We have distinguished four groups of mutation frequencies (Table 1). In group A (most common mutations), F508del was present in 67.18% of all CF French chromosomes, varying from 80% (Celtic population of Brittany) to 61% (populations living in South of France near the Mediterranean coast). Only three other mutations had relative frequencies ≥1%, G542X (2.86%), N1303K (2.10%), 1717-1G>A (1.31%). A second group (B) comprised 11 mutations whose respective frequencies ranged from 0.98% to 0.40% (Table 1). Altogether, these 15 mutations accounted for 80.92% of 7,420 CF alleles. Relative frequencies of 140 mutations in group C ranged from 0.39% to 0.10% (31 mutations), 0.09%– 0.08% (11 mutations), or were less than 0.07% (99 mutations, observed on two to five chromosomes each). Finally, 154 mutations included in group D and accounting for 2.07% of CF alleles were identified only once. By analyzing data on area of residence of families, we stressed regional differences in the relative frequencies of the most common CF mutations (Fig. 2). A trend of decreasing frequency of F508del from Northwest (70%) to Southeast (61%) was generally observed; the second most common mutation was G542X in the Southwest (6.7%) or in the South (5.6%), whereas it was I507del in

CFTR MUTATIONS IN 3,710 CF AND 800 CBAVD PATIENTS

147

Distribution of 310 mutations responsible for CF in a sample of 3,710 patients living in France, as reported in January 1999. The CFTR gene is shown as a dotted line, with each exon numbered in bold. Intronic splice site mutations are separated from exonic mutations by a short dotted line. Mutations listed as polymorphisms in the CFGAC (http:// genet.sickids.on.ca) are in italic and bold. The three large deletions (CF25kbdel, CFdel17b, CF40kbdel) identified in this study have not been included in the figure. a: Complex alleles (two different mutations reported in the same gene); b: Mutation 1898+3A>G has been found associated in cis with mutation 186-13C>G. FIGURE 1.

Normandy (2.7%). Some mutations such as 394delTT (1.61%), R553X (2.15%), or 2789+ 5G>A (2.87%) were more frequent in the North. Founder effects were likely for the splice mutation 1811+1.6kbA>G, whose frequency reached 5.5% in the region of Bordeaux and for the deletion 1078delT in Brittany (2.8%). In this series of 7,420 CF alleles, 158 have been reported to be of Arab descent, originating essentially from Tunisia, Algeria, or Morocco. The most common mutations in this group were F508del (31.01%), 711+1G>T (11.39%), W1282X (6.33%), N1303K (5.7%), G542X (5.06%), and R1162X (3.8%), a distribution which seems different from the global French population.

Distribution of CF genotypes.

3,630/3,710 patients (97.84%) had at least one CF mutant allele identified, including a total of 3,312 patients (89.27%) with two mutations and 318 patients (8.57%) with only one mutation. In the group of CF patients with two mutations, we detected a total of 481 different mutation genotypes (data not shown). Five genotypes are responsible for 57.31% of cases of CF in France: F508del/F508del (47.75 %), F508del/G542X (3.4%), F508del/N1303K (2.7%), F508del/1717-1G>A (2.02%), and F508del/2789+5G>A (1.43%). In 80 patients (2.16%), no CFTR mutation was reported at the time of the study. Overall, 3,215 patients out of 3,710 (86.65%) carried at least one F508del allele.

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TABLE 1.

Distribution of 310 CF Mutations in France With Respect to Relative Frequencies (Total Number of CF Chromosomes = 7,420)

Group

Mutations

A

F508del G542X N1303K 1717-1G>A G551D 2789+5G>A W1282X R553X I507del 1078delT 2183AA>G 711+1G>T R1162X Y1092X 3849+10kbC>T 12 mutationsa 19 mutationsb 11 mutationsc 11 mutationsd 15 mutationse 23 mutationsf 50 mutationsg 154 mutationsh 6,942

B

C

D

Number of alleles

%

4,985 212 156 97 73 72 68 66 52 49 48 33 33 30 30 29 to 15 (239) 14 to 8 (190) 7 to 6 (71) 5 (55) 4 (60) 3 (69) 2 (100) 1 (154)

67.18 2.86 2.10 1.31 0.98 0.97 0.91 0.89 0.70 0.66 0.64 0.44 0.44 0.40 0.40 0.39–0.20 0.19–0.10 0.09–0.08 0.06 0.05 0.04 0.02 0.01 93.56

Cum. %

73.45

7.47

10.57

2.07

a

3659delC, R347P, 3272-26A>G, R334W, W846X, 621+1G>T, G85E, R1066C, L206W, 394delTT, 4055+1G>A, R347H. 3905insT, 1811+1.6kbA>G, S945L, S1251N, Y122X, 2711delT, R117H, E60X, 2184insA, E585X, L558S, S1235R, D1152H, K710X, Q493X, A455E, G178R, I148T, 574delA. c 4016insT, G1244E, R1158X, 3120+1G>A, 1677delTA, I1234V, E831X, 5T, Q220X, E92K, G91R. d G149R, S489X, S492F, S549R, 1898+1G>A, 2622+1G>A, G970R, R1066H, W1204X, 3850-1G>A, Q1313X. e M1V, R75X, L165S, F311L, R560K, 1898+1G>C, 1949del84, 2113delA, 2184delA, R792X, W846X2, 3121-1G>A, H1054D, 3737delA, D1270N+R74W. f 306insA, W79X, R117C, P205S, L227R, I336K, 1248+1G>A, 1609delCA, 1717-8G>A, S549R(T>G), S549N, 1812-1G>A, P574H, 2176insC, R709X, E827X, D836Y, 3007delG, L1065P, L1077P, H1085R, M1101K, 4021insT. g D44G, 300delA, W57X, 405+1G>A, D110H, E116K, 541del4, 542del7, L137R, 621+2T>G, I175V, H199R, H199Y, C225X, V232D, Q290X, E292X, G314V, T338I, 1221delCT, W401X, Q452P, I502T, 1716+2T>C, G544S, R560S, A561E, V562I, Y569D, 1898+3A>G, 1898+5G>A, G628R(G>A), 2143delT, G673X, R851X, Q890X, S977F, 3129del4, 3154delG, 3271+1G>A, G1061R, R1066L, R1070W, 3601-17T>C, S1196X, 3732delA, G1249R, 3898insC, 4374+1G>A, del25kb. h M1K, K14X, W19X, 211delG, G27E, R31C, 237insA, 241delAT, Q39X, 244delTA, 296+2T>C, 297-3C>T, W57X+F87L, 306delTAGA, P67L, A72D, 347delC, R75Q, 359insT, 394delT, 405+4A>G, Q98R, 457TAT>G, R117H+5T, R117H+I1027T, R117L, R117P, H139R, A141D, M152V, N186K, D192N, D192del, E193X, 711+1G>A, 711+3A>G, 712-1G>T, L206F, W216X, C225R, Q237E, G241R, 852del22, 876-14del12, 905delG, 993del5, E292K, Y304X, F311del, 1161delC, R347L, R352Q, W361R, 1215delG, S364P, S434X, D443Y, S466X, C491R, T501A, I506T, F508C, I507del+F508C, F508del+L467F, 1774delCT, R553G, 1802delC, 1806delA, A559E, Y563N, 1833delT, Y569C, Y569H, Y569X, G576X, G576A, T582I, 1898+3A>G+186-13C>G, 1918delGC, R600G, L610S, G628R, 2043delG, 2118del4, E664X, 2174insA, Q689X, K698R, K716X, L732X, 2347delG, 2372del8, R764X, 2423delG, S776X, 2634insT, 2640delT, C866Y, 2752-1G>T, W882X, Y913C, V920M, 2896insAG, H939D, H939R, D979V, D985H, D993Y, 3120G>A, I1005R, 3195del6, 3293delA, 3320ins5, W1063X, A1067T, 3359delCT, T1086I, W1089X, Y1092X+S1235R, W1098X, E1104X, R1128X, 3532AC>GTA, 3548TCAT>G, M1140del, 3600G>A, R1162L, 3667ins4, 3732delA+K1200E, S1206X, 3791delC, S1235R+5T, Q1238R, Q1238X, 3849+4A>G, T1246I, 3869insG, S1255P, R1283K, F1286S, 4005+1G>T, 4006-8T>A, 4015delA, N1303H, N1303I, 4172delGC, 4218insT, 4326delTC, Q1382X, 4375-1C>T, 4382delA, D1445N, CF40kbdel4-10, Cfdel17b. Bold, mutations not published previously. b

A total of 74 patients were found to be homozygous for non F508del mutations (Table 2). This information may be useful for studies concerning correlations between genotypes and phenotypes or functional consequences of given amino acid residues. More than 60% of patients in this group were from Turkish or North African descent. The high rate of allellic homozygosity in these populations may be explained by the relatively high rate of consanguinity among parents.

Spectrum of CFTR Mutations Responsible for CBAVD

A total of 800 patients aged from 23 to 58 years have been analyzed for CFTR mutation detection in a context of male sterility due to CBAVD. CFTR Mutations in CBAVD

This sample reports 137 different CFTR mutations scattered over the whole CFTR gene, includ-


149

Geographic distribution of the most common mutations responsible for CF in 12 regions in France, as given by the area of residence of parents and/or grandparents.

FIGURE 2.

ing missense (61.31%), frameshift (9.49%), nonsense (10.22%), splice site mutations (14.60%), deletions of one amino acid (3.65%), and a sequence change in the promoter (0.73%) (Fig. 3). Seventy-three (53.28%) of these mutations have been found in the CF sample while 64 others (46.72%) were detected in CBAVD only (Fig. 3). Eleven mutations were reported as ‘‘complex alleles,’’ particularly in chromosomes carrying the 5T allele, although several changes (G576A, R668C, A1067T) are considered as neutral polymorphisms (CFGAC). Overall, 959 of 1,600 (59.94%) CBAVD alleles were identified at the time of the study. The most frequent were F508del (21.75%),

the 5T allele (16.31%), and R117H (4.37%), followed by D1152H (1.19%), and D443Y (0.93%). CFTR genotypes in CBAVD.

Two CFTR mutations (including the 5T allele) were present in 381 (47.63%) and one mutation in 197 (24.63%) patients, while no mutation was reported in the remaining 222 patients (27.75%). 348/800 (43.5%) patients with CBAVD carried one F508del allele and 254/800 (31.75%) had at least one 5T allele. Compound heterozygosity was the rule, but 10 patients were found to be homozygotes for one CFTR mutation [5T/5T (n=7), R117H/R117H (n=2), R74W+D1270N/R74W+

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Non-F508del Mutations Found as Homozygous in a Sample of 3,710 Patients With Cystic Fibrosis

TABLE 2.

Mutation

n

711+1G>T G542X N1303K 2183delAA>G W1282X G551D 3905insT R334W R347P 1078delT 1811+1.6kbA>G 2113delA Y1092X R1162X 306insA E92K G178R L227R 1677delTA 1717-1G>A 1717-8G>A R553X S549R(T>G) R560S V562I Y569D 2711delT S945L R1158X I1234V 3849+10kbC>T Q1313X del25kb E831X I175V G314V L1077P

8 7 7 5 4 3 3 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

D1270N (n=1)]. Altogether, at least one CFTR mutation was identified in 72.25% of 800 individuals with CBAVD. We could identify a total of 327 patients who have been more extensively investigated until the two mutations are found and/or whose entire coding/flanking sequences have been scanned. In this subset of patients, 516 of 654 (78.90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% of patients without any detectable CFTR mutation. The most common compound heterozygous genotypes were F508del/5T (28.44%) and F508del/R117H (5.6%). Allele frequencies at IVS8(T)n and M470V polymorphisms in CBAVD.

Data on IVS8(T)n alleles were reported for 677 of 800 patients (Table 3). F508del was found on a 9T background in 293 chromosomes and on a 7T background in one case. 109 of 137 mutations were associated in cis with a 7T allele, 24 mutations were

associated with a 9T, and four were detected on both 7T or 9T backgrounds (see legend to Fig. 3). Six sequence changes listed as mutations (CFGAC or personal communication) were detected on a chromosome carrying the 5T allele (G550R, A800G, T1053I, A1067T, S1235R, and 17173T>G). All R117H alleles whose data at locus IVS8(T)n were communicated to us were found on a 7T background. Overall, 30.82% of CBAVD chromosomes carried a 9T allele, 50.07% a 7T, and 19.17% a 5T allele. The alleles of polymorphism M470V in exon 10 were indicated for 288 patients: M470 (A at nucleotide 1540) was found in 62.5% and V470 (1540G) in 37.5% of their CFTR genes (Table 3). Most of the 5T alleles (79/98 = 80.61%) were found associated with V470. Different Spectrum of Genotypes in CF and CBAVD

In 3,303 patients with CF and 381 patients with CBAVD, the two CFTR mutations were identified. We detected a total of 481 different mutation genotypes in CF and 131 in CBAVD. CF and CBAVD genotypes were classified according to the expected effect of their mutations on CFTR protein (Fig. 4). We used combined information available from: a) the grouping of CFTR mutations into six classes based on the primary mechanism responsible for reduced chloride channel function [Welsh and Smith, 1993; Mickle and Cutting, 1998]; b) genotype-phenotype correlation studies [for review, see Estivill, 1996; Kerem and Kerem, 1996; Mickle and Cutting, 1998]; and c) data on mutant alleles whose consequences on CFTR function and/or regulation have been extensively investigated through heterologous in vitro expression systems [for review, see Seibert et al., 1996a; Seibert et al., 1996b; Seibert et al., 1997; Ko and Pedersen, 1997; Riordan, 1999; Sheppard and Welsh, 1999]. Mutations were defined as ‘‘severe’’ according to the following criteria: 1) they make no protein (nonsense, frameshift, or splicing defects introducing a premature termination = class I); 2) they do not efficiently reach the membrane (class II); 3) they reach the membrane but do not respond to stimulus (class III); 4) they are located in sites of CFTR known to generate severe mutants (such as NBDs, Walker motifs, or internal cytoplasmic loops); and/or 5) they have been described only in severe CF phenotypes with pancreatic insufficiency. Mutations were considered as ‘‘mild’’ or ‘‘variable’’ if: 1) they reach the membrane and respond to stimulus but do not conduct chloride properly or efficiently (class IV); 2) they


151

Distribution of 137 mutations responsible for CBAVD in a sample of 800 French patients with CBAVD, as reported in February 1999. The CFTR gene is shown as a dotted line, with each exon numbered in bold. Intronic splice site mutations are separated from exonic mutations by a short dotted line. Mutations listed as polymorphisms in The Cystic Fibrosis Genetic Analysis Consortium (http://genet.sickids.on.ca) are in italic and bold. Asterisks denote mutations found both in CF and in CBAVD patients. Twenty-four non F508del mutations were found associated with the 9T allele: 394delTT, L90S, D110H, R117G, 621+1G>T, V232D, A455E, G542X, R851L, T908N, 2789+5G>A, 2896insAG, H939R, 3007delG, I980K, I1027T, R1066H, A1067T, D1154G, 3737delA, R74W+D1270N, N1303I, N1303K, D1377H. Four mutations were detected on a 7T or a 9T background: L206W, R347H, D1152H, 3272-26A>G.

FIGURE 3.

produce a small quantity of functional protein as a result of a variable proportion of normal CFTR mRNA transcripts in addition to the abnormal ones (class V); 3) they are located in sites known to generate less severe mutants (external loops, residues lining the pore); and/or 4) they have been observed in CF with pancreatic sufficiency, CBAVD, and/or CF-related attenuated phenotypes only. Some mutants are misprocessed (class II) but generate channels that retain significant activity which is sufficient to confer a milder clinical phenotype (for instance, A455E or P574H). We classified such mutants in the group of ‘‘mild’’ or ‘‘variable’’ alleles. Some mutants remained undefined because available information sources were inadequate, particularly when we found them both TABLE 3.

in severe CF and CBAVD but correlations with the genotype have not been published yet (for instance, N1303I). The distribution of mutation genotypes clearly differed between the two patient populations. In CF, 87.77% of patients carried two severe mutations, whereas 11.33% had a severe mutant in trans of a mild or variable allele (including IVS8-5T) and only 0.24% inherited two mild mutations. None of CBAVD patients was homozygote for F508del, and none was compound heterozygote for F508del and a nonsense or frameshift mutation or for two severe mutations (Fig. 4). They had rather a severe and a mild/variable mutation (87.89%), or two mild/variable mutations (11.57%). A total of 22 different mutational genotypes (18 includ-

Allele Frequencies of Polymorphisms IVS8(T)n in Front of Exon 9 and M470V in Exon 10 of the CFTR Gene in Patients With Congenital Bilateral Absence of Vas Deferens (CBAVD) F508del

Other mutation

No mutation

A. Frequencies of IVS8(%)n alleles in 683 patients with CBAVD 9T 293 61 – 7T 1 228 – 5T – 6 255 F508del

Other mutation

5T

B. Frequencies of M470V alleles in 288 patients with CBAVD M 141 59 19 V 1 36 79

Unknown

Total

%

67 455 –

421 684 261

30.82 50.07 19.71

Unknown

Total

%

141 100

361 216

62.5 37.5

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FIGURE 4. Spectrum of CFTR mutation genotypes in patients with CF or CBAVD. Genotypes were classified according to the expected effect of their mutations on CFTR protein.

ing compound heterozygosity for F508del and a splicing or missense mutation) were found both in CF and in CBAVD (Table 4). Distribution of Mutations Within the CFTR Gene

Due to the high frequencies of F508del and G542X, 75.22% of French CF alleles were mutated in exons 10 and 11, encoding for the first nucleotide binding domain of CFTR (NBD1) or their flanking intronic sequences (Fig. 5). Eight additional exons had a mutant allele frequency close to 1%: exons 3,4,7, and 17b encoding transmemCFTR Mutation Genotypes Identified Both in Cystic Fibrosis (CF) and in Congenital Bilateral Absence of the Vas Deferens (CBAVD)

TABLE 4.

F508del/5T F508del/2789+5G>A F508del/3272-26A>G F508del/R117H* F508del/R117C F508del/L206W F508del/R347H F508del/R347L F508del/D443Y F508del/Y569C F508del/P574H F508del/G628R(G>A) F508del/V920M F508del/R1070W F508del/D1152H F508del/S1235R F508del/T1246I F508del/D1270N+R74W F508delN1303I 3659delC/R347H G542X/T338I R347H/R1066H

CF

CBAVD

3 53 17 10 2 12 10 1 1 1 3 2 1 2 6 3 1 2 1 1 2 1

143 1 4 39 2 4 5 1 5 1 1 1 1 3 8 1 1 3 1 1 2 1

*The only case with CF whose alleles at IVS8(T)n were reported had mutation R117H associated with a 5T allele. In the group of CBAVD patients, 26 were reported with a 7T allele associated with mutation R117H.

Percentage of total mutant alleles identified in each exon and flanking intronic sequences of the CFTR gene after extensive scanning by denaturing gradient gel electrophoresis (DGGE) assay in CF or in CBAVD.

FIGURE 5.

brane domains, exon 13 (R domain), exons 19, 20, and 21 encoding NBD2. The distribution of mutant alleles is clearly different between CF and CBAVD, and suggests that a substantial proportion of CFTR gene mutations in men with CBAVD consists of sequence alterations not detected by routine panels designed for the classic CF population. We present in Figure 5 a sequential DGGE scanning strategy which allows for the most complete identification of mutant alleles in CF or CBAVD. In this optimized strategy, the sequential order of exon scanning is dictated by a function of both the relative frequency of each mutation and the number of different mutations contained within each exon and adjacent intronic nucleotides. DISCUSSION

The purpose of this study was to determine the respective frequencies of CF and CBAVD mutations in France and to compare CF and CBAVD genotypes. Heterogeneity of Mutations Responsible for CF in the French Population

Updated as of February 1999, a total of 310 different mutations have been reported in CF. The highest allelic heterogeneity has been detected in the Mediterranean region, as previously outlined in other countries [Estivill et al., 1997; Casals et al., 1997]. Considering the prevalence of some mutations in different regions, the northern part appears subdivided into at least three zones, from West to East: Brittany, colored by a Celtic settlement (G551D); Nord-Pas-de-Calais, settled by Scandinavians (394delTT); and, in the northeast, a Germanic area (R553X). The southwest (1811+


1.6kbA>G) is markedly different from the rest of France whereas the Mediterranean coast (G542X) shows similarity between the western and eastern parts, with some difference from the central part. This study demonstrates that even within one country, wide variations exist among the frequencies of specific mutations. Other European countries show more substantial genetic homogeneity than France [Cuppens et al., 1993; Bonizzato et al., 1995; Schwarz et al., 1995; Tümmler et al., 1996]. Strategies for Detecting CFTR Mutations

Detailed information on currently used mutation detection strategies and evaluation of CFTR gene mutation testing methods have been reported in the last European external quality assessment [Dequeker and Cassiman, 1998]. Although it is now recommended to determine the precise sequence abnormalities in order to select the appropriate therapeutic approach which could be addressed in a genotype-specific manner [Zeitlin, 1999], no consensus has been reached regarding which mutations should be evaluated or the cost of each type of screening. From our data, it can be extrapolated that commercial kits for routine screening of about 30 known mutations will cover 83% of mutant alleles in the CF population. In the CBAVD group, analysis of the most common CFTR mutations associated with CF in addition to assessment for IVS8-5T will lead to the identification of approximately 62% of mutant genes. A large proportion of CBAVD alleles will escape detection if an extensive scanning of CFTR exons is not performed. A similar conclusion was recently reported for a sample of 64 men with CBAVD who had been comparatively investigated for CF mutation detection and extensive exon scanning [Mak et al., 1999]. Thus, the laborious but powerful manual DGGE technique represents so far the best approach for mutation detection of CF and CBAVD. Screening the General Population for CF Carriers?

Combining detection of the F508del with a few other mutations yielded a 95% sensitivity only in Celtic Bretons. In other parts, expanding the panel of screened mutations is expected to achieve only marginal gains in the sensitivity. Given the heterogeneity of CFTR mutations in France, population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available. We believe that a DNA mi-

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cro-array for CFTR mutation detection and/or resequencing all 27 exons/flanking regions should be the only acceptable technique for carrier screening in the general population. The CFTR Gene Is Not Involved in the Aetiology of All Cases of CBAVD

After scanning of the whole coding and flanking CFTR sequences, there is still a substantial fraction of patients who have only one mutation (including the 5T) or no identifiable abnormalities in the CFTR gene. Some mutations may be undetected, because they are located in regions of the gene that are not analyzed (for instance, 3´ and 5´ UTRs or intronic regions), or because they cannot be detected through PCR-based techniques (deletion encompassing one or more exons). It would be interesting to study extensively CBAVD patients carrying only one CFTR mutation for genital phenotype, in order to see if they constitute a separate phenotypic group of sterile men in whom carrying only one CFTR mutation is a risk factor for sterility. In up to 20% of patients, CBAVD is associated with urinary tract malformations and in these cases the aetiology of CBAVD seems not related to defects in the CFTR gene [Augarten et al., 1994; Schlegel et al., 1996]. It is likely that a sub-group of patients might have undetected, subtle renal abnormalities, while in other patients CBAVD might be due to defects in other genes. Genetic Counselling

In all CBAVD patients, in addition to extensive CFTR mutation screening, the following are strongly recommended: sweat electrolyte evaluation; examination by a physician with experience in CF; and renal ultrasound for a more precise assessment of the genetic risk linked to CF [Dumur et al., 1996]. If a CF mutation is found in the CBAVD patient, each sibling has a 50% risk of being a carrier. Thus genetic counselling should be offered to male siblings of CBAVD patients, who may similarly be affected, and to other family members who may be carriers of a CF mutation. New hopes for men with CBAVD (or CF) to father their own children can be offered after sperm retrieval and assisted reproduction. Accurate genetic counselling of the CBAVD couple also depends upon adequate mutation screening of the partner. As it is presently impossible to screen the whole CFTR gene, any mutational testing will result in a probability that the individual tested, although negative, does carry an undetected mu-

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tation. The residual risk of an individual being a carrier after a negative screening for a fraction a of CFTR mutations in his ethnic background will be Z=q(1-a)/(1-aq), with q the prior carrier risk [ten Kate, 1990]. The predicted phenotypes of offspring resulting from combinations of CFTR mutations in couples with a CBAVD male have been described [Lissens et al., 1996]. If both male and female carry a severe CF mutation, then the risk of a CF or CBAVD child approaches 1/2. If the male carries a severe CF mutation and the female partner is negative after a test that detects 90% of CF mutations, then the risk to this couple of having a CF child is estimated as 1/964, assuming a prior risk for q=1/25 [(chance of male with CBAVD transmitting the CF mutation) x (probability of female partner being a CF carrier if negative) x (chance of this female transmitting a CF mutation) = (1/2) × (1/241) × (1/2) = 1/964]. The presence of a 5T allele in the male is thought to result in CBAVD for any male offspring, with a risk ranging from 1/1721 to 1/1607 assuming a penetrance of 0.56 [Chillon et al., 1995] or 0.60 [Zielenski et al., 1995], respectively, in Caucasian populations. However, considering the wide range of manifestations associated with the genotype ‘‘CF mutation/5T allele,’’ which can range from healthy fertile or CBAVD males to individuals with typical CF [Kerem et al., 1997; and this study], it is not advised to predict the clinical outcome of the pregnancies. Identification of haplotypes associated with the 5T alleles through familial analyses might be of interest, as it was recently suggested that the combination of particular alleles at some of the more common polymorphic loci in the CFTR gene (IVS8-Tn-TGm, and M470V in exon 10) can affect the final quantity and/or quality of CFTR and modulate the partial penetrance of the 5T allele [Cuppens et al., 1998]. This finding could explain why haplotype (TG12-T5-V470) was preferentially found to be associated with CBAVD, whereas haplotype (TG11-T5-M470) was observed in healthy fathers (with genotype ‘‘CFmutation/ 5T’’) of CF children [Cuppens et al., 1998; De Meeus et al., 1998]. Such ‘‘polyvariant mutant genes’’ most probably contribute, through cis and trans effects, to the partial penetrance and the severity of the 5T allele and to heterogeneity in both CFTR Cl- channel conductance and disease phenotype among individuals with the same CFTR genotype [Cuppens et al., 1998]. Because of the incomplete penetrance of mild or CBAVD mutations for some combinations of CFTR genes in these couples (for instance, a male

with a rare missense and a female with a severe mutation), it will be very difficult or even impossible to predict the phenotypic consequences for their children. The option of preimplantation diagnosis (and selection of embryos not carrying the maternal CF mutation) should be discussed, considering all the medical and psychological burdens of assisted reproductive procedures. Causing Disease or Not?

It is possible that some of the missense mutations detected here and by others [Dork et al., 1997] may not be CF or CBAVD-causing. Criteria for disease causality are: 1) mutation resulting in incomplete protein; 2) mutation changing a species-conserved amino acid; 3) common polymorphism excluded after testing at least 100 normal individuals; 4) no further mutation found after scanning the remainder of the gene; 5) mutation observed in two unrelated proband; 6) co-segregation with phenotype in family; and 7) functional studies at the cellular level [Cotton and Scriver, 1998]. Since CFTR is intrinsically a chloride channel when properly anchored in the cell membrane, its activity is best measured by patchclamp analysis or other similary tedious methods that are rarely used by molecular geneticists to confirm the identity of putative mutations. An example of the difficulties in defining missense mutations and polymorphisms is S1235R. This variant was originally described as a mutation [Cuppens et al., 1993]. However, as it has been detected in a patient who also had a stop mutation (Y1092X) elsewhere in the gene (Claustres et al., unpublished results), it is most likely misclassified as a missense mutation. This observation does not excude that S1235R could be, in combination with some polymorphisms affecting splicing (such as 5T), responsible for a CF or CBAVD-related phenotype. Mutations D1270N and R74W have been independently reported elsewhere to be CF alleles [Anguiano et al., 1992; Claustres et al., 1993]. However, we observed that the double mutant R74W+D1270N was present in trans with a severe CF mutation in two healthy unrelated mothers (with normal sweat test) of affected CF children [Verlingue et al., 1993] (Des Georges et al., unpublished results), suggesting that these missense changes may be not disease-causing. An interesting approach for identifying non-pathogenic CFTR variations among the most common European missense changes has been recently reported [Bombieri, et al., 2000]. In conclusion, the high heterogeneity of CFTR mutations in this country reflects the many demographic changes and migrations in France during centuries.


ACKNOWLEDGMENTS We gratefully acknowledge the thousands of families and their respective physicians who participated in the study. Data were collected and analyzed by Marie Des Georges, Caroline Guittard, and Mireille Claustres.

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