RESEARCH PAPER

First report of cystic fibrosis mutations in Libyan cystic fibrosis patients

Sondess Hadj Fredj1, Slaheddine Fattoum1, Abdelraouf Chabchoub2 & Taieb Messaoud1

1Laboratoire de Biochimie et de Biologie Moleculaire, Hopital d’Enfants de Tunis, Bab Saadoun 1029, Tunis, Tunisie, and2Clinique Alyssa, Berge du Lac 1053, Tunis, Tunisie

Background: There are few data on the molecular basis of Cystic

Fibrosis (CF) in North Africa, probably due to under-diagnosis.

Aim: This is the first study of cystic fibrosis transmembrane

conductance regulator (CFTR) mutations in the Libyan

population.

Subjects and methods: This study analysed the complete coding

region and flanking intronic sequences of the CFTR gene in 10

unrelated Libyan CF patients.

Results: This study identified four mutations (F508del,

c.1670delC, N1303K and E1104X), with a high frequency of the

latter.

Conclusion: Identification of CF mutations facilitates molecular

investigation of cystic fibrosis in the Libyan population and

helps to provide effective genetic counselling among CF

families.

Keywords: Cystic fibrosis, CFTR mutations, Libyan patients

INTRODUCTION

Cystic fibrosis is one of the most common severe autosomalrecessive disorders in European and European-derivedpopulations; one child in every 2500 births is affected(Claustres 1998). The gene responsible for this diseaseencodes a 1480 amino-acid protein called the cystic fibrosistransmembrane conductance regulator (CFTR) (Kerem et al.1989; Riordan et al. 1989) that acts as a cAMP-regulatedchloride channel (Chen and Hwang 2008; Riordan 2008).

Currently, over 1700 CFTRmutations have been reportedto the cystic fibrosis genetic analysis consortium (http:www.genet.sickkids.on.ca/cftr), most of which occur rarely;however, the F508del mutation is the most common CFallele worldwide; its average frequency has been estimated as66% in Caucasian CF chromosomes ranging from 87% inDenmark to 26% in Algeria (Estivill et al. 1997). Thefrequencies and types of mutations found in populationsvary according to the geographic and ethnic origin of thestudied population. Therefore, a study of the mutant allelesin a population and their relative frequencies are essential to

determine accurate risk estimation and to improve the valueof genetic counselling.

To date, there is little information about CF in manypopulations of African origin where the frequency and theincidence of the disease remain unknown, including in Libyawhere the type of mutations and prevalence of CF have beenunidentified up to now. A preliminary study has shown thatprevalence of CF in Morocco ranges from 1/1680 to 1/4150(Ratbi et al. 2008).

Determination of CF frequency in African countriespermits the evaluation of the importance of CF as a medicalproblem and the identification of the molecular basis of thedisease allows the development of an appropriate assay forthe detection of CF patients and carriers.

The objective of this study was to characterize the CFTRmutations and genotypes in Libyan CF patients referred tous for investigation.

PATIENTS AND METHODS

PatientsTen unrelated Libyan families who had a child aged between 1month and7 years affectedbyCFwere referred to the children’sHospital in Tunis. Consanguinity was noted in seven of the 10studied families. Diagnosis of CF was based on abnormallyelevated sweat chloride concentrations (.60mmol/l) andclinical symptoms typical for cystic fibrosis: pneumoniarecidivans and/or gastro intestinal disease.

MethodsDNA was isolated from blood cells by salt precipitationmethods (Miller et al. 1988). DNA samples were firstscreened for the F508del mutation by a heteroduplex assayin 10% PAGE (Polyacrylamide Gel Electrophoresis).Samples were further analysed by DGGE (denaturinggradient gel electrophoresis) and/or DHPLC (denaturinghigh pressure liquid phase chromatography) of the entirecoding sequence and flanking intronic sequences. Exons 5,

Correspondence: Taieb Messaoud, Laboratoire de Biochimie et de Biologie Moleculaire, Hopital d’Enfants de Tunis, Bab Saadoun, Tunisie.

Tel: 216 71 562 521. 216 98 508 655. Fax: 216 71 566 463. E-mail: taieb.messaoud@rns.tn

(Received 23 October 2010; accepted 17 January 2011)

Annals of Human Biology, September–October 2011; 38(5): 561–563Copyright q Informa UK, Ltd.ISSN 0301-4460 print/ISSN 1464-5033 onlineDOI: 10.3109/03014460.2011.557090

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17b, 14b and 20 were studied by DGGE. The remainingexons were analysed by DHPLC. Amplification of all 27CFTR exons, including flanking intronic regions, wasperformed using CFTR gene-specific primers, which arecommonly used in studies for CFTR analysis (Fanen et al.1992; Le Marechal et al. 2001).

Samples showing abnormal profiles by DGGE and/orDHPLC were re-amplified from genomic DNA. PCRproducts were purified with the kit Wizard SV Gel andPCR clean up system (Promega, Madison, WI) and weresequenced in both directions using the Big Dye TerminatorCycle Sequencing Reaction Kit (PE Applied Biosystems,Foster City, California, USA). Each single stranded productwas concentrated using a centri-sep column (AppliedBiosystems, Foster City, California, USA).

Mutation nomenclatureNucleotide numbers are derived from cDNA CFTRsequences (GenBank accession no. NM_000492). Mutationsare named according to the numbering used in the CFTRMutation Database (http://www.genet.sickkids.on.ca/cftr/).

RESULTS AND DISCUSSION

CF patients were first screened for the most frequentmutation F508del; this mutation was found in sixchromosomes of the 20 tested CF chromosomes. For thedetection of unknown sequence variants, the combinationof DGGE and DHPLC allowed the identification of threemutations: E1104X, c.1670delC and N1303K, all confirmedby sequencing reaction (Table I).

E1104X, first described in a French-Canadian CFchromosome (Zielenski et al. 1995), was found to be themost common mutation with 40% frequency (Table II);

it was found in four patients in the homozygous state.The clinical phenotypes of these patients are documented:three are pancreatic insufficient and have severe pulmonarydisease suggesting that E1104X is a severe mutation. Theseresults are in concordance with phenotypes described in theTunisian population (Hadj Fredj et al. 2009).

The relatively high incidence of the E1104X mutation inthe Libyan population makes this mutation a candidatemutation for neighbouring populations in the North Africanarea. Indeed, thismutation has been identified inTunisiawitha mean frequency of 16.17%. The high frequency of E1104Xmutation observed in Tunisian and Libyan populations canbe explained by the very commonmigration between the twocountries and the high degree of consanguinity present inthese populations. Also, they may share a common ancestry.The study of haplotypes of this mutation would be useful toprovide further information about its origin and tohelp elucidate the different evolutionary mechanisms(founder effect and heterozygote advantage).

The mutation c.1670delC, originally identified in aFrench CF patient (http:www.genet.sickkids.on.ca/cftr), wasfound in a male patient with meconium ileus. The remainingfully characterized patient carried an heterozygous com-pound genotype: F508del/N1303K. He had severe pulmon-ary disease and a high sweat chloride test (113mmol/l).

Seven patients were homozygous for a CF mutation.Homozygoty was confirmed by the study of both parentsand can be explained by the high consanguinity usuallyobserved in the Libyan population.

Despite a complete scanning of all 27 exons includingflanking intronic regions, three alleles remained uncharacter-ized; these unidentified CF mutations may be localized inintrons or in regulatory regions that are not routinelyinvestigated; also they may correspond to gene rearrange-ments such as large deletions at the heterozygous state,undetected by current PCR-based techniques (Des Georgeset al. 2008).

Some CF mutations, found at relatively high frequenciesin other Maghreb countries (particularly, 711 þ 1G ! T,G542X and W1282X) (Loumi et al. 2008; Hadj Fredj et al.2009) were not found in the present study. The absenceof these mutations could be due to the small size of thesample.

This preliminary study shows the CF mutations in Libyanpopulation. It may be completed by large-scale studies toidentify the clinical pattern and determine the incidence andmolecular basis of CF in Libya.

CONCLUSION

The molecular basis of CF in Arab populations stillremains largely undocumented. Determination of thecommon CFTR mutations in a specific population helpsto confirm the diagnosis of cystic fibrosis, allowing geneticcounselling of CF families, and may yield information onhow different mutations in the CFTR gene are related to theseverity of the disease.

Table II. Frequencies of CFTR mutations identified in 10 unrelatedLibyan CF patients.

Mutations Consequence Localization Frequency (%)

E1104X Glu(Stop 1104 Exon 17b 40%F508del Del Phe507/508 Exon 10 30%c.1670delC Premature stop codon Exon 11 10%N1303K Asn(Lys 1303 Exon 21 5%Unknown – – 15%

Table I. Genotype of 10 Libyan CF patients.

PatientAge atdiagnosis Sex

Sweat test(mmol/l) Genotype

1 6 m M 114 F508del/F508del2 2 m F 96 F508del/F508del3 4 m M 85 E1104X/E1104X4 9 m F 100 E1104X/E1104X5 6 m M 87 E1104X/E1104X6 3 m M 80 E1104X/E1104X7 1 m F 92 c.1670delC/c.1670delC8 2 m M 113 F508del/N1303K9 1 y M 62 F508del/unknown10 3 m M 70 Unknown/unknown

y, years; m, months; F, female; M, male.

562 S. HADJ FREDJ ET AL.

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Declaration of interest: This work was funded by theMinistry of Higher Education, Scientific Research andTechnology in Tunisia. The authors report no conflicts ofinterest. The authors alone are responsible for the contentand writing of the paper.

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