RESEARCH ARTICLE OFFICIAL JOURNAL www.hgvs.org CFTR Mutation Combinations Producing Frequent Complex Alleles with Different Clinical and Functional Outcomes Ayman El-Seedy, 1 Emmanuelle Girodon, 2 Caroline Norez, 1 Julie Pajaud, 1 Marie-Claude Pasquet, 1,3 Alix de Becdeli ` evre, 2 Thierry Bienvenu, 4 Marie des Georges, 5 Fa¨ ıza Cabet, 6 Guy Lalau, 7 Eric Bieth, 8 Martine Blayau, 9 Fr´ ed´ eric Becq, 1 Alain Kitzis, 1,3 Pascale Fanen, 2 and V ´ eronique Ladeveze 1 ∗ 1 Institut de Physiologie et Biologie Cellulaires, Centre National de la Recherche Scientifique Formation de Recherche en Evolution FRE 3511, Universit ´ e de Poitiers, Poitiers, France; 2 AP-HP, Groupe Hospitalier Henri Mondor, Service de Biochimie-G ´ en ´ etique, Cr ´ eteil, France; 3 CHU de Poitiers, Poitiers; France; 4 Laboratoire de Biochimie et G ´ en ´ etique Mol ´ eculaire, Groupe hospitalier Cochin-Saint Vincent de Paul, AP-HP, Paris, France; 5 Laboratoire de G ´ en ´ etique Mol ´ eculaire, IURC, CHU de Montpellier, Montpellier, France; 6 Service d’Endocrinologie Mol ´ eculaire et Maladies rares, Centre de Biologie et Pathologie Est, CHU de Lyon, Bron, France; 7 P ˆ ole de Biochimie et Biologie Mol ´ eculaire, Centre de Biologie Pathologie, CHU de Lille, Lille, France; 8 Service de G ´ en ´ etique M ´ edicale, H ˆ opital Purpan, Toulouse, France; 9 Laboratoire de G ´ en ´ etique Mol ´ eculaire, CHU Pontchaillou, Rennes, France Communicated by Garry R. Cutting Received 13 February 2012; accepted revised manuscript 21 May 2012. Published online 7 June 2012 in Wiley Online Library (www.wiley.com/humanmutation).DOI: 10.1002/humu.22129 ABSTRACT: Genotype–phenotype correlations in cys- tic fibrosis (CF) may be difficult to establish be- cause of phenotype variability, which is associated with certain CF transmembrane conductance regula- tor (CFTR) gene mutations and the existence of com- plex alleles. To elucidate the clinical significance of complex alleles involving p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys, we performed a col- laborative genotype–phenotype correlation study, col- lected epidemiological data, and investigated structure– function relationships for single and natural com- plex mutants, p.[Gly576Ala;Arg668Cys], p.[Gly149Arg; Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala; Arg668Cys]. Among 153 patients carrying at least one of these mutations, only three had classical CF and all carried p.Gly149Arg in the triple mutant. Sixty-four had isolated infertility and seven were healthy individuals with a severe mutation in trans, but none had p.Gly149Arg. Functional studies performed on all single and natural complex mu- tants showed that (1) p.Gly149Arg results in a severe misprocessing defect; (2) p.Asp443Tyr moderately alters CFTR maturation; and (3) p.Gly576Ala, a known splic- ing mutant, and p.Arg668Cys mildly alter CFTR chloride conductance. Overall, the results consistently show the contribution of p.Gly149Arg to the CF phenotype, and suggest that p.[Arg668Cys], p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] are associ- ated with CFTR-related disorders. The present study emphasizes the importance of comprehensive genotype– ∗ Correspondence to: V´ eronique Ladeveze, Institut de Physiologie et Biologie Cellulaires, CNRS FRE 3511, Universit ´ e de Poitiers, 86022 Poitiers, France. E-mail: veronique.ladeveze@univ-poitiers.fr Contract grant sponsor: Association mucoviscidose: ABCF2; the French association Vaincre la Mucoviscidose (to C.N. and F.B.). phenotype and functional studies in elucidating the impact of mutations on clinical phenotype. Hum Mutat 00:1–9, 2012. C  2012 Wiley Periodicals, Inc. KEY WORDS: cystic fibrosis; CFTR; complex allele; struc- ture; function Introduction Around 1,900 mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR; NM_000492.3; MIM# 602421) have been reported in patients with cystic fibrosis (CF; MIM# 219700) [Welsh et al., 2001]. CF is one of the most frequent au- tosomal recessive diseases in the Caucasian population. Milder forms, in which there is evidence of CFTR dysfunction but the diagnostic criteria for CF are not met, are termed CFTR-related dis- orders (CFTR-RD) (Cystic Fibrosis Mutation Database [CFMD], http://www.genet.sickkids.on.ca/cftr/) [Bombieri et al., 2011]. Phe- notype heterogeneity is partly due to CFTR mutational and genotype heterogeneity, interplaying with other genetic factors/modifiers and environmental factors. Generally, patients with the classic form of CF have CF-causing mutations in each copy of the CFTR gene, whereas patients with a CFTR-RD may have a CF mutation in one copy and a mild mutation in the other allele, or mild mu- tations in both copies. However, variable phenotypes have been observed for a given genotype. Little is known about the molec- ular mechanism by which mutations affect CFTR function, and only a minority of mutations has been characterized by functional studies. There has been a steady increase in the number of reports of polymutant variants on the same allele, termed complex alleles [Clain et al., 2001; Claustres et al., 2004; Fanen et al., 1999; Krasnov et al., 2008; Niel et al., 2006; Romey et al., 2000; Savov et al., 1995; CFMD]. This complicates diagnosis and genetic counseling in some cases. Although some variants have been considered neutral because they were identified in the non-CF allele of the parents of CF pa- tients, they seem to be involved in moderate forms or syndromes of late onset, as reported for c.2002C>T, p.Arg668Cys (R668C) and c.1727G>C, p.Gly576Ala (G576A) [Fanen et al., 1992]. These C  2012 WILEY PERIODICALS, INC.