The variability at three microsatellites in the Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) locus has been studied for frequent mutations encountered in an isolated population of »Grande Brière«, a small region located in Southern Brittany. Fluorescent multiplex PCR of these microsatellites were assayed in 16 Cystic Fibrosis (CF) families carrying 5 different mutations. The four most frequent haplotypes on df508 chromosomes were the same as those found in Northern France and Europe but the distribution of these haplotypes provides new enlightenment on the population origin of this insular community

A. Chaventré

A. David

C. Ferec

G. Picherot

J.P. Moisan

M. De Braekeleer

S. Bezieau

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Coll. Antropol. 24 (2000) 2: 281–286UDC 575.116:616-056.7(441.1/.5)Original scientific paperMicrosatellite HaplotypesAssociations with 5 CFTR Mutationsin »Grande Brière«, an IsolateLocated in Southern BrittanyS. Bezieau1,3, G. Picherot2, A. David1, M. De Braekeleer4, C. Ferec5,J.-P. Moisan1,3, and A. Chaventré41 Service de Génétique Médicale, C.H.U, Nantes, France2 Service de Pédiatrie, Centre Hospitalier, Saint-Nazaire France3 Unité INSERM 463, Nantes, France4 Laboratoire de Démographie et d’Anthropologie Génétiques, UFR Sciences Socialeset Psychologiques, Université de Bordeaux II, Bordeaux, France5 Centre de Biogénétique, C.D.T.S, Brest, FranceA B S T R A C TThe variability at three microsatellites in the Cystic Fibrosis Transmembrane Con-ductance Regulator Gene (CFTR) locus has been studied for frequent mutations encoun-tered in an isolated population of »Grande Brière«, a small region located in SouthernBrittany. Fluorescent multiplex PCR of these microsatellites were assayed in 16 CysticFibrosis (CF) families carrying 5 different mutations. The four most frequent haplo-types on df508 chromosomes were the same as those found in Northern France and Eu-rope but the distribution of these haplotypes provides new enlightenment on the popula-tion origin of this insular community.IntroductionCystic Fibrosis (CF) is one of the mostcommon severe autosomal recessive dis-orders in Caucasoid populations and wasdescribed for the first time by Fanconi in19361. Since the cloning of the CFTR2–4,more than 850 mutations causing CF ha-ve been detected (Cystic Fibrosis Muta-tion Database, www.genet.sickkids.on. ca/cftr/), and a geographical distribution hasbeen proposed5. The most common muta-tion, df508, accounts for approximately70% of CF chromosomes in the popula-tion of north Europe and North America6,and 73% in northwest France7. The aimof this study is to analyse the geneticvariability observed at three microsate-281Received for publication January 24, 1999.llite loci associated with mutations en-countered in a small French region of»Grande Brière«. This region is a marshand is mostly formed by different islandsconnected by dike roads. This marsh wasformed from two separate entities:Several islands around a central vil-lage in the middle, encircled by marsh.On the periphery of the marsh, othervillages separated from the central vil-lage by some kilometers (Figure 1). In-habitants, in spite of the industrial revo-lution have constitued an isolat.The aim of this study was to see iffounder effects exist in this population forthe different mutations and haplotypesencountered.Materials and MethodsGenealogical reconstructionThis reconstruction has been very dif-ficult, specially in »central Grande Bri-ère« formed from different islands (Fed-run, Pendille, Mazin,...) because inbreedingrate and endogamy in these islands arequite high as illustrated by the numerousadministrative authorizations given forconsanguinous mariages in the last cen-turies, or by the fact that 6 family namescover 80% of the population (8). On theother hand, the study proved to be rela-tively easy in the »peripheric Brière«. Wehave looked at the remarkably well pre-served registers of births, marriages, anddeath as well as contemporary docu-ments produced by local scholars (mostlyunpublished). The genealogies of 16 CFpatients were reconstructed over 11 to 16generations representing a period of 350to 400 years. All the data have been pro-cessed using Heresis 4 Pro sofware afterhaving rendered anonymous all thoseconcerned. All individuals in the studycame from families that did not have re-cent common ancestors in the pedigree. Avery low migration rate characterizes thepopulation (mostly immigration), and con-tribute to their relative isolation.Mutation detectionThe CF families have been selected bydiagnosis on the basis of medical records,biochemical criteria and Polymerase ChainReaction (PCR) detection. This last inves-tigation was performed as described9 us-ing Amplification Refractory MutationSystem (ARMS) for df508, G551D, andN1717G  A. Mutations S549N and W57Xhave been performed using Single StrandConformation Polymorphism (SSCP)10.282S. Bezieau et al.: Microsatellite Haplotypes Associations, Coll. Antropol. 24 (2000) 2: 281–286Fig. 1. Representation of two different areaslocated in “Grande Brière”. First islandsand central village, and peripheric villa-ges located around, with specific haploty-pes and mutations distributionAnalysis and mutations were confirmedby sequencing.Microsatellite markersIVS8CA (intron 8), IVS17BTA, andIVS17BCA (intron 17)11 were analysed bymultiplex PCR. Genomic DNA (100 ng)was amplified using 200  M of eachdNTP, 5 pmol of each primer AC8R4,AC8D3, AC17R2, AC17D22, AT17R12,and AT17D12 previously described byMorrall et al.12, 50 mM KCl, 10mM Tris(pH 8.3), 1.5 mM MgCl2, and 2 U of Taqpolymerase in a total volume of 25  l. Un-labelled and 6-FAM (fluorescent compo-nent) labelled oligonucleotides were syn-thetised using an Applied Biosystems381A. The DNA thermal Cycler 480 (Per-kin Elmer Cetus) was programmed tocarry out 30 cycles of: 94 °C for 1 min, 50°C for 2 min, and 74 °C for 3 min. A 5 minincubation step at 94 °C was carried outinitially as well as a final step of 10 minat 74 °C.Amplified products were diluted (1/5in deionised water). 1  l from this dilu-tion was mixed with 2  l deionised forma-mide, 0,5  l loading buffer, and 0,5  l la-belled standards (Genescan 500 TAMRA).Samples were denatured at 94 °C for 4min, then loaded on 6% denaturing poly-acrylamide gel and electrophoresed at1000 V for 3h30 on Applied Biosystems373A DNA sequencer. Allele sizes weredetermined automatically by Genescan672.1 software (Applied Biosystems, Fos-ter City, CA 94404) using the second or-der last square method.ResultsThe distribution of the Cystic Fibrosismutations are summarized in Table 1.Out of 32 CF chromosomes, 27, (i.e. 84%),share the dfF508 mutation.The haplotypes, established with mi-crosatellites markers IVS8CA, IVS17BTA,and IVS17BCA are shown in Table 2. Fif-teen different haplotypes have been de-scribed, four of them are linked to thedf508 mutation. Two haplotypes accountfor most of the df508 chromosomes : ha-plotype 23–31–13 for 55%, and haplotype17–32–13 for 29.6%. On the 18 normalchromosomes, 11 haplotypes have beenfound, 8 of them were only found on nor-mal chromosomes and not in mutated CFgene chromosomes.DiscussionIn our population, df508 mutation ac-count for 84% of all mutations versus 73%for the Breton population of Celtic origin(6). This represents highest value everobserved except for the Basque popula-tion (87%) (13). Four other mutations(W57X, S549N, 1717–1G  A, and G551D)are also found showing an heterogeneityof CF chromosomes in this population.Mutation G551D is associated with16–07–17 haplotype as it is in Spain,England, Scotland, Brittany, Czech, andSlovak Republics and Ireland (12, 14, 15,16, 17) and may be explained by the Cel-tic settlement of Brittany.Mutation N1717–1G  A shows thesame haplotype 16–07–17. 16–07–17 wasfound on two normal chromosomes in ourstudy. This was not very surprising be-cause it was the most common haplotypeassociated with normal chromosomes andmutated non df508 chromosomes (18).283S. Bezieau et al.: Microsatellite Haplotypes Associations, Coll. Antropol. 24 (2000) 2: 281–286TABLE 1DISTRIBUTION OF CF MUTATIONS IN EACHFAMILY ANALYSED IN »GRANDE BRIÈRE«Genotypes Number ofprobands Frequencydf508/df508 11 68.75%df508/ W57X 2 12.5%df508/ G551D 1 6.25%df508/ S549N 1 6.25%df508/1717–1G A 1 6.25%TOTAL 16 100%The two W57X chromosomes are asso-ciated with 16–29–13 haplotypes andS549N mutation with 16–31–13. Theyare found in Montoir, a peripheric villageknown to be a place of population flow.These results show an unexpected ap-parent heterogeneity since five differentmutations are found, and for the majorone, i.e. df508, four different haplotypes.As is the case for Breton (6), and Euro-pean populations (11), the Grande Brièrepopulation share the same genetic fea-tures for microsatellites associated withdf508 chromosomes. This observation isnot in agreement with the known high in-breeding rate and endogamy of this re-gion.The »Grande Brière« can be divided intwo different areas (Figure 1) : the islandslocated in the middle marsh and the pe-ripheric villages. In the islands, only thedf508 mutation is found. Furthermore,this mutation is only linked with twohaplotypes 23–31–13, and 17–32–13. The-se mutations come from two couples whosettled in the central islands in the eigh-teenth, and nineteenth centuries. Haplo-types 17–32–13 and the other one, 23–31–13, came from South Brittany.Populations from peripheric villagessharing mutations non df508 and df508associated with less frequent haplotypes17–31–13, and 23–32–13 do not have anycommon features with the characteristicsof the central islands. This observationshows that the two subpopulations didnot mix in the past, and therefore pro-vides another argument showing thestrong isolation of the populations of theislands.The morphology of the ascendants dis-tribution among the central Brière popu-lation for the last four centuries is verydifferent from that of periphery, and moregenerally from panmictic populations.This observation is the logical consequen-ce of the very limited availability of mar-riageable men. The classical distribution284S. Bezieau et al.: Microsatellite Haplotypes Associations, Coll. Antropol. 24 (2000) 2: 281–286TABLE 2DISTRIBUTION OF THE MICROSATELLITE HAPLOTYPES AMONG NORMAL AND CF CHROMO-SOMES FROM THE »GRANDE BRIÈRE« POPULATION. EACH HAPLOTYPE IS NAMED ACCORDINGTO THE NUMBER OF REPEATS AT LOCI IVS8CA, IVS17bTA, AND IVS17bCA, RESPECTIVELYMicrosatelliteshaplotypedf508mutationG551DmutationW57XmutationS549Nmutation1717–1G  AmutationNormalchromosome23–31–13 15 (55.6%)17–32–13 8 (29.6%)17–31–13 3 (11.1%)23–32–13 1 (3.7%) 1 (5.5%)16–07–17 1 (100%) 1 (100%) 2 (11.1%)16–29–13 2 (100%)16–31–13 1 (100%) 2 (11.1%)16–32–13 4 (22%)16–30–13 2 (11.1%)16–35–13 1 (5.5%)16–33–14 1 (5.5%)18–35–13 1 (5.5%)18–34–13 1 (5.5%)17–35–13 2 (11.1%)23–34–13 1 (5.5%)TOTAL 27 1 2 1 1 18for the individual ancestors was an in-verted pyramid (Figure 2a). For the cen-tral »Grande Brière«, this distribution be-came cylindrical (Figure 2b). The reasonfor this was, with a few exceptions, thewomen of the Brière did not choose theirhusband outside from the islands. Menwho were from elsewhere were excludedfrom the matrimonial market more foreconomics rather than socials. These ob-servations explain the fact that we easilyfound the origin for the two microsatelli-tes. This conclusion was supported by thefact that mutations were found only amongtheir own descendants, and on the otherhand because of the high level of consan-guinity in this population. An older pres-ence of the two microsatellites concernedwould have given rise to an homogenousand elevated augmentation of their fre-quency.AcknowledgmentsWe wish to thank all the populationfrom »Grande Brière« for its collaborationin this study.R E F E R E N C E S1. FANCONI, G., Das Coeliakiesyndrom bei ange-boren zysticher Pankreas Fibromatose und Bronchi-ektasien. Wien. Med. Wochenschr 86 (1936) 753. — 2.ROMMENS, J. M., M. C. IANNUZZI, B. KEREM, M.L. DRUMM, G. MELMER, M. DEAN, R. ROZMAHEL,J. L. COLE, D. KENNEDY, N. HIDAKA, M. ZSIGA,M. BUCHWALD, J. R. RIORDAN, L.-C. TSUI, F. S.COLLINS, Science 245 D (1989) 1059. — 3. RIORDANJ. R., J. M. ROMMENS, B. KEREM, N. ALON, R. ROZ-MAHEL, Z. GRZELCZAK, J. ZIELENSKI, S. LOK, N.PLAVSIC, J. L. CHOU, M. L. DRUMM, M. IANNUZZI,F. S. COLLINS, L. C. TSUI, Science 245 (1989) 1066.— 4. KEREM, B., J. M. ROMMENS, J. A. BUCHA-NAN, D. MARKIEWICZ, T. K. COX, A. CHAKRAVAR-TI, M. BUCHWALD, L. C. TSUI, Science 245 (1989)1073. — 5. ESTIVILL, X., C. BANCELLS, C. RAMOS,Hum Mutat 10 (1997) 135. — 6. Cystic Fibrosis Ge-netic Analysis Consortium Am. J. Hum. Genet. 47(1990) 354. — 7. DE BRAEKELEER, M., A. CHAVEN-TRE, G. BERTORELLE, C. VERLINGUE, O. RAGUE-NES, B. MERCIER, C. FEREC, Hum. Genet. 98 (1996)223. — 8. MOYON, A., (1989) Les îles de Brière deHenri IV à Louis XVI. Patronymes et Démographie. —9. FERRIE, R. M., M. J. SCHWARZ, N. H. ROBERT-SON, S. VAUDIN, M. SUPER, G. MALONE, S. LIT-TLE, Am. J. Hum. Genet. 51 (1992) 251. — 10. ORITA,M., Y. SUZUKI, T. SEKIYA, K. HAYASHI, Genomics 5(1989) 874. — 11. MORRAL, N., X. Estivill Genomics13 (1992) 1362. — 12. MORRAL, N., V. NUNES, T.CASALS, M. CHILLON, J. GIMENEZ, J. BERTRAN-PETIT, X. ESTIVILL, Hum. Mol. Genet. 2 (1993) 1015.— 13. CASALS, T., C. VAZQUEZ, C. LAZARO, E. GIR-BEAU, F. J. GIMENEZ, X. Estivill Am J Hum. Genet.50 (2) (1992) 404. — 14 HUGHES, D., A. HILL, A.REDMOND, N. NEVIN, C. GRAHAM, Hum. Genet. 95(1995) 462. — 15 HUGHES, D., A. WALLACE, J. TAY-LOR, M. TASSABEHJI, R. MCMAHON, A. HILL, N.NEVIN, C. GRAHAM, Hum. Mutat. (1996) 229. — 16CASHMAN, S. M., A. PATINO, M. G. DELGADO, L.BYRNE, B. DENHAM, M. DE ARCE, J. Med. Genet.285S. Bezieau et al.: Microsatellite Haplotypes Associations, Coll. Antropol. 24 (2000) 2: 281–286Fig. 2. Comparison of the distribution between individual ancestors in panmicticpopulation (2a) and in the population from central “Grande Brière” (2b)32 (1995a) 972. — 17 CASHMAN, S. M., A. PATINO, A.MARTINEZ, M. GARCIA-DELGADO, Z. MIEDZYBRO-DZKA, M. SCHWARZ, A. SHRIMPTON, C. FEREC, O.RAGUENES, M. JR MACEK, N. MORRAL, M. DE AR-CE, Hum. Hered. 45 (1995b) 6. — 18 MORRAL, N., J.BERTRANPETIT, X. ESTIVILL, V. NUNES, T. CASALS,J. GIMENEZ, A. REIS, R. VARON-MATEEVA, M. JRMACEK, L. KALAYDJIEVA, D. ANGELICHEVA, R.DANCHEVA, G. ROMEO, M. P. RUSSO, S. GARNE-RONE, G. RESTAGNO, M. FERRARI, C. MAGNANI,M. CLAUSTRES, M. DESGEORGES, M. SCHWARTZ,M. SWARZ, B. DALLAPICCOLA, G. NOVELLI, C. FÉ-REC, M. DE ARCE, M. NEMETI, J. KERE, M. ANV-RET, N. DAHL, L. KADASI, Nat. Genet. 7 (1994) 16975.S. BezieauService de Génétique Médicale, C.H.U., Nantes, FrancePOVEZANOST HAPLOTIPOVA MIKROSATELITSKIH S 5 MUTACIJACFTR U »GRANDE BRIERE«, IZOLATU SMJE[TENOMU JU@NOJ BRETANJIS A @ E T A KVarijabilnost tri mikrosatelita regulatornog gena CFTR lokusa cisti~ne fibroze pro-u~avana je zbog u~estalih mutacija u izoliranoj populaciji »Grand Brière«, maloj regijismje{tenoj u Ju`noj Bretanji. Nalaz je prou~en (FM PCR) u 16 obitelji s cisti~nom fibro-zom (CF) nosiocima 5 razli~itih mutacija. ^etiri naju~estalija haplotipa (df508) isti suonim prona|enim u Sjevernoj Francuskoj i Europi, ali njihova razdioba u populacijamapru`a nove spoznaje o porijeklu ove izolirane zajednice.286S. Bezieau et al.: Microsatellite Haplotypes Associations, Coll. Antropol. 24 (2000) 2: 281–286

Associations with 5 CFTR Mutations in »Grande Brière«, an Isolate Located in Southern Brittany

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Associations with 5 CFTR Mutations in »Grande Brière«, an Isolate Located in Southern Brittany

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