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20 No point mutation in Keratin 8 and Keratin 18 genes, in Cystic Fibrosis patients with severe liver disorders
1. Genetics' [ ~ T h e TNF~ receptor TNFRSFIA and genes encoding the amiloride-sensitive sodium channel ENaC as modulators in Cystic Fibrosis F. Stanke 1 , T. Becker2, H. Cuppens 3, V Kumar 1 , J.J. Cassiman 3, S. Jansen 1 , D. Radojkovic 3'4, B. Siebert 1 , J. Yarden 3, D.W Ussery 5, T.E Wienker 2, B. TiJmmler 1 . 1Hannover Medical School, Hannover; 2IMBIE; Bonn, Germany;
3Department of Human Genetics', KU Leuven, Belgium; 4IMGGE, Belgrade, Serbia and Montenegro; 5 CBS, Technical University of Denmark, Denmark Discordant clinical phenotypes of siblings with identical CFTR mutations and the large variability of clinical manifestations of patients who are homozygous for the most common mutation F508del suggest that both environment and genes other than CFTR contribute substantially to CF disease. Candidates for genetic modifiers in CF are elements of host defence, such as the TNFo, receptor, and of ion transport, such as the amiloride-sensitive epithelial sodium channel ENaC, both of which are encoded side by side on 12p13 (TNFRSF1A, SCNN1A) and 1@12 (SCNN1B, SCNN1G). Thirty-seven families with F508del-CFTR hornozygous siblings exhibiting extreme clinical phenotypes from the European CF Twin and Sibling Study [1] were genotyped at 12p13 and 1@12 markers. The ENaC was identified as a modulator of CF by transmission disequilibrium at SCNN1G and association with CF intrapair discordance at SCNN1B [2]. Family-based and case~ontrol analyses and sequencing of SCNN1A and TNFRSF1A uncovered an association of the TNFRSF1A intron 1 haplotype with disease severity [2]. Carriers of risk haplotypes were underrepresented, suggesting a strong impact of both loci on survival. The finding that TNFRSF1A, SCNN1B and SCNN1G are clinically relevant modulators of CF disease supports current concepts that the depletion of airway surface liquid and inadequate host inflammatory responses trigger pulmonary disease in CE
$5
[ ] I n f l u e n c e of tumor necrosis factor gene polymorphisms on lung disease progression in children with Cystic Fibrosis j. Brouard1,2, H. Corvol 1,3, RY. Boelle 3'4, N. Knauer 1,4, C. Vallet 1,3, A. HenrionCaude 1 , M. Boule 1'3, B. Fauroux 1'3, E Ratjen4, H. Grasemann4, A. Clement 1'3.
I INSERM U719 and Department of Paediatric Pneumology, Armand Trousseau Hospital, Paris'; 2Department of Paediatrics, Georges Clemenceau Hospital, Caen; 3 Universitd Pierre et Marie Curie Paris' and Assistance Publique H@itaux de Paris'; 4Department of Biostatistic, INSERM U707, St-Antoine Hospital, Paris', France; 5Children ~' Hospital, University of Duisburg-Essen, Essen, Germany Background: Polymorphisms in cytokine genes can influence inflammation and tissue damage and, consequently, may affect disease progression in cystic fibrosis (CF), a multiorgan disorder characterized by an altered inflammatory response. Methods: We analyzed single-nucleotide polymorphisms in the genes for tumor necrosis factor-f3 (TNF-f3)? at position +252A/G and TNF-o, at positions 857C/T and 308G/A in a population of 381 young patients with CE Genotypes were tested for an association with changes in lung function tests, infection with Pseudomonas aeruginosa and nutritional status by multivariable analysis. Results: Haplotype analysis revealed that the most frequent haplotype TNF-f3 +252A/TNF-o, 857C/TNF-o, 308G was associated with a significantly slower decline in both forced expiratory volume in one second and forced vital capacity (p 0.01, respectively). Conclusion: The TNF-f3 +252A/TNF-~, 857C/TNF-~, 308G haplotype is a marker of less rapid deterioration of lung function in children with CE
References [1] E Mekus et al. Twin Res. 2000:277 293. [2] E Stanke et al. Hum. Genet. 2006: in press.
[•
Analysis of the CLCA-gene cluster as a modulator of Cystic Fibrosis
E.W Kolbe 1 , B. Siebert 1 , U. Laabs 2, I. Leverk6hne 3, A. ,z Barneveld 1 , M. Ballmann 1 , S. Jansen 1 , M. Ritzka 1 , B. Tiimmler 1 , E Stanke 1 . 1Klinische
Forschergruppe, OE 6710, Medizinische Hochschule Hannove~ Hannover; 2EMBL, Heidelberg; 3Institut fiir Veteringirpathologie, FU Berlin, Germany F508del homozygous CF patients differ widely in disease manifestation, suggesting that environmental factors and genes other than CFTR significantly influence course and prognosis. Since the basic defect manifests in defective epithelial chloride transport, alternative chloride conductances have been suggested to partially account for the heterogeneity of clinical disease. The CLCA locus encoding 4 homologous proteins has been shown to influence non-CFTR mediated chloride conductance in intestinal epithelium and hence is a promising candidate for a modulator of the basic defect in CF [1]. We wanted to identify the gene sequences within the locus that are associated with the expression of this non-CFTR mediated chloride conductance. Our study population of predominantly AF508 homozygous individuals from the European Twin and Sibling study consists of 25 two-generation families with 32 CF patients. Their electrophysiological phenotype resolved by intestinal current measurement of rectal suction biopsies was differentiated into one group without any residual chloride conductance (n 21) and another group with DIDS-sensitive alternative chloride conductance (n 11). Eleven informative SNPs spanning the whole CLCA locus of about 210kbp were employed to compare the distribution of 2-marker haplotypes in the two cohorts with contrasting chloride conductance phenotypes. The localization of the putative modulator within the CLCA-gene cluster was narrowed by haplotype mapping to a 30 kbp fragment that is currently being refined by sequencing of contrasting haplotypes. References [1] Ritzka et. al. Hum Genet (2004) 115:483 491.
~No
point mutation in Keratin 8 and Keratin 18 genes, in Cystic Fibrosis patients with severe liver disorders E. Masson 1'3, M.R Audr~zet 1'2'3, C. FSrec 1'2'3. IINSERM U613, Gdndtique Moldculaire et Gdndtique Epiddmiologique, Brest; 2Centre Hospitalier Universitaire, Laboratoire de Gdndtique moldculaire, Brest; 3 Universitd de Bretagne Occidentale, Brest, France Cystic fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). An abnormal CFTR affects the epithelials cells of many organs such as the airways, pancreas, liver, etc. The clinical expression of the disease is heterogeneous. Only approximately 8% of patients have liver disease. Because cystic fibrosis patients bearing the same CFTR genotype can express a very different liver phenotype, studies on modifier gene have emerged. At this date, many genes primarily involved in the cycle of inflammation have been identified as potential modifiers. The aim of our study is to assess the role of the two novel candidate genes, the Keratin 8 (K8) and Keratin 18 (K18) genes, as potential liver risk factors in cystic fibrosis. Mutations in these genes have been reported to present risk factors in other multiple liver diseases like chronic hepatitis. Moreover, a recent study has shown that cells transfected by AF508-CFTR expressed more K8 and K18 than cells transfected by the wild-type CFTR. For this study, samples from 27 CF patients suffering from severe liver disorders have been analysed. The coding regions of K8 and K18 genes were sequenced for these 27 CF patients. Only several polyrnorphisms have been found in the two candidate genes. In K18, a variant p.S229T described as a polymorphism was found in one patient. In K8, an association between two new variations p.R341H and g.1320+1 ldelC has been identified in 2 of 27 patients and in 3 controls DNA. By this study, no relation between liver disease in CF patients and mutations in K8 and K18 genes has been established.
3Department of Human Genetics', KU Leuven, Belgium; 4IMGGE, Belgrade, Serbia and Montenegro; 5 CBS, Technical University of Denmark, Denmark Discordant clinical phenotypes of siblings with identical CFTR mutations and the large variability of clinical manifestations of patients who are homozygous for the most common mutation F508del suggest that both environment and genes other than CFTR contribute substantially to CF disease. Candidates for genetic modifiers in CF are elements of host defence, such as the TNFo, receptor, and of ion transport, such as the amiloride-sensitive epithelial sodium channel ENaC, both of which are encoded side by side on 12p13 (TNFRSF1A, SCNN1A) and 1@12 (SCNN1B, SCNN1G). Thirty-seven families with F508del-CFTR hornozygous siblings exhibiting extreme clinical phenotypes from the European CF Twin and Sibling Study [1] were genotyped at 12p13 and 1@12 markers. The ENaC was identified as a modulator of CF by transmission disequilibrium at SCNN1G and association with CF intrapair discordance at SCNN1B [2]. Family-based and case~ontrol analyses and sequencing of SCNN1A and TNFRSF1A uncovered an association of the TNFRSF1A intron 1 haplotype with disease severity [2]. Carriers of risk haplotypes were underrepresented, suggesting a strong impact of both loci on survival. The finding that TNFRSF1A, SCNN1B and SCNN1G are clinically relevant modulators of CF disease supports current concepts that the depletion of airway surface liquid and inadequate host inflammatory responses trigger pulmonary disease in CE
$5
[ ] I n f l u e n c e of tumor necrosis factor gene polymorphisms on lung disease progression in children with Cystic Fibrosis j. Brouard1,2, H. Corvol 1,3, RY. Boelle 3'4, N. Knauer 1,4, C. Vallet 1,3, A. HenrionCaude 1 , M. Boule 1'3, B. Fauroux 1'3, E Ratjen4, H. Grasemann4, A. Clement 1'3.
I INSERM U719 and Department of Paediatric Pneumology, Armand Trousseau Hospital, Paris'; 2Department of Paediatrics, Georges Clemenceau Hospital, Caen; 3 Universitd Pierre et Marie Curie Paris' and Assistance Publique H@itaux de Paris'; 4Department of Biostatistic, INSERM U707, St-Antoine Hospital, Paris', France; 5Children ~' Hospital, University of Duisburg-Essen, Essen, Germany Background: Polymorphisms in cytokine genes can influence inflammation and tissue damage and, consequently, may affect disease progression in cystic fibrosis (CF), a multiorgan disorder characterized by an altered inflammatory response. Methods: We analyzed single-nucleotide polymorphisms in the genes for tumor necrosis factor-f3 (TNF-f3)? at position +252A/G and TNF-o, at positions 857C/T and 308G/A in a population of 381 young patients with CE Genotypes were tested for an association with changes in lung function tests, infection with Pseudomonas aeruginosa and nutritional status by multivariable analysis. Results: Haplotype analysis revealed that the most frequent haplotype TNF-f3 +252A/TNF-o, 857C/TNF-o, 308G was associated with a significantly slower decline in both forced expiratory volume in one second and forced vital capacity (p 0.01, respectively). Conclusion: The TNF-f3 +252A/TNF-~, 857C/TNF-~, 308G haplotype is a marker of less rapid deterioration of lung function in children with CE
References [1] E Mekus et al. Twin Res. 2000:277 293. [2] E Stanke et al. Hum. Genet. 2006: in press.
[•
Analysis of the CLCA-gene cluster as a modulator of Cystic Fibrosis
E.W Kolbe 1 , B. Siebert 1 , U. Laabs 2, I. Leverk6hne 3, A. ,z Barneveld 1 , M. Ballmann 1 , S. Jansen 1 , M. Ritzka 1 , B. Tiimmler 1 , E Stanke 1 . 1Klinische
Forschergruppe, OE 6710, Medizinische Hochschule Hannove~ Hannover; 2EMBL, Heidelberg; 3Institut fiir Veteringirpathologie, FU Berlin, Germany F508del homozygous CF patients differ widely in disease manifestation, suggesting that environmental factors and genes other than CFTR significantly influence course and prognosis. Since the basic defect manifests in defective epithelial chloride transport, alternative chloride conductances have been suggested to partially account for the heterogeneity of clinical disease. The CLCA locus encoding 4 homologous proteins has been shown to influence non-CFTR mediated chloride conductance in intestinal epithelium and hence is a promising candidate for a modulator of the basic defect in CF [1]. We wanted to identify the gene sequences within the locus that are associated with the expression of this non-CFTR mediated chloride conductance. Our study population of predominantly AF508 homozygous individuals from the European Twin and Sibling study consists of 25 two-generation families with 32 CF patients. Their electrophysiological phenotype resolved by intestinal current measurement of rectal suction biopsies was differentiated into one group without any residual chloride conductance (n 21) and another group with DIDS-sensitive alternative chloride conductance (n 11). Eleven informative SNPs spanning the whole CLCA locus of about 210kbp were employed to compare the distribution of 2-marker haplotypes in the two cohorts with contrasting chloride conductance phenotypes. The localization of the putative modulator within the CLCA-gene cluster was narrowed by haplotype mapping to a 30 kbp fragment that is currently being refined by sequencing of contrasting haplotypes. References [1] Ritzka et. al. Hum Genet (2004) 115:483 491.
~No
point mutation in Keratin 8 and Keratin 18 genes, in Cystic Fibrosis patients with severe liver disorders E. Masson 1'3, M.R Audr~zet 1'2'3, C. FSrec 1'2'3. IINSERM U613, Gdndtique Moldculaire et Gdndtique Epiddmiologique, Brest; 2Centre Hospitalier Universitaire, Laboratoire de Gdndtique moldculaire, Brest; 3 Universitd de Bretagne Occidentale, Brest, France Cystic fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). An abnormal CFTR affects the epithelials cells of many organs such as the airways, pancreas, liver, etc. The clinical expression of the disease is heterogeneous. Only approximately 8% of patients have liver disease. Because cystic fibrosis patients bearing the same CFTR genotype can express a very different liver phenotype, studies on modifier gene have emerged. At this date, many genes primarily involved in the cycle of inflammation have been identified as potential modifiers. The aim of our study is to assess the role of the two novel candidate genes, the Keratin 8 (K8) and Keratin 18 (K18) genes, as potential liver risk factors in cystic fibrosis. Mutations in these genes have been reported to present risk factors in other multiple liver diseases like chronic hepatitis. Moreover, a recent study has shown that cells transfected by AF508-CFTR expressed more K8 and K18 than cells transfected by the wild-type CFTR. For this study, samples from 27 CF patients suffering from severe liver disorders have been analysed. The coding regions of K8 and K18 genes were sequenced for these 27 CF patients. Only several polyrnorphisms have been found in the two candidate genes. In K18, a variant p.S229T described as a polymorphism was found in one patient. In K8, an association between two new variations p.R341H and g.1320+1 ldelC has been identified in 2 of 27 patients and in 3 controls DNA. By this study, no relation between liver disease in CF patients and mutations in K8 and K18 genes has been established.