Comparison of the CFTR Mutation Spectrum in Three Cohorts of Patients of Celtic Origin from Brittany (France) and Ireland

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1 HUMAN MUTATION Mutation in Brief #629 (2003) Online MUTATION IN BRIEF Comparison of the CFTR Mutation Spectrum in Three Cohorts of Patients of Celtic Origin from Brittany (France) and Ireland Virginie Scotet 1, David E. Barton 2, James B.G. Watson 3, Marie-Pierre Audrézet 1, Trudi McDevitt 2, Shirley McQuaid 2, Cathy Shortt 3, Marc De Braekeleer 1,4,5, Claude Férec 1,6*, and Cédric Le Maréchal 6 1 INSERM EMI 01-15, Laboratoire de Génétique Moléculaire, Brest, France; 2 National Centre for Medical Genetics and Department of Medical Genetics, University College Dublin, Our Lady s Hospital for Sick Children, Crumlin, Dublin, Ireland; 3 Cork Regional Hospital, Southern Health Board, Cork, Ireland; 4 Laboratoire de Cytogénétique, Brest, France; 5 Institut National d Etudes Démographiques, Paris, France; 6 EFS-Bretagne, Brest, France *Correspondence to: Claude Férec, M.D. Ph.D., INSERM EMI 01-15, Laboratoire de Génétique Moléculaire, C.H.U. Morvan, 46 rue Félix Le Dantec, BP 454, Brest Cédex, France; Tel: ; Fax: ; claude.ferec@univ-brest.fr Communicated by Mark H. Paalman This study aims to compare the spectrum of the mutations identified in the gene responsible for cystic fibrosis in three cohorts of patients of Celtic origin from Brittany and Ireland. It included 389 patients from Brittany, 631 from Dublin and 139 from Cork. The CFTR gene analysis relied on the detection of the most common mutations, followed by a complete gene scanning using DGGE or D-HPLC. High mutation detection rates were obtained in each cohort: 99.6%, 96.8%, and 96.0% respectively. A high frequency of the c.1652_1655 del3 mutation (F508del: 74.8% to 81.3%) and of the Celtic mutation (c.1784g>a (G551D): 3.7% to 9.7%) was observed in each population. Apart from this, the mutation spectrums differed. In Brittany, the most common abnormalities were: c.1078delt (3.6%), c.4041c>g (N1303K: 1.4%), c.2670g>a (W846X 2 : 1.0%) and c g>a (1.0%), whereas in the cohort of Dublin, the main mutations were: c.482g>a (R117H: 3.0%), c.1811g>c (R560T: 2.4%) and c.621+1g>t (1.7%). Finally, in the Cork area, only the c.482g>a mutation (R117H) reached a frequency of 1%. Two previously-unreported mutations were identified in the Dublin cohort: c a>g and c.3446t>g (M1105R). This collaborative study highlights the similarities of the CFTR alleles in the Breton and Irish populations, but also the disparities that exist between these populations, despite their common origin. Each population has its own history, with its mixture of founder effects and genetic drifts, which are at the origin of the current mutation distribution. The molecular study of the CFTR gene provides new tools for retracing European populations histories Wiley-Liss, Inc. KEY WORDS: cystic fibrosis; CFTR; mutation spectrum; Brittany; Ireland; Celtic. INTRODUCTION Cystic fibrosis (CF; MIM# ) is a severe monogenic disorder of autosomal recessive inheritance, which Received 28 January 2003; accepted revised manuscript 11 April WILEY-LISS, INC. DOI: /humu.9157

2 2 Scotet et al. is frequent in Caucasian populations (incidence: 1/3500) (Welsh et al., 2001). Characterised by a chronic obstructive pulmonary disorder and pancreatic insufficiency, the disease is caused by mutations in the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) gene (MIM# ), which is located on chromosome 7 and encodes a camp regulated chloride channel (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989). This gene presents a large allelic heterogeneity: since its identification 14 years ago, more than one thousand different mutations have been identified world-wide (Tsui, 2003). This allelic diversity led Welsh and colleagues to propose a classification of those mutations according to the type of alteration they induced on the CFTR protein (Welsh et al., 2001). Data on CFTR mutations are collected by an international Consortium (Cystic Fibrosis Genetic Analysis Consortium), which establishes the spectrum of the abnormalities identified throughout the world. This spectrum is noteworthy because it includes a main mutation, accounting for 70% of the mutated alleles world-wide (the deletion F508del), four other mutations observed with a frequency over 1% (G542X: 2.4%, G551D: 1.6%, N1303K: 1.3%, W1282X: 1.2%) and a multitude of private abnormalities (Tsui, 2003). However, the frequencies of the mutations observed at the international level hide regional disparities. Indeed, these frequencies differ according to the geographical and ethnic origin of the patients (Bobadilla et al., 2002; C.F.G.A.C., 1994). The allelic heterogeneity observed in CF, added to these geographical disparities, makes the genotyping of patients a complex task. It is important to take account of allele frequencies and population differences when designing mutation screening strategies, to improve test sensitivity, but such data are not always available. However, the efforts made to identify abnormalities in the CFTR gene and to determine their geographical distribution have enabled the implementation of efficient strategies of diagnosis, notably with the development of commercialised kits (Ferrie et al., 1992). The techniques available for scanning the CFTR gene have greatly improved over the last years. Some of them now enable the entire coding sequence to be scanned in less than one week (Le Maréchal et al., 2001) and, therefore, excellent mutation detection rates can be achieved. The aim of the present study was to compare the spectrum of the CFTR mutations identified in three cohorts of Celtic origin and to analyse how this distribution was influenced by history. To achieve this, we completely analysed a cohort of patients from Brittany, France and two cohorts from the Republic of Ireland (Dublin Centre and Cork area). POPULATION AND METHODS Description of the studied regions The populations of Brittany and Ireland have a same Celtic origin, i.e. they belong to the areas on the Atlantic fringe of north-western Europe where a Celtic language was originally spoken. Brittany is a region of nearly three million inhabitants situated in the western part of France (Fig. 1). Its population is relatively homogeneous and has been subject to little population mixing. The high frequency of cystic fibrosis in this region led to the implementation of a CF neonatal screening program in 1989 (Scotet et al., 2000). Cork and Dublin are two harbour regions of the Republic of Ireland (Fig. 1). Dublin, the capital (920,000 inhabitants), is located on the eastern coast of the Republic, whereas Cork (130,000 inhabitants) is located in the south west of the country. With its agglomeration, Dublin contains a quarter of the whole population of Ireland and has therefore seen much population mixing. Because this city has increased in size 4-fold over the past 100 years, while the population of Ireland has remained quite stable, it is likely that its population represents a mixture of people from all over Ireland. Patients In order to assess the actual frequency of private mutations, we included in this study only unrelated CFaffected patients. In this way, this study included 389 unrelated patients from Brittany (selected from the retrospective registration of the patients born in this region since 1960 we recently conducted (Scotet et al., 2002)), 631 patients from the Dublin Centre, which were referred both from Dublin clinics and other clinics around the country, and 139 patients from the Cork area, representing a total sample of more than 2000 CF chromosomes. Patients from Cork clinics referred to the Dublin Centre were removed from the Dublin cohort and added to the Cork cohort. Only index cases with a sweat chloride greater or equal to 60 mmol/l and/or Pseudomonas colonisation of the lungs were included.

3 CFTR Mutation Spectrum in Breton and Irish Patients 3 Dublin Centre Cork Area 278 CF alleles 10 mutations F508del 81.3% G551D 9.7% R117H 1.4% 1262 CF alleles 35 mutations F508del 76.5% G551D 6.5% R117H 3.0% R560T 2.4% 621+1G>T 1.7% Brittany 778 CF alleles 62 mutations F508del 74.8% G551D 3.7% 1078delT 3.6% N1303K 1.4% W846X 2 1.0% G>A 1.0% Figure 1. Main mutations found in the cohorts from Brittany, Dublin Centre and Cork area. CFTR gene analysis The analysis of the CFTR gene (CFTR cdna accession number: M28668) was performed in two steps in the Dublin cohort. Firstly, the National Centre for Medical Genetics, Dublin performed an analysis of the most common CFTR mutations, using the ARMS test (Ferrie et al., 1992), which enables the detection of the following mutations: F508del, R117H, I507del, G542X, G551D, R560T, N1303K, R352Q, G>A and 621+1G>T. This molecular study was completed with the analysis of the kbC>T mutation by PCR-RFLP (Restriction Fragment Length Polymorphism), and of the intron 8 poly-t-tract by PCR and direct sizing. The clinicallyconfirmed CF cases whose genotype remained incomplete were tested by the OLA kit covering 31 mutations (Applied Biosystems); some other exons were also tested by the single strand conformation polymorphism method (SSCP). The samples with remaining unidentified alleles were then sent to the genetics laboratory in Brest, which proceeded to scan the entire CFTR gene, using first denaturing gradient gel electrophoresis (DGGE) (Audrézet et al., 1993; Férec et al., 1992) and then denaturing high performance liquid chromatography (D-HPLC), a new technique which, as we have recently shown, has excellent sensitivity (Le Maréchal et al., 2001). In Brittany, the CF patients were genotyped by these two latter methods. Samples from the Cork area were also sent directly to the laboratory in Brest for genotyping.

4 4 Scotet et al. Statistical analysis We determined the spectrum of the CFTR mutations identified in the three cohorts of patients and compared their respective frequencies by a Chi square test. We verified whether the Hardy-Weinberg equilibrium was respected. RESULTS AND DISCUSSION Table 1 represents the spectrum of all the mutations identified in each population, whereas Tables 2 and 3 show only the most common ones. High mutation detection rates were obtained in these three cohorts (from 96.0% to 99.6%). The number of different abnormalities identified in each population varied: a total of 62 mutations was identified in the cohort from Brittany (for 778 mutated alleles), whereas only 35 abnormalities were detected in the Dublin cohort (for 1262 alleles) and ten in the Cork cohort (for 278 alleles). The number of private mutations was also higher in Brittany (n=9) than in the cohorts of Dublin (n=6) or of Cork (n=3). High mutation detection rates were obtained in these three cohorts (from 96.0% to 99.6%). The frequency of the main mutation, F508del, was elevated in these three populations when compared to worldwide data. It was associated with 74.8% of the mutated alleles in Brittany, with 76.5% in Dublin and 81.3% in Cork. Besides the F508del deletion, the G551D mutation was also observed with an elevated frequency in each cohort: it was found in 9.7% of the CF alleles in Cork, in 6.5% in Dublin and in 3.7% in Brittany. It appeared significantly higher in the cohort from Cork than in the two others (χ²=14.6 p=0.0007). The G551D mutation was the most frequent molecular anomaly after the deletion F508del in the two Irish cohorts. Nevertheless, in Brittany, another mutation appeared as frequently: the 1078delT, which was present in 3.6% of the CF alleles; it has not been identified in the two Irish cohorts.

5 CFTR Mutation Spectrum in Breton and Irish Patients 5 Table 1. Spectrum of the CFTR Mutations Identified in the Cohorts from Brittany, Dublin Centre, and Cork Area Nucleotide Amino acid Brittany Dublin Cork change * change Exon Number Frequency Number Frequency Number Frequency 211delG % 310G>T E60X % 4 0.3% 347C>A A72D % 368G>A W79X % 386G>A G85E % 3 0.2% 403G>A G91R % 482G>A R117H % % 4 1.4% 498T>A Y122X % 574delA % 577G>A G149R % 621+1G>T int % % 790C>T Q220X 6a 1 0.1% 875+1G>C int 6a 1 0.4% 905delG 6b 1 0.1% 1065C>G F311L % 1078delT % 1132C>T R334W % 1172G>A R347H % 1172G>T R347L % 1172G>C R347P % 1187G>A R352Q % 2 0.7% 1208A>G Q359R % 1154insTC % 1221delCT % G>A int % delTA int % 1334G>A W401X % 1461ins % 1471delA % 1607C>T S492F % 1609C>T Q493X % 1648_1653delATC I507del % % 1 0.4% 1652_1655del 3 bp F508del % % % 1690G>T V520F % G>A int % 9 0.7% 1756G>T G542X % 8 0.6% 1779T>G S549R % 1784G>A G551D % % % 1789C>G R553G % 1789C>T R553X % 1 0.1% 1806delA % 1811G>A R560K % 1811G>C R560T % 2 0.7% 1819T>A Y563N % 1853C>A P574H % G>A int % 2184delA % 1 0.1% 2184insA % G>A int % 2 0.2% G>T int % A>G ** int % 2670G>A W846X 2 14a 8 1.0% G>T int 14a 1 0.1% A>G int 14a 2 0.2% G>A int 14b 6 0.8% 2966C>T S945L % 3007delG % 3040G>C G970R % 3062C>T S977F % G>A int % A>G int 17a 4 0.5% 2 0.2% 2 0.7% 3320dupli(CTATG) 17b 1 0.1% 3329G>A R1066H 17b 1 0.1% 3340C>T R1070W 17b 1 0.1% 3408C>A Y1092X 17b 7 0.9% 3442G>T E1104X 17b 1 0.1% 3446T>G ** M1105R 17b 1 0.1% 3586G>C D1152H % T>C delC int % 3616C>T R1162X % 2 0.2% 3659delC % 3832A>G I1234V % A>G int % kbC>T int % 3877G>A G1249R % 3884G>A S1251N % 3898insC % 3905insT % 3978G>A W1282X % G>A int % 4016insT % 4041C>G N1303K % 5 0.4% 4136T>C L1335P % 1 0.4% 4279insA % Unidentified Unidentified % % % * All nucleotide changes correspond to cdna numbering. ** New mutations. The most common mutations are in bold characters. Total % % %

6 6 Scotet et al. Table 2. Distribution of the Main CFTR Mutations Observed in Brittany (Basse and Haute-Bretagne) Nucleotide change Amino acid change Basse-Bretagne Haute-Bretagne Number Frequency Number Frequency Brittany * 1652_1655del 3 bp F508del % % % 1784G>A G551D % % % 1078delT % 3 1.1% % 4041C>G N1303K 3 0.6% 8 3.0% % 2670G>A W846X % 1 0.4% 8 1.0% G>A 5 1.0% 3 1.1% 8 1.0% 3408C>A Y1092X 1 0.2% 6 2.2% 7 0.9% G>A 2 0.4% 4 1.5% 6 0.8% G>A 5 1.0% 1 0.4% 6 0.8% 310G>T E60X 3 0.6% 2 0.7% 5 0.6% 621+1G>T 2 0.4% 3 1.1% 5 0.6% 1172G>A R347H 5 1.0% 5 0.6% 1756G>T G542X 4 0.8% 1 0.4% 5 0.6% 482G>A R117H 3 0.6% 1 0.4% 4 0.5% A>G 2 0.4% 2 0.7% 4 0.5% 1648_1653delATC I507del 1 0.2% 2 0.7% 3 0.4% 1789C>T R553X 3 0.6% 3 0.4% 3978G>A W1282X 2 0.4% 1 0.4% 3 0.4% Unidentified Unidentified 3 0.6% 3 0.4% Total Total % % % The analysis concerning Basse and Haute-Bretagne contains two CF chromosomes less than the analysis by administrative division (776 instead of 778). These two chromosomes concern a patient born in a commune which, administratively, belongs to Brittany, but which, ecclesiastically, did not belong to Brittany (it belonged to the neighbouring bishopric of Nantes). Consequently, this patient (F508del/F508del) was excluded from the analysis by bishopric. Despite a similar high frequency of the F508del and G551D mutations, the other most common mutations observed in each region were not the same (Table 1, Figure 1). In Brittany, the four mutations found with a frequency above 1% were: 1078delT (3.6%), N1303K (1.4%), W846X 2 (TGG TGA) (1.0%) and G>A (1.0%). In the cohort from Dublin, the three mutations presenting a frequency greater or equal to 1% were different: R117H (3.0%), R560T (2.4%) and 621+1G>T (1.7%). In the cohort from Cork, only one mutation other than F508del and G551D reached a frequency of 1%: R117H (1.4%). Finally, the main differences observed between these cohorts were the high frequency of the 1078delT mutation in Brittany (3.6%) and of the R560T in the Dublin cohort (2.4%).

7 CFTR Mutation Spectrum in Breton and Irish Patients 7 Table 3: Distribution of the Main CFTR Nutations Observed in the Irish Cohorts (Dublin and Cork) Nucleotide change Amino acid change Dublin cohort Cork cohort Number Frequency Number Frequency Ireland 1652_1655del 3 bp F508del % % % 1784G>A G551D % % % 482G>A R117H % 4 1.4% % 1811G>C R560T % 2 0.7% % 621+1G>T % % 1648_1653delATC I507del % 1 0.4% % G>A 9 0.7% 9 0.6% 1756G>T G542X 8 0.6% 8 0.5% 1187G>A R352Q 3 0.2% 2 0.7% 5 0.3% 1461ins % 5 0.3% 4041C>G N1303K 5 0.4% 5 0.3% 310G>T E60X 4 0.3% 4 0.3% 1690G>T V520F 4 0.3% 4 0.3% 3007delG 4 0.3% 4 0.3% A>G 2 0.2% 2 0.7% 4 0.3% 386G>A G85E 3 0.2% 3 0.2% kbC>T 3 0.2% 3 0.2% Unidentified Unidentified % % % Total Total % % % The 62 mutations detected in Brittany combined to give 81 different genotypes in CF patients. In the cohort from Dublin, there were 55 different genotypes and in Cork, 14. The Hardy-Weinberg equilibrium was respected in these series. Differences appeared in the spectrum of genotypes between the three cohorts. In Brittany, more than half of the patients was homozygous F508del (57.6%). Just three genotypes were responsible for two thirds of CF cases in this region: F508del/F508del (57.6%), F508del/G551D (4.9%) and F508del/1078delT (4.6%). In the Dublin Centre, the F508del/F508del genotype was observed in 61.0% of patients. The other most frequent genotypes were: F508del/G551D (9.5%), F508del/R117H (3.5%) and F508del/R560T (2.7%). Finally, in the Cork area, about 70.0% of patients were homozygous for the main mutation, 13.7% were F508del/G551D and 2.9% were F508del/R117H. The frequency of the F508del/G551D genotype differed significantly between the cohorts (χ²=12.2 p=0.0022). It was more frequently found in the cohorts from Cork and Dublin than in that from Brittany. A similar result was observed for the F508del/F508del genotype (χ²=6.4 p=0.041). This collaborative study examines the spectrum of CFTR mutations identified in three cohorts of CF patients with a same Celtic origin: one cohort from Brittany, France and two cohorts from the Republic of Ireland (Cork and Dublin). This study highlights, on one hand, the common background of these populations with a common origin, through the high frequency of the Celtic mutation (G551D) resulting from the expansion of the Celts which was at its height between the IV th and III th centuries BC, and, on the other hand, the specificities of each cohort, reflecting the influence of their own history. As mentioned above, the populations of Brittany and Ireland have a same Celtic origin. This results from the invasion of the Celts at the end of the Neolithic period, who, at this time, left the Indo-European cradle located in Ukraine and in the South of the current Russia, and made their way towards the West of Europe. Around 1000 years BC, they occupied a large territory covering the major part of Gaule. From there, they hived off towards Brittany (at the time called Armorique), but also towards Spain, North of Italy and the British Isles. Moreover, between the IV th and the VII th centuries, the Bretons of the British Isles invaded Brittany, expelled by the Angles and Saxons. These Bretons came mainly from Wales and Cornwall, and in a lesser measure, from Ireland. However, some historians and archaeologists currently question the degree of extension of the Celtic populations towards the western part of Europe. According to them, this extension might not have been so important than initially reported.

8 8 Scotet et al. We noted similar high frequencies of the F508del and G551D mutations in the three cohorts studied. This last molecular anomaly is mainly found in Celtic populations (Cashman et al., 1995a; 1995b; Hamosh et al., 1992). It has been associated with 5.5% of CF chromosomes in Scotland, 3.8% in Czech Republic and 3.7% in Northern Ireland (Bobadilla et al., 2002). We, in collaboration with others, have shown by studying the haplotypes for three intragenic microsatellites markers (IVS8CA, IVS17bTA and IVS17bCA) that the G551D alleles of Irish, Scottish, English, Breton and Czech subjects were associated with a unique haplotype (marker names ). This testifies to the diffusion of this mutation by the Celtic populations (Cashman et al., 1995b). The genealogical reconstruction performed in the families carrying this mutation in Brittany revealed that it appeared to be concentrated along the coasts of the north-western part of the region, more particularly in the bishopric named Léon where it was present in 7.7% of the CF chromosomes, i.e., a frequency closer to that observed in Ireland (Scotet et al., 2002). Besides the F508del and G551D mutations, disparities appeared in the spectrum of mutations between the three cohorts, reflecting the influence of the past of each population, with their own populations movements, genetic drifts, founder effects, (Tables 2 and 3). To illustrate this, we can note that the 1078delT mutation appeared frequently in Brittany (3.6%), whereas it was not observed in Ireland. The genealogical reconstruction showed its high frequency in South Finistère, more particularly in Pays Bigouden and Cap Sizun, resulting probably from a founder effect (Scotet et al., 2002). If this mutation is not observed in Ireland, it has also been found in Wales (2.2%) (Cheadle et al., 1993), which is witness of the exchanges that existed during the past between Brittany and Wales. The mutations frequently observed in the cohort from Dublin are not the same as those observed in the other studied populations, which is the consequence of a different history. The R560T, frequent in this cohort, has also been found in Northern Ireland (2.6%) (Bobadilla et al., 2002), whereas the 621+1G>T has also been observed with an abnormally high frequency in Saguenay-Lac-Saint-Jean, Quebec (23%), giving evidence of a founder effect (Rozen et al., 1992). This mutation appears also frequent in Wales (6.6%) (Cheadle et al., 1993) and in Greece (5.0%) (Bobadilla et al., 2002). Generally, we observed a lower number of different mutations and of private ones in the Republic of Ireland than in Brittany. This observation is not surprising for an island, which has therefore seen less population mixing. These disparities in the distribution of CFTR mutations can be observed at a more precise geographical level, as it is the case in Brittany (Table 2). Indeed, in a previous study, we compared the distribution of CFTR mutations between the western and eastern parts of the region (Scotet et al., 2002). We concluded that western Brittany presented a specific mutation spectrum (1078delT, G551D, G>A, W846X 2 ), whereas the eastern part of the region showed a spectrum of mutations more similar to that generally observed in France (N1303K, Y1092X, G>A, etc). These disparities should result from differences in the degree of isolation and of populations movements between these two parts of the region. Western Brittany presented a homogeneous population, in which there were high rates of consanguinity and endogamy and low rates of migrations, whereas eastern Brittany saw higher population mixings. This study leads to a precise knowledge of the CFTR mutation spectrum, which enables the improvement of the diagnostic strategies, a precise determination of the sensitivity of the molecular diagnosis test and the refinement of genetic counselling in families. In these Breton and Irish populations, the CFTR gene has been analysed with the best techniques available at this time, which have excellent sensitivity (DGGE and D-HPLC) (Audrézet et al., 1993; Férec et al., 1992; Le Maréchal et al., 2001). The entire gene has been sequenced and high detection rates have been reached in each population. Similar high detection rates have been obtained in some other small populations, for example in the Jewish Ashkenazi population (97%) (Abeliovich et al., 1992) or in Saguenay-Lac- Saint-Jean, Quebec (100%) (De Braekeleer et al., 1998). Other regions, which have seen more significant population mixing during history, such as the Mediterranean countries for example, present much lower detection rates. In conclusion, this collaborative study, which has determined the distribution of the CFTR mutations in a sample of more than 2000 CF chromosomes of Celtic origin, illustrates that cohorts with a shared origin can nevertheless show genetic diversities. The spectrums of CFTR mutations show disparities, even if G551D is the most frequent mutation after the deletion F508del in each of the studied cohorts. Each population has its own history, with its mixture of founder effects and genetic drifts, which are at the origin of the current distribution of CFTR mutations. This highlights that the current distribution of CFTR mutations reflects past and migratory movements.

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