Clin Genet 2007 Printed in Singapore.

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# 2007 The Authors Journal compilation # 2007 Blackwell Munksgaard

CLINICAL GENETICS doi: 10.1111/j.1399-0004.2007.00820.x

Short Report

Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF
Schneider M, Hirt C, Casaulta C, Barben J, Spinas R, Buhlmann U, Spalinger J, Schwizer B, Chevalier-Porst F, Gallati S. Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF. Clin Genet 2007. # Blackwell Munksgaard, 2007 Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disorder in Caucasians, and is associated with at least one mutation on each CF transmembrane conductance regulator (CFTR) allele. Some patients, however, with only one identifiable point mutation carry on the other allele, a large deletion that is not detected by conventional screening methods. The overall frequency of large deletions in patients with CF is estimated to be 13%. Using the CFTR Multiplex Ligation dependent Probe Amplification Kit (MRC-Holland, Amsterdam, Netherlands) that allows the exact detection of copy numbers from all 27 exons in the CFTR gene, we screened 50 patients with only one identified mutation for large deletions in the CFTR gene. Each detected deletion was confirmed using our real-time polymerase chain reaction (PCR) assay and deletion-specific PCR reactions using junction fragment primers. We detected large deletions in eight patients (16%). These eight CF alleles belong to four different deletion types (CFTRindel2, CFTRdele14b17b, CFTRdele17a17b and CFTRdele 29) whereof the last is novel. Comparing detailed clinical data of all these patients with CF and the molecular genetic findings, we were able to elaborate criteria for deletion screenings and possible genotypephenotype associations. In conclusion, we agree with other authors that deletion screenings should be implemented in routine genetic diagnostics of CF.
M Schneidera, C Hirta, C Casaultab, J Barbenc, R Spinasd, U Buhlmanne, J Spalingerf, B Schwizerg, F Chevalier-Porsth and S Gallatia
Division of Human Genetics, and Division of Pneumology, Childrens University Hospital, Inselspital, Bern, Switzerland, cDivision of Pneumology/ Allergology, Childrens Hospital, St. Gallen, Switzerland, dDivision of Pneumology, Childrens University Hospital, Zurich, Switzerland, ePediatric Clinic, Triemli Hospital, Zurich, Switzerland, fDivision of Gastroenterology, Childrens Hospital, Lucerne, Switzerland, gDivision of Pneumology, Cantonal Hospital, Lucerne, Switzerland, and hBiochimie Pediatrique, Hopital Debrousse, Lyon, France
b a

Key words: CFTRdele29 cystic fibrosis hot spot region Ex17b large deletion MLPA quantitative real-time PCR Corresponding author: Professor Sabina Gallati, PhD, Division of Human Genetics, Childrens University Hospital, Inselspital, 3010 Bern, Switzerland. Tel.: 14131 632 94 93; fax: 14131 632 94 84; e-mail: sabina.gallati@insel.ch Received 30 November 2006, revised and accepted for publication 2 March 2007

Cystic fibrosis (CF), the most common lifeshortening autosomal recessive disorder in Caucasians with an estimated incidence of one in 2000 to one in 2500 newborns and a carrier frequency of about one in 22 to one in 25, is primarily caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Besides the most common mutation, F508del, more than 1500 different mutations (www.genet.sickkids.on.ca/cftr) have been reported to the Cystic Fibrosis Genetic Analysis Consortium so far. CF is caused by at

least one mutation (mainly point-mutations) in each CFTR gene copy. However, in a proportion of patients with typical or CF-like symptoms, the second mutation remains unidentified. Among others, large deletions (spanning single or multiple exons) may lie behind these unidentified mutations. The overall frequency of large deletions in the CFTR gene is estimated to be 13% (1) in patients diagnosed with CF. In well-defined study cohorts, e.g. patients with one unidentified CFTR allele and typical CF clinics, the detection rate of
1

Schneider et al.

large deletions reported so far varies between 15% and 25% (14), but might be even higher (5). The first systematic examination of rearrangements in the CFTR gene was performed in 2004. Since then, only a few additional screenings have been accomplished leading to the identification of more than 30 different large deletions and 4 large duplications (www.genet.sickkids.on.ca/cftr). We present here the first systematic CFTR deletion/duplication screening using the CFTR Multiplex Ligation dependent Probe Amplification (MLPR) Kit (MRC-Holland, Amsterdam, Netherlands) that allows the detection of the copy number of all 27 exons in a single reaction. As the first description of MLPA in 2002 (6), the number of studies dealing with the detection of deletions and duplications by MLPA is permanently increasing, and it has been proven to be a reliable and cost-efficient method. The aim of our study was to contribute to the search of unidentified mutations in the CFTR gene in patients who are clinically highly suspected to suffer from CF but carrying only one detectable mutation after our extensive single strand conformation polymorphism/hetero duplex (SSCP/HD) screening (7). Another important goal of this study was the presentation and analysis of detailed clinical data of each patient, which allowed us to establish criteria for a deletion screening strategy on the one hand and to elaborate possible genotype phenotype correlations on the other hand.
Materials Patient samples and controls

patients with CF with known large deletions either provided by two French research groups (eight samples) or by our own lab (Table 1). Deletion screening of the entire CFTR gene by MLPA We used DNA samples from a group of 50 unrelated patients (,40 years) with typical or atypical CF. The clinical manifestations of these patients varied from monosymptomatic disorder such as infertility or bronchiectasis to classic CF with meconium ileus, failure to thrive, pancreatic insufficiency and chronic obstructive pneumopathy. In exception of one patient with no detectable mutation, all patients presented with only one mutation detectable by our SSCP/HD screening method (7) with a sensitivity of 9798%. Normal controls In each experiment DNA from at least one healthy control individual was included. Altogether, we used DNA samples from five different control individuals. Clinical data of patients with newly detected large deletions In order to discuss possible genotypephenotype correlations and more importantly criteria for the implementation of a large deletion screening, we collected detailed clinical data from five different hospitals in Switzerland including age at diagnosis, results of sweat tests, course of disease in brief, complications, actual therapy and family history.
Methods DNA extraction

Deletion screening in patients with CF with known large deletions The reliability of the CFTR MLPA Kit was confirmed by testing nine DNA samples from

DNA was extracted from peripheral blood lymphocytes using either the Phenol method or

Table 1. Overview of individuals screened using the CFTR MLPA Kit (MRC-Holland) Origin of sample Division of Human Genetics, Bern Patient group: 50 patients, ,40 years Description of patients/characterisation of deletion 49 patients with only one detected mutation in the CFTR gene and typical or CF-like clinical signs. In 5 out of these 49 patients only a T5/TG11 (33) or T5/TG12 (23) allele in intron 8 could be detected. One patient with no detected mutation. CFTRdele23 Five healthy individuals CFTRindel4 (c.2745#938_489 1 2#011del8#165ins41) CFTRindel46a (c.273 1 7#983_743 1 362del18#654insACCTCG) CFTRindel1116 (c.1#584 1 10T.C; c.1584 1 12_2#988 1 403del47.5kbins35bp; homozygous) CFTRdele2223 (c.3#86478_4#242 1 577del1532) CFTRdele210 (c.53 1 4#533_1#5857#322del95#738) CFTRdele410 (c.27410#538_1#393 1 277del39#572) CFTRindel1617b (c.2#909636_3#3681#611del6#965ins32) CFTRindel2224 (c.3#9643#8904#413 1 3143del9#454ins5)

Mutation control individual Normal controls Ferec et al. (9) (Brest, France) four mutation controls

Chevalier-Porst et al. (1) (Lyon, France) four mutation controls

CF the life-shortening autosomal recessive disorder

the Qiagen Blood Maxi Kit (Qiagen, Basel, Switzerland) according to the suppliers protocol. Concentrations were measured by Spectrophotometry (Eppendorf BioPhotometer, Switzerland OD 260), and samples were diluted to a final concentration of 25 ng/ml.
Deletion screening with the CFTR MLPA Kit

(P091) does not detect the CFTRindel1 (c.4_53 1 69del119ins299), we accomplished the screening of exon 1 by the use of our real-time PCR assay. We screened 35 patients younger than 30 years for this large deletion.
Confirmation of large deletions by classic PCR using junction fragment primers

Analyses were performed according to the manufacturers recommendations (MRC-Holland). In addition to the patients samples, we included in all experiments the same mutation control (CFTRdele23) and at least one control from a healthy individual. Fragments were separated on an ABI 3100 Capillary Sequencer (Applied Biosystems, Rotkreuz, Switzerland) using Genescan-ROX 500 size standards and analyzed using GENEMAPPER (Applied Biosystems) version 3.5 software. Dosage quotients of potentially deleted exons were calculated by two different methods recommended by MRC-Holland and leedsdna.info, e.g. the so-called population analysis and the calculation using internal control probes.
Confirmation of deletions detected by MLPA and deletion screening of exon 1 by relative quantitative real-time PCR

Breakpoints of all large deletions detected in our screening were confirmed by direct sequencing (ABI 3100, Applied Biosystems) of allele-specific PCR products obtained from amplification with known or newly established junction fragment primers (Table 2).
Identification of new deletion breakpoints

To identify the deletion breakpoints of CFTRindel2 and CFTRdele29, we used two different approaches. Whereas in case of CFTRindel2 the so-called primer walking method (Fig. 1) was used, we performed a quantitative real-time PCR assay in case of the CFTRdele29. Identification of the deletion breakpoints by quantitative real-time PCR is illustrated in Fig. 2.

Results

All large deletions found by MLPA were confirmed by our real-time polymerase chain reaction (PCR) assay (8), which allows the exact copy number detection of all 27 exons of the CFTR gene. Because the CFTR MLPA Kit
Table 2. Primer sequences for the junction fragments Deletion type CFTRindel2 c.545813_164 1 2188del8112ins186 (maximal length of insertion) c.545811_164 1 2186del8108ins182 (minimal length of insertion) CFTRdele29 c.53 1 9713_1392 1 2669del61633 CFTRdele14b17b c.2752674_3499 1 198del9858 CFTRdele17a17b c.3121977_3499 1 248del2515
a

In the first step, we tested the CFTR MLPA Kit with DNA samples from patients with known large deletions provided by two French research groups. Overall, we confirmed nine different deletion types representing 22 of 27 exons.

Primer sequences Amplification of a 1013 bp long junction fragment: I01aU(1.41U): ATT AGG GTG CCT GTA TGG TTG I03aL(1.2L): GAG CCA GAT AGG TTA ATG TTC TTC Primer sequences for an allele-specific 211 bp long PCR product: I01&I03U: AAT TAG TAA CAA AGG AAT GAA AGA I02&I03L: CCA ATT AAA ACT TAG ATA TGG GA I01U: CAG GAA TAA GTT GTA ATA TGG GA I09L: CAT CCA GTG ATG CAA AAT TC I14aU: ATT TGT TCC TTA GCC AAT GTa I17bL: GGA ATC TAT TAA TTT TGA AGC Ta I16U: CTG ATA ATG GTC CCT TTC AGb I17bL: AAA CTT ACC GAC AAG AGG Ab

Chevalier-Porst et al. (1). (T. Doerk). For CFTRdele2 the breakpoints were identified by amplifying an 8939 bp (wild-type allele), respectively, a 1013 bp (allele with deletion) large junction fragment.

Schneider et al.
Fig. 1. Identification of deletion breakpoints (5#-DB and 3#-DB, respectively) using the primer walking method. Briefly, in case of the primer walking method one primer, e.g. the 3#-downstream primer (1.1R), remains the same while the other, e.g. the 5#-upstream primer, is shifted (walking) in steps of 0.15 kb towards it. In the second step, the closest forward primer (1.4F) remains the same, and the reverse primer is shifted in 5# direction. This procedure is repeated until the resulting polymerase chain reaction (PCR) product (c1d) is reduced to a size of 8001000 bp. Sequencing of the PCR product permits the identification of the deletion breakpoints.

In the next step, we screened 50 persons for large deletions using the CFTR MLPA Kit. We detected eight CF alleles with four different deletion types in eight patients (16%). Whereas, CFTRdele14b17b and CFTRdele17a17b were detected in three patients with CF each, CFTRindel2 and the novel CFTRdele29 (c.53 1 9713_1392 1 2669del61633) both were present in only one patient. In order to determine the breakpoints and the size of the deletion found in P1, we used the primer walking method presented in Fig. 1. Our findings showed that P1 bears the same large deletion as described by Ferec et al. (9). Finally, deletion breakpoints of the novel deletion CFTRdele29 were identified by narrowing quantitative real-time PCR (Fig. 2). Applying this procedure allowed sequencing of a 1.2 kb junction fragment (see Table 2 for primer sequences), and the identification of the breakpoints defining the deletion as to be non-complex spanning 61633 bp and causing a reading frameshift.

In our Swiss CF population, including 574 patients with CF with two identified mutations, large deletions were found in 1.0% (11/1148) of all CFTR chromosomes. Allele frequencies of the respective deletion types were 0.52% for CFTRdele14b17b, 0.26% for CFTRdele17a17b, and each 0.09% for CFTRdele29 and CFTRindel2. With the exception of patients 1 and 7, we additionally performed specific mutation analysis in the parents to determine the parentage of the deletions. No additional deletions were detected by the specific screening of exon 1 in 35 patients below age 35 using our quantitative real-time PCR assay. Table 3 summarizes both molecular genetic findings and clinical hallmarks of the eight patients carrying large deletions in one copy of the CFTR gene.
Discussion

One of the principal aims of this study was the contribution to the detection of unidentified

Fig. 2. Identification of deletion breakpoints using a relative quantitative polymerase chain reaction approach. Determination of the dosage quotient of an amplification product positioned in the middle of a gene segment (a), e.g. an intron, allows fast narrowing of the region of interest. In each step (n) the range for the localization of a deletion breakpoint can be halved. It is sufficient to narrow (x 300600 bp) one of the deletion breakpoints because in the junction fragment deletion breakpoints are juxtaposed.

Table 3. Clinical data of the eight patients (P1P8)a

Patient 2 19.02.1984 Patient 3 08.08.1999 Patient 4 11.02.1999 Patient 5 07.07.1994 Patient 6 08.10.2003 Patient 7 25.10.2001 Patient 8 11.12.1984

Patient 1 04.10.1990

Genotype [3] c.152561 A.G; c.1540 A.G; L997F 3 days Meconium ileus 4 months Persistent thriving disorder and recurrent infections of the upper and lower airways since birth 9 months Recurrent infections of the upper and lower airways since birth with an increased frequency after 5 months. At the same time persistent weight (below P3) and height (P3) although normal intake of food [3] c.1001 1 11 C.T; c.152561 A.G; c.1540 A.G [2] c.152561 A.G; c.1540 A.G [3] c.1001 1 11 C.T; c.152561 A.G; c.1540 A.G

R553X (E11)/ CFTRindel2

G542X (E11)/ CFTRdele29

DF508 (E10)/ CFTRdele14b17b

DF508 (E10)/ CFTRdele14b17b

P5L (E1)/ CFTRdele14b17b

DF508 (E10)/ CFTRdele17a17b

R553X (E11)/ CFTRdele17a-17b

DF508 (E10)/ CFTRdele17a17b

Polymorphisms [number of polymorphisms]

[1] c.1540 G.G

Age at diagnosis 2 months Clinics at diagnosis Obstruction of the distal bowel at the age of 2 months with resection of 15 cm from the ileum. Weight and height were normal (both P90). X-ray of the chest showed signs of mild pulmonary hyperinflation

Sweat testsb (age) Osmolality 246 mosmol/kg Pulmonary situation: recent data obtained from lung function tests revealed only mild lung disease Cl2 97; Cl2 113 mmol/l Pulmonary situation: detection of P. aeruginosa and Staphylococcus aureus in pharyngeal swabs already at the age of 1 year. Increasing hyperinflation of the lungs with persistent problems of secretion and ventilation of the middle and lower lobe of the right lung Cl2 105 mmol/l (14 months) Growth: under regularly adapted and optimized therapy normal thriving (height (P75) and weight P75 P90). Pulmonary situation: rare episodes of prolonged infections of the airways because of bacterial infections that were consequently treated with oral and inhalative antibiotics. Chronic colonization with P. aeruginosa since the age of 18 months. Recent data obtained from lung function tests revealed normal values

Cl2 152 mmol/l

Course of disease in brief

Growth: normal gain in weight and height (P5075 and P5075, respectively). Pulmonary situation: recurrent bacterial infections of the upper and lower airways without longer periods of exacerbation. Improvement after initiation of an inhalation therapy with corticosteroids. Before leaving Switzerland in 5/1999 the girl showed only mild obstructive pneumopathy

[5] c.125 G.C; c.152561 A.G; c.1540 T.A; c.1898 1 152 T.A; c.2694 T.G 7.5 years 2 weeks 6 months Decreased weight Recurrent bacterial Obstruction of the gain at the age of 4 small intestine (no infections of the months. classic meconium upper and lower Simultaneously ileus) with airways during frequent defecation childhood. Normal necessary laparotomy 1 week with voluminous, thriving fatty and stinky after birth. Histology from the stools. Only 2 months later ileum showed neither signs of an episode with acute inflammatory obstructive bronchitis after process nor changes typical for a period of recurrent rhinitis. At M. Hirschsprung the same time stop of weight gain Cl2 73; Cl2 88 Cl2 120; Cl2 111 Osmolality 214 mmol/l mmol/l mosmol/kg Pulmonary Growth: Normal Growth: even without substitution gain of weight and situation: colonization with P. height (P2550, of pancreas aeruginosa in respectively P50 enzymes no pharyngeal swabs 75). Pulmonary thriving disorder. situation: detection already at the age Pulmonary of 9 months. situation: only once of S. aureus and Eradication of Haemophilus detection of P. aeruginosa until influenza in colonization with P. aeruginosa at the pharyngeal swabs age of 5 years successful. at the age of 10 age of 8.5 years. months. In Additionally repeated detection progress, she suffered no severe of S. aureus. No signs of obstructive infection of the airways or restrictive lung disorder

[5] c.125 G.C; c.152561 A.G; c.1540 T.A; c. 1898 1 152 T.A; c.2694 T.G 9 months Persistent rhinitis and cough already at the age of 2 weeks. Recurrent episodes with cough even with no simultaneous infection of the airways. Additionally recurrent problems with defecation (thin and frequent dejections). Stop of weight gain at the age of 7 months Cl2 125; Cl2 142 mmol/l Growth: normal gain of weight and height during early childhood (P310, respectively P310). Pulmonary situation: recent tests of the lung function showed an obstructive ventilation disorder of intermediate degree

CF the life-shortening autosomal recessive disorder

6
Patient 2 19.02.1984 Patient 3 08.08.1999 Patient 4 11.02.1999 Patient 5 07.07.1994 Patient 6 08.10.2003 Patient 7 25.10.2001 Patient 8 11.12.1984 G542X (E11)/ CFTRdele29 1 year 1.5 years no detection of P. aeruginosa since she was 6 years old n.a. 8 months DF508 (E10)/ CFTRdele14b17b DF508 (E10)/ CFTRdele14b17b P5L (E1)/ CFTRdele14b17b DF508 (E10)/ CFTRdele17a17b R553X (E11)/ CFTRdele17a-17b DF508 (E10)/ CFTRdele17a17b n.a. FEV1 89%, Tiffeneau 80%, and RV 147% 1 Intestines: meconium ileus. Pancreas: reduced glucose tolerance. Nose: chronic rhinosinusitis, polypectomy in 1996 Metabolic: several episodes of electrolyte imbalance with hyokalemic, hypochloremic alkalosis, and needing hospital care Nasal polyps 1 1 2 1 Unique detection of P. aeruginosa in the sputum at the age of 5 years with subsequent eradication n.a. FVC 96%, FEV1 FVC 91%, FEV1 94%, and Tiffeneau 94%, and Tiffeneau 105% 90%, normal flowvolume curves 1 Hepatopathy and Nasal polyps FVC 93%, FEV1 55%, Tiffeneau 58%, RV 192%, and MEF 50 18% 1 Nasal polyps Inhalation with salbutamol in sodium cromoglycate twice per day, pancreatinum, omeprazolum, multivitamins, budenoside (nasal) Therapy at the age of 4.5 years: inhalation with salbutamol in 0.9% NaCl twice per day, physiotherapy, substitution with pancreatinum and multivitamins Inhalation with salbutamol twice per day, pancreatinum, multivitamins. Additionally, professional physiotherapy every 2 week Inhalation with salbutamol and ipratropium in 0.9% NaCl, bromide twice per day. The infection with P. aeruginosa. was treated with tobramycin inhalations and ciprofloxacin p.o. Pancreatinum, inhalation with salbutamol in sodium cromoglycate, respiratory physical therapy, multivitamins Inhalation twice per day with salbutamol, pancreatinum, multivitamins, calcium and magnesium Twice per day inhalation with salbutamol/ ipratropium bromide and newly also with budenoside per day, n-acetylcysteine, pancreatinum, multivitamins

Table 3. Continued

Schneider et al.

Patient 1 04.10.1990

Genotype

R553X (E11)/ CFTRindel2

Age at first colonization with Pseudomonas aeruginosa

8.5 years

Lung functionc

Pancreatic insufficiency Additional complications

At the age of 9 years: FVC 99.4%, FEV1 74.4%, Tiffeneau 75.7%, and RV 183% 1

Actual therapyd

Intestines: obstruction of the distal intestines at the age of 3 3=4 years. Hepatopathy: signs of a discrete hepatopathy with slightly elevated liver enzymes already in early childhood. Temporary treatment with Ursofalk Therapy at the age of 9 years: inhalation with budenoside and salbutamol twice per day, nacetylcysteine, pancreatinum, cisapride, ursodeoxycholic acid and mutlivitamins

Table 3. Continued
Patient 2 19.02.1984 Patient 3 08.08.1999 Patient 4 11.02.1999 Patient 5 07.07.1994 Patient 6 08.10.2003 Patient 7 25.10.2001 Patient 8 11.12.1984

Patient 1 04.10.1990

Genotype Both parents from Italy No cases of CF in the family/parents are both Swiss Father with recurrent rhinitis, otherwise inconspicuous/ parents are both Swiss No cases of CF in the family. Father with recurrent rhinitis. On the mothers side several cases of autoimmune diseases (SLE and MS). The boys sister is 2 years older, carries the same two mutations but shows a different clinical course (recurrent rhinitis and abdominal pain attacks of unknown origin)/parents are both Swiss No cases of CF in the family. There is a cousin with possible failure to thrive/parents are both Swiss

R553X (E11)/ CFTRindel2

G542X (E11)/ CFTRdele29

DF508 (E10)/ CFTRdele14b17b

DF508 (E10)/ CFTRdele14b17b

P5L (E1)/ CFTRdele14b17b

DF508 (E10)/ CFTRdele17a17b

R553X (E11)/ CFTRdele17a-17b No cases of CF in the family. Mother with allergic eczema during childhood, sister with allergy against cow milk/parents are both Swiss

DF508 (E10)/ CFTRdele17a17b No cases of CF in the family. A grandfather (ms) suffered from coeliacia, an uncle (ms) suffered from allergic rhinoconjunctivitis/ parents are both Swiss

Family history/ nationality

n.a./both parents originate from Italy

CF, cystic fibrosis; FVC, forced vital capacity; MEF, mean expiratory flow; MS, multiple sclerosis; n.a., data not available; RV, residual volume; SLE, systemic lupus erythematosus. a The grey highlights represent maternal allele. b Normal and pathologic values for Cl2 according to National Committee on Clinical Laboratory Standards guidelines (,40 mmol/l, normal; 4060 mmol/l, borderline; and .60 mmol/l, pathologic). Normal and pathologic values for osmolality: ,180 mosmol/kg, normal; 180200 mosmol/kg, borderline; and .200 mosmol/kg, pathologic. c Most recent lung function if not described further. d Most recent therapy if not described further.

CF the life-shortening autosomal recessive disorder

Schneider et al.

CFTR mutations. For this reason, we screened 50 unrelated patients with only one identified mutation for large deletions in the CFTR gene using the CFTR MLPA Kit. The identification of large deletions in eight patients (16%) corresponds to the detection rates of previous studies. Actually, the calculated allele frequency with a value of 1.0% was even lower compared with the studies from Niel et al. (3) (1.3%) and Bombieri et al. (4) (1.33%), respectively.

deletion CFTRdele17a17b. Of course, we are aware of the fact that the observed genotype phenotype associations have to be confirmed by other research groups and/or other methods (such as CFTR transcript analysis) because of the limited number of patients with CF carrying these types of large deletions. Additionally, it may be that our findings reflect more the status of differently expressed modifier genes than being the result of a specific CFTR genotype.

Possible genotypephenotype associations

Another goal of our study was the examination of possible genotypephenotype associations (Table 3). As expected, all of our patients with CF and with large deletions showed pathologic sweat test values, early (,1 year) diagnosis (except for patient 5), and both pulmonary and pancreatic (except for patient 5) disease manifestations. Among the eight patients with CF with large heterozygous deletions, there are two pairs with the same genotypic constellation, namely F508del/dele14b17b (P3/4) and F508del/ dele17a17b (P6/8), respectively. Both CFTRdele14b17b and CFTRdele17a 17b are out of frame mutations but differ in size of the deletion (9868 bp and 2515 bp, respectively). Whereas the two patients with the larger frameshift deletion of exons 14b to 17b were colonized with Pseudomonas aeruginosa already in the first 2 years (P3: 1 year; P4: 1.5 years) patients P6 and P8 carrying the smaller frameshift deletion of exons 17a and 17b have not been colonized with P. aeruginosa so far. Hepatobiliary disease (treatment with ursodeoxycholic acid) appeared only in patient 1 and 7 presenting with the nonsense mutation R553X on the non-deleted allele. Finally, the presence of the non-synonymous base substitution L997F and the dele29 on the same CFTR chromosome of P2 points out that not the missense mutation L997F acts as the disease causing mutation but in fact the deletion spanning exons 2 to 9. These findings are in accordance with a recently published case (10) reporting that L997 is rather a polymorphism than a pathogenic mutation. In summary, the clinical data from our deletion patients show that all patients carrying large deletions have typical clinical manifestations of CF (except for the P5 with the mild P5L mutation) and are diagnosed at an early age. Furthermore, patients with the large deletion CFTRdele14b 17b seem to show a more severe pulmonary disease compared with patients with the shorter
8

Suggestions for indications for CFTR deletion/ duplication screenings

Based on the screening results, we were able to establish the following criteria indicating a screening for large heterozygous deletions: Patients with only one identified mutation or with two mutations whereof at least one is very mild, presenting with the following: (1) Classic or CF-like clinical manifestations, and who are aged 30 years: We did not detect any large deletion in patients older than 30 years (9/50). In conclusion, it is not reasonable to search for large deletions in patients older than 30 years, except for patients presenting with congenital bilateral absence of the vas deferens (CBAVD) and only one detected (mild) point mutation. (2) A positive (or borderline) sweat test: All patients in whom we found large deletions had at least one positive sweat test. Of the remaining 42 patients only 10 had either a positive (5) or borderline sweat test (5). (3) Fewer than six polymorphisms: In our study patients with a positive or borderline sweat test, but without large deletions, carry in average more polymorphisms (5.5) than patients with large deletions (2.75). Taking these three criteria into account allows a straightforward screening strategy and significantly increases the detection rate of large deletions. In our study, the exclusion of all patients not fitting these three criteria would have increased the detection rate to 19.5% for (1), 44.4% for (2) or 20.5% for (3) alone. Considering all three criteria would have increased the detection rate to 61.5%. In conclusion, our study shows that deletion screening should be integrated in the mutation analysis strategy for patients with typical CF, especially for those patients with only one detectable mutation after extensive screening of the whole CFTR gene. Although an allele frequency

CF the life-shortening autosomal recessive disorder

of 1% for large deletions seems rather low, it must be kept in mind that .99% of all known point mutations have allele frequencies that are below 0.1%. Our criteria are mainly considered as help for genetic laboratories who intend to perform deletion screenings in large patient groups and need to make a pre-selection of the patients. In our experience, MLPA is the method of choice for the search of deletions and duplications in large patient cohorts because it is very cost-efficient and reliable at the same time.
Acknowledgements
We thank Professor C Ferec (Genetique Moleculaire et Genetique Epidemiologique, Brest, France) for providing samples from patients carrying large deletions of the CFTR gene.

References
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4. Bombieri C, Bonizzato A, Castellani C, Assael BM, Pignatti PF. Frequency of large CFTR gene rearrangements in Italian CF patients. Eur J Hum Genet 2005: 13: 687689. 5. Hantash FM, Redman JB, Starn K et al. Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening. Hum Genet 2006: 119: 126136. 6. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002: 30: e57. 7. Liechti-Gallati S, Schneider V, Neeser D, Kraemer R. Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease. Eur J Hum Genet 1999: 7: 590598. 8. Schneider M, Joncourt F, Sanz J, von Kanel T, Gallati S. Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler. Clin Chem 2006: 52: 20052012. 9. Ferec C, Casals T, Chuzhanova N et al. Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms. Eur J Hum Genet 2006: 14: 567576. 10. Derichs N, Schuster A, Grund I et al. Homozygosity for L997F in a child with normal clinical and chloride secretory phenotype provides evidence that this cystic fibrosis transmembrane conductance regulator mutation does not cause cystic fibrosis. Clin Genet 2005: 67: 529531.