CFTR mutations in the Algerian population

Journal of Cystic Fibrosis 7 (2008) 54 – 59 www.elsevier.com/locate/jcf

CFTR mutations in the Algerian population O. Loumi a,⁎, C. Ferec b,⁎, B. Mercier b , J. Creff b , B. Fercot b , R. Denine c , J.P. Grangaud d a

Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, Bab-Ezzouar Alger, Algérie b INSERM U613, Laboratoire de Génétique Moléculaire, 46 rue Félix Le Dantec – 29200 Brest, France c Hôpital ISSAD HASANI Beni-messous, Laboratoire de Biochimie, Algérie d Faculté de Médecine, Université d’Alger, Algérie Received 22 March 2006; received in revised form 19 April 2007; accepted 24 April 2007 Available online 14 June 2007

Abstract The nature and frequency of the major CFTR mutations in the North African population remain unclear, although a small number of CFTR mutation detection studies have been done in Algeria and Tunisia, showing largely European mutations such as F508del, G542X and N1303K, albeit at different frequencies, which presumably emerged via population admixture with Caucasians. Some unique mutations were identified in these populations. This is the first study that includes a genetic and clinical evaluation of CF patients living in Algeria. In order to offer an effective diagnostic service and to make accurate risk estimates, we decided to identify the CFTR mutations in 81 Algerian patients. We carried out D-HPLC, chemical-clamp denaturing gradient gel electrophoresis, multiplex amplification analysis of the CFTR gene and automated direct DNA sequencing. We identified 15 different mutations which account for 58.5% of the CF chromosomes. We used a quantitative PCR technique (quantitative multiplex PCR short fragment fluorescence analysis) to screen for deletion/duplication in the 27 exons of the gene. Taking advantage of the homogeneity of the sample, we report clinical features of homozygous CF patients. As CFTR mutations have been detected in males with infertility, 46 unrelated Algerian individuals with obstructive azoospermia were also investigated. © 2007 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Denaturing gradient gel electrophoresis; D-HPLC; Luminex™; ARMS™; QMPSF; Mutations; Polymorphisms; Algeria

1. Introduction Since the cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1,2], which is responsible for cystic fibrosis (CF), more than 1300 CF mutations have been reported by the members of the Cystic Fibrosis Genetic Analysis Consortium. There are marked variations in the frequency of some non-F508del CF mutations with respect to geographical location and ethnic group. In ⁎ Corresponding authors. O. Loumi is to be contacted at Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, Bab-Ezzouar Alger, Algérie. C. Ferec, INSERM U613, Laboratoire de Génétique Moléculaire, 46 rue Félix Le Dantec–29200 Brest, France. E-mail addresses: [email protected] (O. Loumi), [email protected] (C. Ferec).

Algeria, no information is available about the incidence of CF and, in general, there are few data on the molecular basis of CF in the Maghrib, probably due to underdiagnosis [3–5]. F508del is found on only 20% of the CF chromosomes in Algeria [6] and Tunisia. Up to now, about 30 other mutations have been reported, most of which were previously described in other populations. Still, 3 mutations may be specific to the Algerian [7–9] population (A141D, L227R, and N1303H) and 2 to the Tunisian population (T665S and 2766del8). In order to carry out an effective diagnostic service, we have undertaken a complete screening of the 27 exons of the CFTR gene in Algerian CF chromosomes by D-HPLC, denaturing gradient gel electrophoresis (DGGE) and QMPSF analysis. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters is presented.

1569-1993/$ - see front matter © 2007 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2007.04.004

O. Loumi et al. / Journal of Cystic Fibrosis 7 (2008) 54–59

2. Materials and methods 2.1. Sample composition A total of 81 related and unrelated children from Algeria presenting a reminiscent clinic picture of cystic fibrosis were investigated. The patients were referred to us by different paediatric units of Algeria. Cystic fibrosis diagnosis was based on sweat tests: normal values are b 40 mM; positive values are N 60 mM. Borderline values are between 40 and 60 mM. The results were considered as being discordant when repeated sweat chloride analyses yielded inconsistent results. Sweat tests carried out at the ISSAD HASSANI hospital of BeniMessous (Algiers) gave the following results: 36 patients showed a positive sweat test (with clinical pulmonary and/or gastrointestinal findings), 19 patients showed a discordant sweat test and 26 patients a normal sweat test affected by dilatation of bronchi and chronic obstructive pulmonary and digestive disease. Forty-six unrelated azoospermia men with normal FSH values and normal testicular volume were referred to us by the endocrinology unit of Mustapha Hospital (Algiers). We had no more clinical information.

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(QMPSF) to resolve a significant fraction of previously uncharacterized CFTR alleles. Mutation detection and characterization were performed as previously described [15–19]. 2.2.2. Automatic DNA sequencing Each electrophoretically altered fragment detected by DGGE, D-HPLC was reamplified from genomic DNA. PCR products were purified on Microcon 100 columns (Millipore) and sequenced by Sanger dideoxy-mediated chain termination, using the ABI PRISM™ BigDye™ Terminator Cycle Sequencing Kit (PE Applied Biosystems) using walking primers. The sequences of rearranged PCR products were compared with the CFTR reference genomic sequence (GenBank Accession Number NT_007933.13 REGION: 42296307y42485805). 3. Results Initially, the 3-bp deletion that results in deletion of a phenylalanine residue at position 508 of the CFTR protein (F508del) was detected on a non-denaturing 12% polyacrylamide gel.

2.2. Mutation analysis 3.1. Classic CF (N = 36) Blood samples were collected from the affected children, their parents and azoospermia men. Genomic DNA was extracted from peripheral blood leucocytes by standard methods. All DNA samples were first screened for the F508del mutation as previously described [10]. The samples of 36 children, without identified CF mutations were analysed by D-HPLC, DGGE of the entire coding sequence and flanking intronic sequences as described elsewhere [11,12,8]. For D-HPLC analysis, we chose a Touchdown PCR protocol as already described [14] and by OLA assay [13]. We used a quantitative PCR technique (quantitative multiplex PCR short fragment fluorescence analysis) to screen for deletion/duplication in the 27 exons of the gene [15–19]. In addition to F508del, 30 mutations were screened in the 19 children with borderline sweat test and in the 26 children with normal sweat test, by means of the amplification refractory mutation system (ARMS™) using a commercial kit allowing the detection of 30 mutations in the CFTR gene [20] (ELUCIGENE™ CF 30). Genomic DNA from the 46 obstructive azoospermia men was screened for 29 mutations by using Luminex™ technology based on fluorescent detection using a flow cytometer, microbeads dyed with multiple fluorescent colours and laser detection [21,22].We also analysed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein [23]. 2.2.1. QMPSF analysis and molecular characterization of the genomic rearrangements In the present study, we used the technique of quantitative multiplex PCR amplification of short fluorescent fragments

The allele frequency of the F508del mutation observed in the 72 CF chromosomes was 16.7% (12/72) (Table 1). Among the 60 CF chromosomes carrying an unidentified mutated allele, 14 different mutations were identified, as reported in Table 1: N1303K(6/72), 711+1G→T (6/72), V754M(1/72), 1812−1G→A(2/72), 2183AA/G(3/72), [4332delTG−621+3A/G](1/72), G542X(1/72), V562I(2/ 72), 1609delCA(2/72), 4271delC(1/72), W1282X(3/72), S977F(1/72), 21Kbdel( 1/72). Our screening of the entire coding sequence of the CFTR gene identified 9 DNA sequence variations shown in Table 2. A female CF patient of Berber origin was a compound heterozygote V754M/1812−1G→A. She was admitted at the age of 3 years to the Department of Paediatrics (Ain Taya Hospital, East Algeria) for bronchiolitis, a high sweat chloride test (300 mEq/l) and sputum colonized by Pseudomonas aeruginosa. The V754M (G to A at position 2392) mutation has previously been reported to the Cystic Fibrosis Genetic Analysis Consortium by Roger Mountford and seems to confer moderate disease when it is associated either with 1812−1G→A or G542X. Moreover, we have identified two complex mutations: R74W–D1270N in the mother of an Algerian F508del heterozygous CF patient, and none of these variations were inherited by the child. Using intragenic polymorphisms, we have confirmed that R74W and D1270N were not inherited by the F508del heterozygous CF child. This child has inherited the 2183AA/G maternal mutation which, when associated with F508del, confers a CF phenotype [24]. As this has also been reported in Tunisian CF patients [5], and

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Table 1 CFTR mutations detected in 36 Algerian patients (N = 72 CF chromosomes) Mutations

Substitution nucleotide

Substitution amino acid

Localisation

N

%

Cum. fr.

hF508del N1303K 711+1G→T 2183AA/G

del CTT C→G 4041 G→T711+1 del A2184 A→G 2183 delCA G→A 1812−1 G→A 1816 G→A 2392 G→A 3978 A→G 621+3 delTG4332 G→T 1756 del A 4271 C→T 3062 21-kb del C→T 352 G→A 3940

Del phe 507/508 Asn→Lys 1303 MRNA splicing defect Frameshift

Exon 10 Exon 21 Intron 5 Exon 13

12 6 6 3

16.7 8.3 8.3 4.2

16.7 25.0 33.3 37.5

Frameshift mRNA splicing defect Val→Ile 562 Val→Met 754 Trp→Stop 1282 mRNA splicing defect Frameshift Gly→Stop 542 Frameshift Ser→Phe 97 Del AA Arg→Trp 74 Asp→Asn 1270

Exon 10 Intron 11 Exon 12 Exon 13 Exon 20 Intron 4 Exon 23 Exon 11 Exon 23 Exon 16 E2–E3 Exon 3 Exon 20

2 2 2 1 3 1

2.8 2.8 2.8 1.4 4.2 1.4

40.3 43.1 45.9 47.3 51.5 52.9

1 1 1 1 0 0 43

1.4 1.4 1.4 1.4 0 0 58.5

54.3 55.7 57.1 58.5

1609delCA 1812−1G→A V562I V754M W1282X 621+3A/G a 4332delTGa G542X 4271delC S977F 21Kb del R74W D1270N Total

N = number of chromosomes; Cum. fr. = cumulative frequency. a Mutations hawked by the same chromosome 7.

given that the Algerian mother remains presently unaffected, we suggest that this complex mutation should mostly be classified as mild or benign. This compound heterozygote mother had an asymptomatic form of the disease. This was confirmed by two positive sweat tests (85–90 mEq/l). The second complex mutation was found in one CF patient with pulmonary insufficiency. During the screening, abnormal profile D-HPLC of his PCR product was identified in exons 23, 13 and intron 4. The direct sequencing of these regions revealed the following molecular defects: 4332delTG, 2183AA/G and 621+3A→G. In order to establish their respective transmission to the affected child, we analysed only the father’s DNA. As the frameshift mutation 2183AA/G was the only one to be identified in the father’s genotype, we deduced that the other two (4332delTG+621+3A/G) were on the same haplotype and transmitted by his mother. These mutations have been independently reported elsewhere to the Consortium to be CF alleles, disease-causing mutations. Additional clinical data including a sweat test for the mother will be collected in order to exclude a mild or severe CF phenotype. The 4332delTG (deletion of TG at position 4332) mutation does not modify any restriction site. It has previously been reported by Claustres in a CBAVD patient associated with the R117H mutation [25] and seems to confer a CF phenotype when it is associated with 2183AA/G and 621+3A/G mutations in our case. The 1609delCA allele was only found in the Algerian CF patient [3,4], who was homozygous for this mutation. This deletion occurs in the first nucleotide binding fold of the CFTR gene and creates several termination codons; the first one is located ten codons downstream of the CA deletion. All these data indicate that the 1609delCA mutation is most probably a CF disease mutation.

Taking advantage of the homogeneity of the sample, the genotype–phenotype correlation can then be established. In our study, we identified 9 homozygous CF patients: four F508del/F508del, one N1303K/N1303K, two 711+1G→T/ 711+1G→T, one 1609delCA/1609delCA and one W1282X/ W1282X. All these patients had pancreatic insufficiency. The N1303K/N1303K homozygous patient was the 4-year-old daughter of related parents and presented pancreatic insufficiency and serious pulmonary disease with P. aeruginosa colonization. Three positive sweat chloride tests confirmed the CF diagnosis. The F508del homozygote was the son of related parents. He had pancreatic exocrine insufficiency, severe pulmonary disease and a high sweat chloride test. He died at the age of 11 years. The 1609delA homozygote was a girl aged 4.5 years from Miliana (west Algeria). She suffered from chronic cough, respiratory insufficiency and diarrhoea. The child was hospitalised twice at 7 and 13 months for treatment of staphylococcal pneumonia. The diagnosis of CF was confirmed by sweat chloride levels of 140.57 mEq/l. Table 2 Variants detected in 36 Algerian patients (N = 72 chromosomes)

T854T M470V Q1463Q 1001+11C/T L997F 875+40A/G 5T E528E P1290P dup1716+51→61

Variants

Localisation

N

%

T→G 2694 A→G 1540 G→A 4521 C→T 1001+11 G→C3123

Exon 14a Exon 10 Exon 24 Intron 6b exon 17a Intron 6a Intron 8 Exon 10 Exon 20 Intron 10

20 11 9 7 2 2 2 2 1 0

27.7 15.2 12.5 9.7 2.7 2.7 2.7 2.7 1.3 0

G→A 1716 A→G 4002 dup of 11 bp at 1716+51

N = number of chromosomes.

O. Loumi et al. / Journal of Cystic Fibrosis 7 (2008) 54–59

The patient died within 1 year of diagnosis from respiratory insufficiency and cor pulmonale. The 1609delCA mutation was therefore the probable cause of the severe CF symptoms [4]. The duplication of 11 bases [1716+(51→61)] was only found in the father of a compound heterozygote V562I/S997F CF patient. The duplication has previously been found in 3 random Algerian individuals [3]. This duplication does not create a potential splice donor or splice acceptor sequence. All these data indicate that the dup 1716+(51→61) mutation is most likely a polymorphism or a variant. At this time we have no reason to believe that this is a disease mutation.

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Table 4 CFTR mutations and variants detected in 46 Algerian obstructive azoospermia men (N = 92 chromosomes) N

%

Mutations ΔF508 N1303K 711+1G→T

2 1 2

2.17 1.08 2.17

Variants 1001+11G/T T854T 5T

13 18 8

14.13 19.56 8.69

Cum. fr. 2.17 3.25 5.43

N = number of chromosomes; Cum. fr. = cumulative frequency.

3.2. Mild CF-like symptoms Concerning the 26 patients with a normal sweat test (b40 mM), analyzed by the technique of ARMS™ we identified the F508del and 711+1G/T mutations (Table 3). The heterozygous patient for F508del was diagnosed with bronchial dilatation at 9 years of age. His sweat chloride concentration was 39 mEq/l. The heterozygous 711+1G/T child was diagnosed with repeated bronchitis at five months, with a sweat chloride concentration of 35 mEq/l. On the 19 children with discordant sweat test analyzed also by ARMS™ technique, one heterozygous F508del was identified. Only this patient has been analyzed by D-HPLC, which allowed to reveal after automatic sequencing the variant L997F. He was diagnosed at 26 months of age with chronic cough, digestive disease and discordant sweat test (58 mEq/l; 22 mEq/l; 75 mEq/l). Several sequences variants were identified by DGGE assay (Table 3). In reference to the previously suggested classification of the phenotype [26], whereby pancreatic sufficiency is attributable to the presence of at least mild dominant CF allele we show that the 711+1G→T mutation is associated with the pancreatic insufficiency phenotype. The 711+ 1G→T homozygous patient, from Tebessa (west of Algeria), was admitted at the age of 5.5 years to the Department of Paediatrics at the Bologhine Hospital in Algiers because of failure to thrive, bronchiolitis and chronic diarrhoea (3–4 greasy stools/day). Laboratory test indicated Klebsiella pneumoniae in a bronchial sample. Diagnosis of CF was confirmed by sweat chloride levels of 126, 119 and 168 mEq/l.

Table 3 CFTR mutations and variants detected in 19 patients (Na) and 26 patients (Nb) respectively with discordant and normal sweat test Na = 19

%

Mutations ΔF508 711+1G→T

1 0

2.63 0

Variants 1001+11G/T T854T L997F

4 9 1

10.53 23.68 2.63

Nb = 26 1 1

– – –

% 1.92 1.92

– – –

This child died at the age of 6 years from respiratory insufficiency. His younger sister was seen at the age of 3 months for chronic coughing. She had sweat chloride levels of 302 mEq/l and she is homozygous for the 711+1G→T. The mutation was identified by OLA assay [13]. 3.3. Obstructive azoospermia Among the 46 men with obstructive azoospermia that we analysed, we identified 3 CFTR mutations by Luminex™ technology and three variants by DGGE (Table 4). Five patients had one identified CFTR mutation while 41 did not present any mutation. Only the 5 heterozygotes have been analysed by D-HPLC and QMPSF, one of them presented a combination of sequence variation as F508C, M470V and 1525−61A/G, classified as polymorphisms in the CFGAC [27]. 4. Discussion In the classic CF group, after the complete analysis of the CFTR gene by the different mentioned techniques, all the CF mutations (except 21 kb deletions) are located in 9 exons and 3 introns and account for 58.5% of Algerian CF chromosomes. 41.5% of mutations remain to identify after a complete CFTR exon screen. Compared to the Tunisian population, the same informativity of alleles was observed for 5 mutations [5]. The F508del mutation represents 17.9% of CFTR alleles, while it represents 16.6% in our Algerian population [5]. This frequency is lower than that observed for North European populations but is comparable to reported data for Turkish, Jewish and Tunisian populations [5]. The N1303K mutation has already been reported to be significantly more common in Southern European populations than in Northern European populations (Cystic Fibrosis Consortium). The 711+1G→T mutations identified in 3 Algerian CF patients was first described with a high frequency in CF families living in Quebec. To our knowledge, however, no homozygous case has been observed in this population. It has not been reported in many European countries except in some CF patients in France in families of “Arab” extraction, and in Spain. It is the third most common

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mutation in Tunisia (6.9%) [28–31], after F508del (17.9%) and G452X (8.5%). Our data provide additional evidence for a higher frequency of this splicing mutation in the Maghrib. This information is of interest in designing the appropriate strategy for genetic testing of patients in Algeria, and in European countries where immigration from the Maghrib is common. Moreover, 2 additional rare mutations have been identified in an Algerian CF patient. Surprisingly, none of the defined mutations (R553X, G551D, 1717−1G→A, G542X), which occur relatively frequently in exon 11 in Caucasian populations, was identified in our Algerian population except the G542X that has been identified once. This was also described in the Spanish population with a 4.1% frequency. We can consider it as endemic in Mediterranean countries. The previously reported 21-kb deletion, which removes exons 2–3 [32], seems frequent at the Slavic populations. This last has been found however once in one Algerian CF patient (1/72) using QMPSF analysis. The 21-kb deletion seems to confer severe disease when it is associated with the G542X mutation. Current data show that the CFTRdele2,3 (21 kb) mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has sporadically been observed in Canada, USA, France, Spain, and Turkey [32]. The precise phenotypic consequences of the various CFTR mutations are important to establish for the clinical prognosis of the patients and genetic counselling of the families. Since most CFTR mutations are rare, information about their severity depends on the study of a large number of cases and/or the analysis of patients that are homozygous for these mutations. Because of the high rate of consanguinity in North African populations, homozygous forms can be observed even for rare mutations. The course of the disease in the 5.5-year-old child homozygous for the 711+1G→T mutation allows us to assess the clinical features due to this mutation and thus to establish the genotype–phenotype relationship. It suggests that this mutation is a severe allele with regard to lung disease. The greasy diarrhoea further suggests its association with pancreatic insufficiency. This 711+1G→T mutation affects the splice donor consensus sequence in intron 5 [9], resulting in the skipping of exon 5 and the in-frame joining of exon 4 to exon 6a [33,34]. Although the reading frame appeared to be preserved in the transcript, a significant portion of the first membrane-spanning domain would be deleted from the mutant CFTR polypeptide, if made. Concerning the compound heterozygote F508del/L997F, the L997F variant has previously been reported to the CFGAC by Kabra and Castellani [35]. It was observed in a 5-year-old North Indian boy, heterozygous, born to consanguineous parents migrated from Pakistan. He had a borderline high sweat chloride value and features highly suggestive of cystic fibrosis. But in our case the L997F seems

to result in severe disease when associated with F508del. The incidence of CFTR mutations in children with normal and discordant sweat chloride levels is unknown. A few mutations that might be associated with these sweat chloride values have now been described [36]. In the case of patients with the negative sweat test we reported the case of two heterozygote children presenting one of the two mutations 711+1G→T or F508del and showing pulmonary problems or bronchial dilatation. This leads us to think, according to the literature [37], that the presence of one mutation would be a predisposition to a chronic pulmonary illness. Further, it has been reported that the heterozygotes for CFTR mutations would either have some pulmonary problems or be carriers of a dilatation of bronchi. In this case it would be necessary to study parents of CF patients to determine the severity and impact of these chronic pulmonary affections. Interestingly three of the identified variants (M470V, T854T and Q1463Q) are frequently informative in our Algerian families and could be useful for indirect molecular diagnosis of CF. The Luminex method that detects 29 mutations permitted to have 10.87% of obstructive azoospermia men who presented a mutation. For the remaining 89.13%, it would be necessary to complete the analysis by other techniques, because the mutation detection method used here does not have 100% sensitivity, only the 5 heterozygotes benefited from an analysis completed by D-HPLC and QMPSF. 5. Conclusion In conclusion, to our knowledge, this is the first study that includes a genetic and clinical evaluation of Algerian CF patients living in Algeria. The information provided by our study on the distribution profile of mutations in this sample of CF Algerian patients is of interest in designing an appropriate strategy for genetic testing of patients in Algeria and in Europe countries where immigration from the Maghrib is common. The sweat test remains the key test of diagnosis of the cystic fibrosis; however, one must underline that a normal sweat test does not exclude the cystic fibrosis. Acknowledgements We would like to thank all the CF families and their respective physicians for their full collaboration in these investigations. We would like to thank Prof. Hireche (Hospital Parnet), Semrouni M. (hôpital Mustapha), Baghriche M. (Hôpital Bouloghine), Cherif N. (clinique AMP) for providing Algerian families. We wish to thank Creff J. Fercot B and Raguénès O for excellent technical assistance. We thank all those who participated to this study especially Elion J. (Hôpital Robert Debre), Bienvenu T. (Hôpital Cochin), Audrezet M.P. (Hôpital Morvan, Brest). This work was supported by the Algerian Government (Ministère de l’enseignement supérieur et de la recherche scientifique).

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