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A new large CFTR rearrangement illustrates the importance of searching for complex alleles

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A new large CFTR rearrangement illustrates the importance of searching for complex alleles
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    HUMAN MUTATION Mutation in Brief #902(2006) Online  MUTATION IN BRIEF  © 2006   WILEY-LISS, INC. Received 24 Nov 2005; accepted revised manuscript 20 March 2006. A New Large CFTR  Rearrangement Illustrates the Importance of Searching for Complex Alleles F Niel 1 , M Legendre 1 , T Bienvenu 2 , E Bieth 3 , G Lalau 4 , I Sermet 5 , D Bondeux 6 , R Boukari 7 , J Derelle 8 , P Levy 9 , P Ruszniewski 9 , J Martin 1 , C Costa 1 , M Goossens 1 , and E Girodon 1* 1 Service de Biochimie-Génétique, Hôpital Henri Mondor AP-HP, Créteil, France; 2 Service de Biochimie-Génétique,  Hôpital Cochin AP-HP, Paris, France; 3  Laboratoire de Génétique Médicale, Hôpital Purpan, Toulouse, France; 4  Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Calmette, Lille, France; 5 Service de Pédiatrie, Hôpital  Necker-Enfants-Malades AP-HP, Paris, France; 6   Département de Pédiatrie, Hôpital Porte Madeleine, Orléans, France; 7  Service de Pédiatrie, Hôpital Noureddine El Atassi, Alger, Algérie; 8   Médecine Infantile, Hôpital d’Enfants, Vandoeuvre-les Nancy, France; 9 Service de Gastro-Entérologie, Hôpital Beaujon AP-HP, Clichy, France *Correspondence to: Emmanuelle Girodon, Service de Biochimie-Génétique, Hôpital Henri Mondor AP-HP, Créteil, France, Tel.: 33 1 49 81 28 57; Fax: 33 1 49 81 28 42; E-mail: Emmanuelle.Girodon@im3.inserm.fr Communicated by Richard G.H. Cotton   The p.Val754Met variant, described in 1996 in a CF patient, has been considered a CF mutation. However, biochemical aspects, results of functional studies and, finally, the identification of a complex deletion removing exons 3 to 10 and 14b to 16 in  cis  of p.Val754Met in a CF patient, argue against a strong deleterious effect. An inventory through the French CF network of patients carrying p.Val754Met led to the registration of seven patients (CF: n=4; idiopathic chronic pancreatitis: n=3) and six healthy individuals, all heterozygous for the variation. Extensive CFTR  gene analysis was carried out, including the search for large rearrangements and other possible mutations. The complex deletion, whose breakpoints are described here, was found only in the four CF patients, in association with the same haplotype. This data, added to the fact that the p.[Phe508del]+[Val754Met] genotype was found in a healthy individual, bring further arguments against the association of p.Val754Met with CF. We thus suggest looking for a possible complex allele whenever p.Val754Met is detected and considering it neutral regarding genetic counseling when found in isolation. © 2006 Wiley-Liss, Inc.   KEY WORDS: CFTR ; ABCC7; CF; cystic fibrosis; gene rearrangement; complex deletion; complex allele; p.Val754Met INTRODUCTION Cystic fibrosis (CF; MIM# 219700) is one of the most common autosomal recessive hereditary diseases in the Caucasian population. Up to 1400 sequence variations of the cystic fibrosis transmembrane conductance regulator ( CFTR; also symbolized  ABCC7  ; MIM# 602421) gene have been reported to the CF international consortium mutation data base (www.genet.sickkids.on.ca/cftr ). The discrimination between pathogenic mutations and DOI: 10.1002/humu.9431  2 Niel et al.  polymorphisms is difficult or subtle in some cases, in particular for missense mutations (Cotton and Scriver, 1998). Of these, some have been described as polymorphisms because they were identified in the non CF allele of CF  patients’ parents, but also seem to be involved in moderate forms of CF or syndromes of late onset, such as the 1342-12(T)5 or IVS8(T)5 variant (c.1210-12(T)5 according to the approved nomenclature, www.hgvs.org/mutnomen/) (Chillon et al., 1995a; Costes et al., 1995; Pignatti et al., 1996; Wang et al., 2000),  p.Leu997Phe (Pignatti et al., 1995; Girodon et al., 1997), 1716A>G (c.1584A>G according to the approved nomenclature) (Cuppens and Cassiman, 1995). Conversely, other mutations were initially reported as mutations  because they were identified in CF patients, but are now reclassified as polymorphisms, as is the case for  p.Ile148Thr (Rohlfs et al., 2002; Claustres et al., 2004). The existence of at least two mutations or sequence variations on the same allele, named complex alleles (Savov et al., 1995; Fanen et al., 1999; Romey et al., 1999; Clain et al., 2001; Rohlfs et al., 2002; Clain et al., 2005), somewhat complicates genetic counseling in certain situations. The p.Val754Met variant (2392G>A or c.2260G>A according to the approved nomenclature), first described by A.Wallace in 1996 in a CF patient (www.genet.sickkids.on.ca/cftr ) and subsequently identified in other CF patients, has therefore been considered a CF mutation. Healthy individuals who were found to carry this variation were thus considered CF carriers. This may happen when additional screening using scanning techniques is required besides testing for frequent mutations, depending on individuals’ geographical or ethnic background. However, there have been several lines of evidence against a strong deleterious effect of p.Val754Met: 1) the conservative nature of the Val>Met substitution; 2) the presence of a Met residue in place of Val in some species and the benign nature of the substitution predicted by the SIFT ®  and Polyphen ®  softwares; 3) results of functional studies which suggest that residue Val754 does not belong to the functional “R” domain (Chen et al., 2000); 4) finally, the recent identification by semi-quantitative fluorescent multiplex PCR of a complex deletion, CFTR dele3_10,14b_16, removing exons 3 to 10 and 14b to16 in cis  of p.Val754Met in a CF patient (Niel et al., 2004). The aim of this study was to determine whether we should still consider p.Val754Met as a possible disease-associated mutation or reclassify it as a neutral polymorphism. We thus made an inventory of patients and individuals with p.Val754Met, screened for the complex deletion and for other possible CFTR  anomalies, and studied the associated CFTR  haplotypes. Here, we gather lines of evidence that p.Val754Met should be no longer considered as a CF mutation. PATIENTS AND METHODS Patients and controls Thirteen patients and individuals from diverse srcins and all heterozygous for the p.Val754Met variant, were registered from the French CF network of molecular genetics laboratories (Table 1): seven patients (four having CF and three presenting with idiopathic chronic pancreatitis) and six healthy individuals (an infant, three partners of CF carriers, a CF patient’s relative and a mother of a fetus with bowel anomalies). Detailed phenotypic data are indicated in Table 2. Patient #1, in whom the deletion was first identified, has already been referred to in Niel et al. (Niel et al., 2004). The four CF patients were unrelated and had a classical form of the disease with pulmonary symptoms, pancreatic insufficiency and a positive sweat test. In patients #2 and #4, the diagnosis of CF had been established since birth upon neonatal screening test results. The other three patients had chronic pancreatitis with no evidence of other CF symptoms, but no sweat test could be done. The study of two other genes involved in chronic  pancreatitis,  PRSS1 ( exons 1 to 3) and   SPINK1  (exons 1 and 3), performed in another laboratory, was negative for them. The search for other risk factors, including alcohol consumption, biliary, metabolic or endocrine disorders was negative for patients #5 and 6. Patient #7, deceased at 37 y, had no children but had never been investigated for infertility. He had severe Crohn’s disease and, though recurrent acute pancreatitis has been described in the course of this disease (Weber et al., 1993; Levine and Lukawski-Trubish, 1995; Oishi et al., 2004), the chronic pancreatitis in this patient may, in part at least, have been caused by alcoholism.  A New CFTR  Complex Allele 3   Table 1. Phenotype and genotype data of patients/individuals carrying the p.Val754Met variation Patient Phenotype Origin Allele 1 Allele 2 CFTR  haplotype linked to  p.Val754Met  c  p.Val754Met  b CFTR dele3_10, 14b_16  b  1 a CF Kabylia + + 1812-1G>A (c.1680-1G>A) 22 ; del; del; 7; 17 2 CF Northwestern France + + 3659delC (c.3528delC) 22; del; del; 7; 17 3 CF Algeria + + p.Asn1303Lys 22; del; del; 7; 17 4 CF Turkey + + p.Phe508del 22; del; del; 7; 17 5 Chronic  pancreatitis Portugal + - but p.Phe311Leup.Phe508del 22; 23; 10-9; 7; 17 6 Chronic  pancreatitis  Not known + -  IVS8(TG)12(T)5 (c.1210-34(TG)12(T)5) 21 or 23; 16; 10-7; 7; 17 7 Chronic  pancreatitis  Northern France + -  IVS8(TG)11(T)5 (c.1210-34(TG)11(T)5) 22; 23; 10-9; 7; 17 8 healthy Southwestern France + - p.Phe508del 22; 23; 10-9; 7; 17 9 healthy Northern France + - Wild 22; 23; 10-9; 7; 17 10 healthy Northern France + - Wild 22; 16 or 21; 10-9; 7; 17 11 healthy Northern France + - Wild 22; 23; 10-9; 7; 17 12 healthy Turkey + - Wild 22; 23; 10-9; 7; 17 13 healthy France + - Wild 22; 23; 10-9; 7; 17 The recommendations for mutation nomenclature (www.hgvs.org/mutnomen/   ) were used to name CFTR  gene sequence variations at the protein level. For variations described at the nucleotide level, the A of the ATG translation start codon was numbered as +133 in accordance with the current CFTR  gene numbering based on cDNA sequence (GenBank NM_000492.2) and on the CF mutation database. These variations were also given in parentheses following the approved nomenclature format (A of the ATG translation start codon as +1, “c.” as prefix). a  Patient #1, in whom the deletion was firstly identified, was referred to in Niel et al. (Niel et al., 2004).  b  “+” and “-” refer to the presence or absence of p.Val754Met and CFTR dele3_10,14b_16. c  IVS1(CA); IVS8(CA); IVS8(TG)m(T)n; IVS17b(TA); IVS17b(CA) Mutations and haplotypes in patients for whom segregation study could not be performed are indicated in italics.  4 Niel et al. Table 2. Phenotype data of patients carrying the p.Val754Met variation Patient Age at diagnosis Current age Gender Pulmonary outcome Pancreatic status Other Sweat test a 1 9 y Deceased at 14 y F Severe PI 150 2 birth (neonatal screening) 6 y M Severe PI 80 3 1 y 11 y M Severe PI Failure to thrive 120-150 4 birth (neonatal screening) 17 m M Severe PI 104-107 5 16 y 17 y M None PS Minor epilepsy Nd 6 60 y 62 y F None PS Lymphoid hemopathy  Nd 7 adulthood Deceased at 37 y M None PS Severe, complicated, Crohn’s disease; alcoholism  Nd y: years; m: months; F: female; M: male; PI: pancreatic insufficiency; PS: pancreatic sufficiency; Nd: not done. a  The sweat test was done with the Gibson-Cooke reference method (chloride concentrations in mEq/l; normal values <60mEq/l). The frequency of the p.Val754Met variant in the general population has been estimated low in European  populations, below 0.17%, as it was found neither among 191 healthy European individuals (Bombieri et al., 2000) nor among 115 healthy French individuals (Girodon et al., 2002). As two of our patients carrying p.Val754Met were of Arab srcin and one was Turkish, we studied another control population of 116 healthy individuals from Arab (mostly Northern African) or Turkish extraction, who were referred to our laboratory for carrier screening (56  partners of CF carriers and 60 parents of fetuses with bowel anomalies but not affected with CF). Case #12 was taken from this control study. According to French legislation and the recommendations of the local ethics committee, written consents to the genetic study were obtained from the patients and/or their parents and from the healthy subjects. Mutation screening The recommendations for mutation nomenclature (www.hgvs.org/mutnomen/) were used to name CFTR  gene sequence variations at the protein level. For variations described at the nucleotide level, the A of the ATG translation start codon was numbered as +133 in accordance with the current CFTR  gene numbering based on cDNA sequence (GenBank NM_000492.2) and on the CF mutation database. These variations were also given in  parentheses following the approved nomenclature format (A of the ATG translation start codon as +1, “c.” as  prefix). Genomic DNAs were extracted from whole blood samples collected on EDTA using various protocols. Extensive CFTR  gene analyses were performed in the patients / individuals carrying p.Val754Met: (i) screening for frequent mutations using diverse commercial assays; (ii) scanning of the 27 exons and their boundaries using denaturing gradient gel electrophoresis (DGGE) (Fanen et al., 1992; Costes et al., 1993) or denaturing high pressure liquid  phase chromatography (DHPLC) (Le Marechal et al., 2001), followed by sequencing to characterize the variants; (iii) screening for the intronic splicing 1811+1.6kbA>G mutation (c.1679+1634A>G) (Chillon et al., 1995b); (iv) screening for large CFTR  rearrangements using a semi-quantitative fluorescent multiplex PCR (QFM-PCR) assay recently developed in our laboratory and which enabled the detection of 20% of the rearrangements among  previously unidentified alleles in CF patients (Niel et al., 2004). The healthy subjects tested for genetic counseling purposes were screened for frequent mutations (CF OLA assay, Abbott, Rungis, France, www.abbott.com   ) and for other mutations according to their geographical or ethnic  background (Claustres et al., 2000; Kilinc et al., 2002), including DHPLC analysis of exon 13 where p.Val754Met is located.  A New CFTR  Complex Allele 5   CFTR  haplotype analysis To determine the haplotypes associated with p.Val754Met in isolation and in cis  with the complex deletion, five intragenic multiallelic markers were studied: IVS1(CA) [185+10167(CA)17_26] ([c.53+10167(CA)17_26 IVS8(CA) [1342-307(GT)14_24] ([c.1210-307(GT)14_24 IVS8(TG)m(T)n [1342-34(TG)9_13(T)5_9] ([c.1210-34(TG)9_13(T)5_9 IVS17b(TA) [3499+200(TA)7_57] ([c.3367+200(TA)7_57]) and IVS17b(CA) [3499+428(CA)11_19] ([c.3367+428(CA)11_19]). Molecular characterization of the complex deletion CFTR dele3_10,14b_16 A combination of QFM-PCR and long-range PCR experiments using the Expand Long Template TM  PCR system and the Expand 20 kb PLUS  PCR System (Roche Diagnostics, Mannheim, Germany, www.roche-diagnostics.com   ) were used to delineate the extent of the complex CFTR dele3_10,14b_16 deletion, initially named 297-?_1716+?del; 2752-?_3120+?del (c.[165-?_1584+?del; 2620-?_2988+?del]) (Niel et al., 2004), which removed at least 50 kb from exons 3 to 10 and 4 kb from exons 14b to 16. Resulting fragments obtained from long-range PCRs were sequenced to define precisely the deletion breakpoints. The sequences were compared with the CFTR  reference genomic sequences (GenBank accession numbers AC000111.1 and AC000061.1). RESULTS Among the 13 patients / individuals carrying p.Val754Met, only the four patients affected with classical CF carried the complex deletion (Fig. 1), and were thus compound heterozygotes for two severe CF mutations (Table 1). In all these cases, p.Val754Met was in cis  of the deletion. Severe CF mutations were found in two other cases. Patient #5, already heterozygous for p.Phe508del, was found to carry the known p.Phe311Leu CF mutation. In case #8, the healthy infant carried p.Phe508del and p.Val754Met in trans , but not the deletion. Chronic pancreatitis  patients #6 and 7 carried the IVS8(T)5 variant, albeit linked to different (TG) repeats, but we could not confirm that it was in trans  of p.Val754Met. Complete scanning of the coding regions failed to detect other CFTR  mutations in the patients and healthy individuals. ex8 ex14b F9 reference genereference gene DSCR1 ex13ex12 ex10ex7ex16F9 reference gene ex15 ex9   ex8 ex14b F9 reference genereference gene DSCR1 ex13ex12 ex10ex7ex16F9 reference gene ex15 ex9   ex8 ex14b F9 reference genereference gene DSCR1 ex13ex12 ex10ex7ex16F9 reference gene ex15 ex9   Figure 1. Electropherogram from a semi-quantitative fluorescent multiplex PCR experiment in patient 1, carrying at the heterozygous state the complex deletion CFTR dele3_10,14b_16 (blue), superimposed with that of a normal control (red). The x axis represents the computed length of the PCR products in base pairs as determined by using an internal lane standard, indicated in black. The y axis shows fluorescent intensities in arbitrary units. Normalization of the profiles was performed using F9  (chromosome X) or  DSCR1  (chromosome 21) reference genes (Niel et al., 2004). An exon deletion is evidenced by a two-fold reduction in fluorescence intensity in the patient as compared to the normal control. The size difference for exon 9 between the control and the patient is attributable to the IVS8 (TG)m(T)n polymorphism, the two being homozygous for different alleles. Analysis of five intragenic CFTR  microsatellites in the patients / individuals carrying p.Val754Met and their families evidenced the same haplotype linked to the complex allele, [IVS1(CA) 22 ; IVS17b(TA) 7 ; IVS17b(CA) 17 ], extended to IVS8(CA) 23  and IVS8(TG) 10 (T) 9  in cases 8, 11 and 12 (Table 1). The same haplotype could be linked to p.Val754Met also in cases #5, 7, 9 and 13, despite the absence of a segregation study. However, the haplotype
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