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Human Molecular Genetics, Vol. 1, No. 8 647—648
A nonsense mutation in exon 4 of the cystic fibrosis gene frequent among the population of the Reunion Island F.Chevalier-Porst*, J.C.Chomel1, D.Hillaire23, A.Kitzis1, J.C.Kaplan1, R.Goutaland, M.Mathieu and D.Bozon Centre d'Etudes des Maladies Metaboliques, Laboratoire de Biochimie, Batiment D, HSpital Debrousse, 29 Rue Soeur Bouvier, 69322 Lyon Cedex 05, 1 Laboratoire de Biochimie Genetique, C.H.U. Cochin, 24 Rue du Faubourg Saint Jacques, 75014 Paris, 2Centre de Transfusion Sanguine, Centre Hospitalier Regional F. Guyon, BP 207, 97405 Saint Denis, He de la Reunion and 3Unite de Recherches sur les Handicaps Genetiques de I'Enfant, INSERM, Hopital des Enfants Malades, 149 Rue de Sevres, 75743 Paris Cedex 15, France Received July 23, 1992; Revised and Accepted September 22, 1992
* To whom correspondence should be addressed
G A T c
Figure 1. Direct genomic sequencing of exon 4 from a heterozygous patient showing a A—T substitution at position 498 (antisense).
Figure 2. Identification of Y122X on a 10% acrylamide gel electrophoresis after Msel digestion of exon 4 amplified product showing the segregation in a CF family: the affected child is homozygous for this mutation.
F508 + Y122X) account for 68% of the CF chromosomes is probably due to the settlement of the population in this island. Y122X has been found only on CF chromosomes of white Caucasians known as 'petits blancs' who are the descendants of
Downloaded from http://hmg.oxfordjournals.org/ at Indiana University Library on November 26, 2013
The gene involved in Cystic Fibrosis (CF), the most common genetic disease among Caucasians, has been isolated and sequenced (1, 2, 3). The protein encoded by this gene, named CFTR (for Cystic Fibrosis Transmembrane Conductance Regulator) contains 1480 amino acids, has 2 membrane associated domains, two ATP Binding Folds (NBF), and a large highly charged domain (R) containing phosphorylation sites for protein kinases A and C. The most common mutation responsible for Cystic Fibrosis is a deletion, in exon 10 (first NBF domain), of a triplet coding for a phenylalanine at position 508 (delta F508) in the CFTR protein. This mutation has been found with a frequency of 68% in 13000 CF chromosomes tested, with however marked variations in populations of different geographical origin (4). We report a nonsense mutation located in exon 4 of the CFTR gene, found with a high frequency in the Caucasian population of the Reunion Island. Direct sequencing of the PCR product of exon 4 (5, 6) of a CF patient shows (Figure 1) a T to A substitution at nucleotide position 498 of the cDNA sequence (3). This mutation creates a stop codon (TAA) at amino acid position 122 of CFTR instead of a tyrosine (TAT) and thus was named Y122X. Y122X is located in the first transmembrane spanning region and is predicted to create a truncated, non functional, polypeptide. This nonsense mutation introduces a new Msel site, therefore digestion of the PCR product should yield 4 fragments (299-71 - 3 5 - 3 3 base pairs) for the normal sequence and 5 fragments (181 — 118 — 71—35—33 base pairs) for the mutated one (Figure 2). This mutation was originally identified in a white Caucasian patient who carries delta F508 on his CF paternal chromosome. The maternal CF chromosome bearing Y122X carries the C haplotype defined by the extragenic DNA markers XV2C and KM 19 (7). This patient has a classical form of CF with pancreatic insufficiency and was born in the Reunion Island, a french province in the Indian Ocean. The screening for Y122X in this particular population by Msel digestion showed that 19 CF chromosomes out 66 are positive, 4 patients being homozygous. In this population delta F508 represents 39% of the CF alleles and Y122X 29%. In our sample of 1000 CF chromosomes from mainland France, delta F508 accounts for 68 % of the CF alleles and none of them tested by Msel digestion carry Y122X. This difference of distribution in CF alleles between mainland France and the Reunion Island and the fact that 2 mutations (delta
648 Human Molecular Genetics, Vol. 1, No. 8
ACKNOWLEDGMENTS We are gratefiil to all the patients and their families. We thank Dr J.F.Lesure, Dr F.Renouil, Dr F.Pierson from the Reunion Island and Pr Gilly and Dr Chazalette from France for their cooperation. We gratefully acknowledge the help of Mr L.Tesson and Mrs C.Musenger. This work was supported by AFLM (Association Francaise de Lutte contre la Mucoviscidose).
REFERENCES 1. Kerem,B., Rommens,J.M., Buchanan,J.A., Markiewicz,D., Cox.T.K., Chakravarti.A., Buchwald.M. and Tsui.L.C. (1989) Science 245, 1073-1080. 2. Rommens.J.M., Iannuzzo,M.C, Kerem.B., Drumm.M.L., Melmer.G., Dean,M., Rozmahel.R., Cole,J.L., Kennedy,D., Hidaka.N., Zsiga,M., Buchwald.M., RiordanJ.R., Tsui.L.C. and Collins,F.S. (1989)Science245, 1059-1064. 3. Riordan.J., Rommens.J., Kerem.B., Alon.N., Rozmahel.R., Grzelczak.Z., ZielenskiJ., Lok,S., Plavisk,N., Chou.J., Drumm, M., Iannuzzi.M., Collins.F. and Tsui.L.C. (1989) Science 245, 1066-1073. 4. Cystic Fibrosis Genetic Analysis Consortium (1990) Am. J. Hum. Genet. 47, 354-359. 5. Zielenski,J., Rozmahel.R., Bozon.D., Kerem,B., Grzelczak,Z., Riordan.J.R., Rommens,J. and Tsui.L.C. (1991a) Genomics 10, 214-228. 6. Zielenski,J., Bozon.D., Kerem,B., Markiewicz.D., Durie,P., Rommens.J. and Tsui.L.C. (1991b) Genomics 10, 229-235. 7. Estivill.X., Farall,M., Scambler.P.J., Bell,G.M., Hawley.K.M.F., Lench N.J. and Bates,G.P. (1987) Nature 326, 840-845. 8. Hillaire.D., Chomel,J.C, Lesure.F., RenouU.M., Musenger,C, Pierson,F., Berthelon,M., Lenoir,G., Gerard,G., Bois,E., Kitzis.A., Munnich.A., Kaplan,J.C. and Feingold.J. (1991) Ann. Genet. 34-1, 5 - 7 .
Downloaded from http://hmg.oxfordjournals.org/ at Indiana University Library on November 26, 2013
the first French Settlers (46 men and 37 women) in the XVIIth century (8). Geographical isolation for about 200 years explains consanguinity among large families which has increased the incidence of genetic diseases (Cystic Fibrosis: 1/1000). More than two hundred different mutations in the CF gene have been reported to the Cystic Fibrosis Genetic Analysis Consortium. Most of them are rare except in particular ethnic groups: the nonsense mutation described in this paper has not been found in the mainland France population but is frequent in the Caucasian population of the Reunion Island. The real origin of Y122X is unknown but the fact that all the Y122X chromosomes are of the same haplotype group (C) suggests a common origin for these chromosomes. In the remaining non delta F508, non Y122X CF chromosomes, the distribution of haplotypes does not differ significantly from the one observed in mainland France, suggesting that several rare mutations must account for these alleles.
Human Molecular Genetics, Vol. 1, No. 8 647—648
A nonsense mutation in exon 4 of the cystic fibrosis gene frequent among the population of the Reunion Island F.Chevalier-Porst*, J.C.Chomel1, D.Hillaire23, A.Kitzis1, J.C.Kaplan1, R.Goutaland, M.Mathieu and D.Bozon Centre d'Etudes des Maladies Metaboliques, Laboratoire de Biochimie, Batiment D, HSpital Debrousse, 29 Rue Soeur Bouvier, 69322 Lyon Cedex 05, 1 Laboratoire de Biochimie Genetique, C.H.U. Cochin, 24 Rue du Faubourg Saint Jacques, 75014 Paris, 2Centre de Transfusion Sanguine, Centre Hospitalier Regional F. Guyon, BP 207, 97405 Saint Denis, He de la Reunion and 3Unite de Recherches sur les Handicaps Genetiques de I'Enfant, INSERM, Hopital des Enfants Malades, 149 Rue de Sevres, 75743 Paris Cedex 15, France Received July 23, 1992; Revised and Accepted September 22, 1992
* To whom correspondence should be addressed
G A T c
Figure 1. Direct genomic sequencing of exon 4 from a heterozygous patient showing a A—T substitution at position 498 (antisense).
Figure 2. Identification of Y122X on a 10% acrylamide gel electrophoresis after Msel digestion of exon 4 amplified product showing the segregation in a CF family: the affected child is homozygous for this mutation.
F508 + Y122X) account for 68% of the CF chromosomes is probably due to the settlement of the population in this island. Y122X has been found only on CF chromosomes of white Caucasians known as 'petits blancs' who are the descendants of
Downloaded from http://hmg.oxfordjournals.org/ at Indiana University Library on November 26, 2013
The gene involved in Cystic Fibrosis (CF), the most common genetic disease among Caucasians, has been isolated and sequenced (1, 2, 3). The protein encoded by this gene, named CFTR (for Cystic Fibrosis Transmembrane Conductance Regulator) contains 1480 amino acids, has 2 membrane associated domains, two ATP Binding Folds (NBF), and a large highly charged domain (R) containing phosphorylation sites for protein kinases A and C. The most common mutation responsible for Cystic Fibrosis is a deletion, in exon 10 (first NBF domain), of a triplet coding for a phenylalanine at position 508 (delta F508) in the CFTR protein. This mutation has been found with a frequency of 68% in 13000 CF chromosomes tested, with however marked variations in populations of different geographical origin (4). We report a nonsense mutation located in exon 4 of the CFTR gene, found with a high frequency in the Caucasian population of the Reunion Island. Direct sequencing of the PCR product of exon 4 (5, 6) of a CF patient shows (Figure 1) a T to A substitution at nucleotide position 498 of the cDNA sequence (3). This mutation creates a stop codon (TAA) at amino acid position 122 of CFTR instead of a tyrosine (TAT) and thus was named Y122X. Y122X is located in the first transmembrane spanning region and is predicted to create a truncated, non functional, polypeptide. This nonsense mutation introduces a new Msel site, therefore digestion of the PCR product should yield 4 fragments (299-71 - 3 5 - 3 3 base pairs) for the normal sequence and 5 fragments (181 — 118 — 71—35—33 base pairs) for the mutated one (Figure 2). This mutation was originally identified in a white Caucasian patient who carries delta F508 on his CF paternal chromosome. The maternal CF chromosome bearing Y122X carries the C haplotype defined by the extragenic DNA markers XV2C and KM 19 (7). This patient has a classical form of CF with pancreatic insufficiency and was born in the Reunion Island, a french province in the Indian Ocean. The screening for Y122X in this particular population by Msel digestion showed that 19 CF chromosomes out 66 are positive, 4 patients being homozygous. In this population delta F508 represents 39% of the CF alleles and Y122X 29%. In our sample of 1000 CF chromosomes from mainland France, delta F508 accounts for 68 % of the CF alleles and none of them tested by Msel digestion carry Y122X. This difference of distribution in CF alleles between mainland France and the Reunion Island and the fact that 2 mutations (delta
648 Human Molecular Genetics, Vol. 1, No. 8
ACKNOWLEDGMENTS We are gratefiil to all the patients and their families. We thank Dr J.F.Lesure, Dr F.Renouil, Dr F.Pierson from the Reunion Island and Pr Gilly and Dr Chazalette from France for their cooperation. We gratefully acknowledge the help of Mr L.Tesson and Mrs C.Musenger. This work was supported by AFLM (Association Francaise de Lutte contre la Mucoviscidose).
REFERENCES 1. Kerem,B., Rommens,J.M., Buchanan,J.A., Markiewicz,D., Cox.T.K., Chakravarti.A., Buchwald.M. and Tsui.L.C. (1989) Science 245, 1073-1080. 2. Rommens.J.M., Iannuzzo,M.C, Kerem.B., Drumm.M.L., Melmer.G., Dean,M., Rozmahel.R., Cole,J.L., Kennedy,D., Hidaka.N., Zsiga,M., Buchwald.M., RiordanJ.R., Tsui.L.C. and Collins,F.S. (1989)Science245, 1059-1064. 3. Riordan.J., Rommens.J., Kerem.B., Alon.N., Rozmahel.R., Grzelczak.Z., ZielenskiJ., Lok,S., Plavisk,N., Chou.J., Drumm, M., Iannuzzi.M., Collins.F. and Tsui.L.C. (1989) Science 245, 1066-1073. 4. Cystic Fibrosis Genetic Analysis Consortium (1990) Am. J. Hum. Genet. 47, 354-359. 5. Zielenski,J., Rozmahel.R., Bozon.D., Kerem,B., Grzelczak,Z., Riordan.J.R., Rommens,J. and Tsui.L.C. (1991a) Genomics 10, 214-228. 6. Zielenski,J., Bozon.D., Kerem,B., Markiewicz.D., Durie,P., Rommens.J. and Tsui.L.C. (1991b) Genomics 10, 229-235. 7. Estivill.X., Farall,M., Scambler.P.J., Bell,G.M., Hawley.K.M.F., Lench N.J. and Bates,G.P. (1987) Nature 326, 840-845. 8. Hillaire.D., Chomel,J.C, Lesure.F., RenouU.M., Musenger,C, Pierson,F., Berthelon,M., Lenoir,G., Gerard,G., Bois,E., Kitzis.A., Munnich.A., Kaplan,J.C. and Feingold.J. (1991) Ann. Genet. 34-1, 5 - 7 .
Downloaded from http://hmg.oxfordjournals.org/ at Indiana University Library on November 26, 2013
the first French Settlers (46 men and 37 women) in the XVIIth century (8). Geographical isolation for about 200 years explains consanguinity among large families which has increased the incidence of genetic diseases (Cystic Fibrosis: 1/1000). More than two hundred different mutations in the CF gene have been reported to the Cystic Fibrosis Genetic Analysis Consortium. Most of them are rare except in particular ethnic groups: the nonsense mutation described in this paper has not been found in the mainland France population but is frequent in the Caucasian population of the Reunion Island. The real origin of Y122X is unknown but the fact that all the Y122X chromosomes are of the same haplotype group (C) suggests a common origin for these chromosomes. In the remaining non delta F508, non Y122X CF chromosomes, the distribution of haplotypes does not differ significantly from the one observed in mainland France, suggesting that several rare mutations must account for these alleles.
