- Email: [email protected]
Searching new loci associated to autosomal dominant hypercholesterolemia
e54
Abstracts / Atherosclerosis 235 (2014) e27–e83
a
Center for Cardiovascular Prevention, Charles University and Thomayer Hospital, Prague 4, Czech Republic; b Division of Cardiovascular Diseases, Mayo Clinic, Rochester, USA; c Center for Cardiovascular Prevention, Charles University and Thomayer Hospital, Prague, Czech Republic; d 2nd Department of Internal Medicine, Charles University Center for Hypertension, Pilsen, Czech Republic; e International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic; f Division of Cardiovascular Diseases, Mayo Clinic, Rochester, USA Objectives: Increased aortic stiffness may be one of the mechanisms by which obesity increases cardiovascular risk independently of traditional risk factors. While body mass index (BMI) is generally used to define excess adiposity, several studies have suggested that measures of central obesity may be better predictors of cardiovascular risk. However, data comparing the association between several measures of central and general obesity with aortic stiffness in the general population are inconclusive. Methods: In 1031 individuals (age 5313 years, 45% men) without manifest cardiovascular disease randomly selected from the population, we tested the association between parameters of central obesity (waist circumference – WC, waist-to-hip-ratio –WHR, waist-to-height ratio –WHtR) and general obesity (BMI) with carotid-femoral pulse wave velocity (cfPWV). Results: In univariate analysis, WC and WHtR were more strongly associated with cfPWV than BMI in both genders, while WHR showed a stronger association with cfPWV only in women. WHtR was more closely associated with cfPVW than WHR. This difference between obesity measures remained after multivariate adjustment. When the fully adjusted hierarchical regression was used, among central obesity measures, WHtR had the largest additive value on top of BMI, while there was no additive value of BMI on top of WHtR. Conclusion: Central obesity parameters are more closely associated with aortic stiffness than BMI. Of central adiposity measures, WHtR has the strongest association with aortic stiffness beyond body mass index and cardiovascular risk factors. Our results suggest that WHtR may be the best anthropometric measure of excess adiposity in the general population. 40 - Inherited dyslipidemias EAS-0323. APOLIPOPROTIEN E VARIANTS IN PRIMARY HYPERLIPIDEMIAS A.M. Bea1, I. Lamiquiz-Moneo1, I. De Castro-Orós1, L. Baila-Rueda1, A. Cenarro1, F. Civeira1 1 Laboratorio Investigación Molecular, Instituto Aragonés de Ciencias de la Salud. Hospital Universitario Miguel Servet, Zaragoza, Spain
Objectives: Apolipoprotein E (ApoE) has an important role in cholesterol and triglycerides metabolism related to its function as chylomicron and LDL receptor ligand. APOE gene has been traditionally associated with type III hyperlipoproteinemia, which is mainly caused by the APOE ε2/ε2 genotype. However, other APOE mutations have been associated with other dyslipidemias. The objective was to identify rare genetics variants in APOE gene. Methods: A total of 442 unrelated consecutive subjects were recruited in the Lipid Unit at Hospital Universitario Miguel Servet with a clinical diagnosis of primary hyperlipoproteinemia. 87 were affected of familial hypercholesterolemia without a pathogenic mutation in LDLR nor APOB genes (FH-), 93 with familial hypercholesterolemia with a pathogenic mutation in LDLR or APOB genes (HF+), 158 with familial combined hyperlipidemia (FCHL), 65 with polygenic hypercholesterolemia (PH) and 39 with primary hypertriglyceridemia (HTG). 65 normolipemic subjects were analyzed as control group. In all subjects, exon 4 in APOE was sequenced and clinical and analytical variables were registered. Results: The following rare variants have been identified: p.Arg154Ser (rs121918393) in 2 FCH subjects; p.Leu167del in 3 HF-, 1 HF+, 3 FCHL and 1 HTG subjects; p.Gly145Asp (rs267606662) in 1 FCHL and 1 HF+ subjects; p.Arg163Cys (rs769455) in 1 HF-, 1 HTG and 1 FCHL subjects. It has been also identified a non previously described variant in 1 HTG subject:
p.Met82Ile. Moreover, the p.Ala217Ala variant (rs72654468) was found in 2 normolipemic subjects besides of 1 HF+ and 1 PH individuals. Conclusion: Five different APOE gene variants were identified in 16 subjects with different dyslipidemias that were not found in controls, so we consider they may be causally associated with their lipid abnormalities. In summary, our results suggest that the genetic variability in APOE gene contributes to the etiology of different dyslipidemias not only to dysbetalipoproteinemia. 40 - Inherited dyslipidemias EAS-0577. USE OF NEXT GENERATION SEQUENCING FOR THE DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLEMIA N. Planaa, R. Figuerasb, E. Estevec, M. Maurid, M. Buenoe, A. Caixàsf, E. Llarguésg, A. Vilah, C. Moralesi, C. Solerj, J. Argimonk, J. Mayosl, J. Graum, A. Zamoran, L. Mataso, L. Vilap, F. Blanco-Vacaq a Medicine, Hospital Sant Joan, Reus, Spain; b Medicine, Hospital de Bellvitge, L'Hospitalet de Llobregat, Spain; c Endocrinology, Hospital Josep Trueta, Girona, Spain; d Medicine, Consorci Municipal de Terrassa, Terrassa, Spain; e Endocrinology, Hospital Arnau de Vilanova, Lleida, Spain; f Endocrinology, Hospital Parc Taulí, Sabadell, Spain; g Medicine, Hospital Granollers, Granollers, Spain; h Medicine, Hospital Figueres, Figueres, Spain; i Medicine, Fundació Althaia, Manresa, Spain; j Medicine, Hospital Santa Caterina, Girona, Spain; k Medicine, Hospital Sagrat Cor, Barcelona, Spain; l Medicine, Hospital Igualada, Igualada, Spain; m Medicine, Hospital Municipal Badalona, Badalona, Spain; n Medicine, Hospital Blanes, Blanes, Spain; o Medicine, Hospital Sant Pau, Barcelona, Spain; p Endocrinology, Hospital Sant Joan Despí, Sant Joan Despí, Spain; q Biochemistry, Hospital Sant Pau, Sant Sant Pau, Spain
Objectives: More than 1500 mutations causing Familial Hypercholesterolemia (FH) have been described worldwide, with a mutation spectrum that varies depending on the country. Classically, FH mutation detection is carried out by Sanger sequencing and MLPA, since Copy Number Variations (CNVs) account for 5-10% of LDLR mutations. Between 2008 and 2012, we have used several versions of a microarray (LIPOchip) for detecting the (around 250) most frequent mutations in the European population. Because of the high mutation heterogeneity, during 2013 we decided to test the Next Generation Sequencing (NGS) Roche 454 based product SEQPRO LIPO RS (Progenika Biopharma SA). We describe here the results obtained up to now with this procedure. Methods: 80 samples of FH patients living in Catalonia were screened with this kit while 55 more are pending. The characteristics and clinical significance of the variations detected were analyzed. The results were correlated the clinical diagnosis information following the Make Early Diagnosis to Prevent Early Death (MedPed) criteria. Results: 25 different variants were detected in 33 of the 80 samples tested (41%). One mutation was on the APOB gene, 2 of them were CNVs of the LDLR gene, while all the other were variants of unknown significance (VUS) (n¼7) or already validated pathogenic mutations (n¼17) of the LDLR gene. From these mutations, 8 were null alleles, 11 were amino acid changes and 5 were splicing mutations. 11 patients showed mutations that would not have been detected by LIPOchip. Average MedPed of patients having a null allele was 10.42, an aminoacid change 8, a splicing mutation 9.75, while patients in which no mutation was found had an average MedPed of 7.11. Patients having an already validated pathogenic mutation had an average MedPed of 10.76 and patients with VUS, 6.71. Conclusion: This NGS-based kit was proven adequate for FH mutation detection and characterization.
40 - Inherited dyslipidemias EAS-0337. SEARCHING NEW LOCI ASSOCIATED TO AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIA A. Cenarroa, T. Tejedorb, M. Stefc, I. de Castro-Orósa, P. Calmarzad, F. Civeiraa
Abstracts / Atherosclerosis 235 (2014) e27–e83
a
Laboratorio de Investigación Molecular, Instituto Aragonés de Ciencias de la Salud Hospital Universitario Miguel Servet, Zaragoza, Spain; b Dpto. Anatomía Embriología y Genética, Universidad de Zaragoza, Zaragoza, Spain; c Dpto. Genómica, Progenika Biopharma, Bilbao, Spain; d Servicio de Bioquímica, Hospital Universitario Miguel Servet, Zaragoza, Spain Objectives: Autosomal dominant hypercholesterolemias (ADH) are familiar defects associated to elevated levels of LDL-cholesterol and high risk of coronary heart disease. Mutations in LDLR, APOB and PCSK9 genes are the most frequent causes of ADH. However, molecular diagnosis of patients with ADH phenotype does not identify a causal mutation in 25% of cases, suggesting that the genetic background of ADH is heterogeneous. The aim of this study was to identify the ADH causal gene in subjects in whom the presence of mutation in LDLR, APOB and PCSK9 genes had been excluded. Methods: Five unrelated subjects were selected, with at least 2 available family members, with high increase of LDL-cholesterol > 99 percentile and triglyceride levels < 300 mg/dL, dominant transmission of hypercholesterolemia, with negative LIPOchip and LDLR sequencing results and normal APOE genotype. Exome sequencing and filtering of results by comparison with genetic variants in databases: dbSNP, HapMap, 1000 genomes and GWAS were carried out. Results: The following variants were obtained: Subject 1: 21 substitutions, 2 insertions/deletions; Subject 2: 21 substitutions, 1 insertion/deletion; Subject 3: 61 substitutions, 10 insertions/deletions; Subject 4: 45 substitutions, 2 insertions/deletions; Subject 5: 40 substitutions, 3 insertions/ deletions. Moreover, we found 15 genes with substitution variants and 3 genes with insertion/deletion variants matching in several subjects. Also, 9 genes related to lipid metabolism with substitution variants matching in several subjects were identified. At present, validation of identified variants with PolyPhen and SIFT programs is being carried out. Conclusion: Exome analysis of subjects with extreme phenotype can identify new genetic variants causing ADH. 40 - Inherited dyslipidemias EAS-0716. PCSK9 AND LDLRAP1 SEQUENCING ANALYSIS IN SUBJECTS WITH FAMILIAL HYPERCHOLESTEROLEMIA AND NEITHER MUTATION IN LDLR NOR APOB GENES F. Civeiraa, T. Tejedorb, M. Stefc, L. Palaciosc, R. Mateo-Gallegoa, I. Lamiquizd, I. de Castro-Orosd, A. Cenarrod a Lipid Unit, Instituto Aragonés de Ciencias de la Salud Hospital Universitario Miguel Servet, Zaragoza, Spain; b Biochemistry, Universidad de Zaragoza, Zaragoza, Spain; c I+D+I, Progenika Biopharma, Derio, Spain; d Laboratorio de Investigación, Instituto Aragonés de Ciencias de la Salud Hospital Universitario Miguel Servet, Zaragoza, Spain
Objectives: Autosomal dominant hypercholesterolemias (ADH) are familiar defects mostly due to mutations in LDLR and APOB. However, DNA sequencing analysis in ADH does not identify a causal mutation in approximately 25% of cases. Gain-of-function mutations in the PCSK9 gene (proprotein convertase subtilisin/kexin type 9), are also associated with ADH, although their frequency in ADH subjects is not known. Autosomal recessive hypercholesterolemia is a rare disease caused by mutations in LDLRAP1. If LDLRAP1 loss-of-function mutations are associated with ADH have been poorly studied. The aim was to identify the contribution of PCSK9 and LDLRAP1 in subjects with ADH in whom the presence of mutation in LDLR and APOB genes had been excluded. Methods: Two hundred and twenty nine unrelated subjects with ADH were selected. Inclusion criteria were: primary isolated high LDL-cholesterol >95 percentile, dominant transmission of hypercholesterolemia, with negative LIPOchipÒ and LDLR sequencing.
e55
PCSK9 and LDLRAP1 sequencing was performed with SEQPRO LIPO RSÒ or SEQPRO LIPO ISÒ platforms. Both platforms are PCR based products designed for the amplification of FH-linked regions (LDLR, APOB, PCSK9 and LDLRAP1 genes) and the inclusion of indexes for the identification of samples when pooled in a same Next Generation Sequencing run. For the sequencing in the Roche-454 Junior bench top device, the CE-marked RS product has been used while for the sequencing in the Illumina MiSeq bench top device the IS product was used. Results: A total of 9 subjects were heterozygous for a gain-of-function mutation in PCSK9. Seven subjects carried p.Leu22_Leu23dup mutation and 2 subjects the p.Leu23del mutation. No LDLRAP1 loss-of-function mutations were found. Conclusion: PCSK9 gain-of-function-mutations are present in approximately 4% of the subjects with ADH and neither mutation in LDLR nor APOB. Two mutations seem responsible for most cases in Spain. LDLRAP1 mutations are not found in this large group of ADH subjects. 40 - Inherited dyslipidemias EAS-0518. NOVEL-GENE-FINDING STRATEGY FOR THE AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIC PHENOTYPE S.W. Fouchier1, J.C. Defesche1 1 Experimental Vascular Medicine, Academic Medical Center, Amsterdam, Netherlands
Objectives: Autosomal Dominant Hypercholesterolemia (ADH) is a heterogeneous common disorder and uncovering molecular determinants that underlie ADH is a major focus of cardiovascular research. Despite rapid technical advances however, efforts to identify novel ADH genes has yet not been very successful and are largely challenged by the rate of nonpenetrance and phenocopies. We aimed to investigated the impact of this heterogeneity on successful novel-gene-finding, which has not been comprehensively investigated. Methods: For the ADH phenotype subjects are considered as affected according to age, gender and plasma cholesterol levels above the 95th percentile, and the disease penetrance is generally set at 0.9. These parameters were evaluated in over 20,000 individuals belonging to 1224 families, tested for true pathogenic APOB and LDLR mutations. Additionally, values of the area under the receiver operating characteristics curve (AUC) of percentiles for TC and LDL were assessed. Results: The use of the above parameters would have been successfully resulted in pinpointing the causal variant in only 40% of all families. An average penetrance at or above 0.9 with a cut-point at the 95th percentile was only observed for LDLR nonsense mutations. For APOB and LDLR missense mutations a disease penetrance equal or above 0.9 is only expected, when TC and LDL cut-points between the 75-90th percentile are chosen to determine the individuals affection status. The mean AUC for pathogenic APOB and LDLR mutations was 0.891 for TC and 0.919 for LDL, corresponding to respectively the 76th and 69th percentile. Conclusion: For the ADH phenotype, pedigree information and mapping data are essential to identify shared haplotypes, phenocopies and nonpenetrance, to be able to select the most distantly related and informative individuals before exome sequencing is applied. Additionally, TC and LDL cut-points need refinement to potentially increase success rates in novel-gen-finding related to ADH. 40 - Inherited dyslipidemias EAS-0826. DYSLIPIDEMIA AND SUSTAINED ELEVATIONS IN TRANSAMINASES FROM EARLY CHILDHOOD ARE COMMON IN LYSOSOMAL ACID LIPASE DEFICIENCY P. Deegana, B. Burtonb, M. Di Roccoc, G. Ennsd, O. Guardmagnae, S. Horslenf, G. Hovinghg, S. Lobrittoh, V. Malinovai, V. McLinj, J. Raimank, S.
Abstracts / Atherosclerosis 235 (2014) e27–e83
a
Center for Cardiovascular Prevention, Charles University and Thomayer Hospital, Prague 4, Czech Republic; b Division of Cardiovascular Diseases, Mayo Clinic, Rochester, USA; c Center for Cardiovascular Prevention, Charles University and Thomayer Hospital, Prague, Czech Republic; d 2nd Department of Internal Medicine, Charles University Center for Hypertension, Pilsen, Czech Republic; e International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic; f Division of Cardiovascular Diseases, Mayo Clinic, Rochester, USA Objectives: Increased aortic stiffness may be one of the mechanisms by which obesity increases cardiovascular risk independently of traditional risk factors. While body mass index (BMI) is generally used to define excess adiposity, several studies have suggested that measures of central obesity may be better predictors of cardiovascular risk. However, data comparing the association between several measures of central and general obesity with aortic stiffness in the general population are inconclusive. Methods: In 1031 individuals (age 5313 years, 45% men) without manifest cardiovascular disease randomly selected from the population, we tested the association between parameters of central obesity (waist circumference – WC, waist-to-hip-ratio –WHR, waist-to-height ratio –WHtR) and general obesity (BMI) with carotid-femoral pulse wave velocity (cfPWV). Results: In univariate analysis, WC and WHtR were more strongly associated with cfPWV than BMI in both genders, while WHR showed a stronger association with cfPWV only in women. WHtR was more closely associated with cfPVW than WHR. This difference between obesity measures remained after multivariate adjustment. When the fully adjusted hierarchical regression was used, among central obesity measures, WHtR had the largest additive value on top of BMI, while there was no additive value of BMI on top of WHtR. Conclusion: Central obesity parameters are more closely associated with aortic stiffness than BMI. Of central adiposity measures, WHtR has the strongest association with aortic stiffness beyond body mass index and cardiovascular risk factors. Our results suggest that WHtR may be the best anthropometric measure of excess adiposity in the general population. 40 - Inherited dyslipidemias EAS-0323. APOLIPOPROTIEN E VARIANTS IN PRIMARY HYPERLIPIDEMIAS A.M. Bea1, I. Lamiquiz-Moneo1, I. De Castro-Orós1, L. Baila-Rueda1, A. Cenarro1, F. Civeira1 1 Laboratorio Investigación Molecular, Instituto Aragonés de Ciencias de la Salud. Hospital Universitario Miguel Servet, Zaragoza, Spain
Objectives: Apolipoprotein E (ApoE) has an important role in cholesterol and triglycerides metabolism related to its function as chylomicron and LDL receptor ligand. APOE gene has been traditionally associated with type III hyperlipoproteinemia, which is mainly caused by the APOE ε2/ε2 genotype. However, other APOE mutations have been associated with other dyslipidemias. The objective was to identify rare genetics variants in APOE gene. Methods: A total of 442 unrelated consecutive subjects were recruited in the Lipid Unit at Hospital Universitario Miguel Servet with a clinical diagnosis of primary hyperlipoproteinemia. 87 were affected of familial hypercholesterolemia without a pathogenic mutation in LDLR nor APOB genes (FH-), 93 with familial hypercholesterolemia with a pathogenic mutation in LDLR or APOB genes (HF+), 158 with familial combined hyperlipidemia (FCHL), 65 with polygenic hypercholesterolemia (PH) and 39 with primary hypertriglyceridemia (HTG). 65 normolipemic subjects were analyzed as control group. In all subjects, exon 4 in APOE was sequenced and clinical and analytical variables were registered. Results: The following rare variants have been identified: p.Arg154Ser (rs121918393) in 2 FCH subjects; p.Leu167del in 3 HF-, 1 HF+, 3 FCHL and 1 HTG subjects; p.Gly145Asp (rs267606662) in 1 FCHL and 1 HF+ subjects; p.Arg163Cys (rs769455) in 1 HF-, 1 HTG and 1 FCHL subjects. It has been also identified a non previously described variant in 1 HTG subject:
p.Met82Ile. Moreover, the p.Ala217Ala variant (rs72654468) was found in 2 normolipemic subjects besides of 1 HF+ and 1 PH individuals. Conclusion: Five different APOE gene variants were identified in 16 subjects with different dyslipidemias that were not found in controls, so we consider they may be causally associated with their lipid abnormalities. In summary, our results suggest that the genetic variability in APOE gene contributes to the etiology of different dyslipidemias not only to dysbetalipoproteinemia. 40 - Inherited dyslipidemias EAS-0577. USE OF NEXT GENERATION SEQUENCING FOR THE DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLEMIA N. Planaa, R. Figuerasb, E. Estevec, M. Maurid, M. Buenoe, A. Caixàsf, E. Llarguésg, A. Vilah, C. Moralesi, C. Solerj, J. Argimonk, J. Mayosl, J. Graum, A. Zamoran, L. Mataso, L. Vilap, F. Blanco-Vacaq a Medicine, Hospital Sant Joan, Reus, Spain; b Medicine, Hospital de Bellvitge, L'Hospitalet de Llobregat, Spain; c Endocrinology, Hospital Josep Trueta, Girona, Spain; d Medicine, Consorci Municipal de Terrassa, Terrassa, Spain; e Endocrinology, Hospital Arnau de Vilanova, Lleida, Spain; f Endocrinology, Hospital Parc Taulí, Sabadell, Spain; g Medicine, Hospital Granollers, Granollers, Spain; h Medicine, Hospital Figueres, Figueres, Spain; i Medicine, Fundació Althaia, Manresa, Spain; j Medicine, Hospital Santa Caterina, Girona, Spain; k Medicine, Hospital Sagrat Cor, Barcelona, Spain; l Medicine, Hospital Igualada, Igualada, Spain; m Medicine, Hospital Municipal Badalona, Badalona, Spain; n Medicine, Hospital Blanes, Blanes, Spain; o Medicine, Hospital Sant Pau, Barcelona, Spain; p Endocrinology, Hospital Sant Joan Despí, Sant Joan Despí, Spain; q Biochemistry, Hospital Sant Pau, Sant Sant Pau, Spain
Objectives: More than 1500 mutations causing Familial Hypercholesterolemia (FH) have been described worldwide, with a mutation spectrum that varies depending on the country. Classically, FH mutation detection is carried out by Sanger sequencing and MLPA, since Copy Number Variations (CNVs) account for 5-10% of LDLR mutations. Between 2008 and 2012, we have used several versions of a microarray (LIPOchip) for detecting the (around 250) most frequent mutations in the European population. Because of the high mutation heterogeneity, during 2013 we decided to test the Next Generation Sequencing (NGS) Roche 454 based product SEQPRO LIPO RS (Progenika Biopharma SA). We describe here the results obtained up to now with this procedure. Methods: 80 samples of FH patients living in Catalonia were screened with this kit while 55 more are pending. The characteristics and clinical significance of the variations detected were analyzed. The results were correlated the clinical diagnosis information following the Make Early Diagnosis to Prevent Early Death (MedPed) criteria. Results: 25 different variants were detected in 33 of the 80 samples tested (41%). One mutation was on the APOB gene, 2 of them were CNVs of the LDLR gene, while all the other were variants of unknown significance (VUS) (n¼7) or already validated pathogenic mutations (n¼17) of the LDLR gene. From these mutations, 8 were null alleles, 11 were amino acid changes and 5 were splicing mutations. 11 patients showed mutations that would not have been detected by LIPOchip. Average MedPed of patients having a null allele was 10.42, an aminoacid change 8, a splicing mutation 9.75, while patients in which no mutation was found had an average MedPed of 7.11. Patients having an already validated pathogenic mutation had an average MedPed of 10.76 and patients with VUS, 6.71. Conclusion: This NGS-based kit was proven adequate for FH mutation detection and characterization.
40 - Inherited dyslipidemias EAS-0337. SEARCHING NEW LOCI ASSOCIATED TO AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIA A. Cenarroa, T. Tejedorb, M. Stefc, I. de Castro-Orósa, P. Calmarzad, F. Civeiraa
Abstracts / Atherosclerosis 235 (2014) e27–e83
a
Laboratorio de Investigación Molecular, Instituto Aragonés de Ciencias de la Salud Hospital Universitario Miguel Servet, Zaragoza, Spain; b Dpto. Anatomía Embriología y Genética, Universidad de Zaragoza, Zaragoza, Spain; c Dpto. Genómica, Progenika Biopharma, Bilbao, Spain; d Servicio de Bioquímica, Hospital Universitario Miguel Servet, Zaragoza, Spain Objectives: Autosomal dominant hypercholesterolemias (ADH) are familiar defects associated to elevated levels of LDL-cholesterol and high risk of coronary heart disease. Mutations in LDLR, APOB and PCSK9 genes are the most frequent causes of ADH. However, molecular diagnosis of patients with ADH phenotype does not identify a causal mutation in 25% of cases, suggesting that the genetic background of ADH is heterogeneous. The aim of this study was to identify the ADH causal gene in subjects in whom the presence of mutation in LDLR, APOB and PCSK9 genes had been excluded. Methods: Five unrelated subjects were selected, with at least 2 available family members, with high increase of LDL-cholesterol > 99 percentile and triglyceride levels < 300 mg/dL, dominant transmission of hypercholesterolemia, with negative LIPOchip and LDLR sequencing results and normal APOE genotype. Exome sequencing and filtering of results by comparison with genetic variants in databases: dbSNP, HapMap, 1000 genomes and GWAS were carried out. Results: The following variants were obtained: Subject 1: 21 substitutions, 2 insertions/deletions; Subject 2: 21 substitutions, 1 insertion/deletion; Subject 3: 61 substitutions, 10 insertions/deletions; Subject 4: 45 substitutions, 2 insertions/deletions; Subject 5: 40 substitutions, 3 insertions/ deletions. Moreover, we found 15 genes with substitution variants and 3 genes with insertion/deletion variants matching in several subjects. Also, 9 genes related to lipid metabolism with substitution variants matching in several subjects were identified. At present, validation of identified variants with PolyPhen and SIFT programs is being carried out. Conclusion: Exome analysis of subjects with extreme phenotype can identify new genetic variants causing ADH. 40 - Inherited dyslipidemias EAS-0716. PCSK9 AND LDLRAP1 SEQUENCING ANALYSIS IN SUBJECTS WITH FAMILIAL HYPERCHOLESTEROLEMIA AND NEITHER MUTATION IN LDLR NOR APOB GENES F. Civeiraa, T. Tejedorb, M. Stefc, L. Palaciosc, R. Mateo-Gallegoa, I. Lamiquizd, I. de Castro-Orosd, A. Cenarrod a Lipid Unit, Instituto Aragonés de Ciencias de la Salud Hospital Universitario Miguel Servet, Zaragoza, Spain; b Biochemistry, Universidad de Zaragoza, Zaragoza, Spain; c I+D+I, Progenika Biopharma, Derio, Spain; d Laboratorio de Investigación, Instituto Aragonés de Ciencias de la Salud Hospital Universitario Miguel Servet, Zaragoza, Spain
Objectives: Autosomal dominant hypercholesterolemias (ADH) are familiar defects mostly due to mutations in LDLR and APOB. However, DNA sequencing analysis in ADH does not identify a causal mutation in approximately 25% of cases. Gain-of-function mutations in the PCSK9 gene (proprotein convertase subtilisin/kexin type 9), are also associated with ADH, although their frequency in ADH subjects is not known. Autosomal recessive hypercholesterolemia is a rare disease caused by mutations in LDLRAP1. If LDLRAP1 loss-of-function mutations are associated with ADH have been poorly studied. The aim was to identify the contribution of PCSK9 and LDLRAP1 in subjects with ADH in whom the presence of mutation in LDLR and APOB genes had been excluded. Methods: Two hundred and twenty nine unrelated subjects with ADH were selected. Inclusion criteria were: primary isolated high LDL-cholesterol >95 percentile, dominant transmission of hypercholesterolemia, with negative LIPOchipÒ and LDLR sequencing.
e55
PCSK9 and LDLRAP1 sequencing was performed with SEQPRO LIPO RSÒ or SEQPRO LIPO ISÒ platforms. Both platforms are PCR based products designed for the amplification of FH-linked regions (LDLR, APOB, PCSK9 and LDLRAP1 genes) and the inclusion of indexes for the identification of samples when pooled in a same Next Generation Sequencing run. For the sequencing in the Roche-454 Junior bench top device, the CE-marked RS product has been used while for the sequencing in the Illumina MiSeq bench top device the IS product was used. Results: A total of 9 subjects were heterozygous for a gain-of-function mutation in PCSK9. Seven subjects carried p.Leu22_Leu23dup mutation and 2 subjects the p.Leu23del mutation. No LDLRAP1 loss-of-function mutations were found. Conclusion: PCSK9 gain-of-function-mutations are present in approximately 4% of the subjects with ADH and neither mutation in LDLR nor APOB. Two mutations seem responsible for most cases in Spain. LDLRAP1 mutations are not found in this large group of ADH subjects. 40 - Inherited dyslipidemias EAS-0518. NOVEL-GENE-FINDING STRATEGY FOR THE AUTOSOMAL DOMINANT HYPERCHOLESTEROLEMIC PHENOTYPE S.W. Fouchier1, J.C. Defesche1 1 Experimental Vascular Medicine, Academic Medical Center, Amsterdam, Netherlands
Objectives: Autosomal Dominant Hypercholesterolemia (ADH) is a heterogeneous common disorder and uncovering molecular determinants that underlie ADH is a major focus of cardiovascular research. Despite rapid technical advances however, efforts to identify novel ADH genes has yet not been very successful and are largely challenged by the rate of nonpenetrance and phenocopies. We aimed to investigated the impact of this heterogeneity on successful novel-gene-finding, which has not been comprehensively investigated. Methods: For the ADH phenotype subjects are considered as affected according to age, gender and plasma cholesterol levels above the 95th percentile, and the disease penetrance is generally set at 0.9. These parameters were evaluated in over 20,000 individuals belonging to 1224 families, tested for true pathogenic APOB and LDLR mutations. Additionally, values of the area under the receiver operating characteristics curve (AUC) of percentiles for TC and LDL were assessed. Results: The use of the above parameters would have been successfully resulted in pinpointing the causal variant in only 40% of all families. An average penetrance at or above 0.9 with a cut-point at the 95th percentile was only observed for LDLR nonsense mutations. For APOB and LDLR missense mutations a disease penetrance equal or above 0.9 is only expected, when TC and LDL cut-points between the 75-90th percentile are chosen to determine the individuals affection status. The mean AUC for pathogenic APOB and LDLR mutations was 0.891 for TC and 0.919 for LDL, corresponding to respectively the 76th and 69th percentile. Conclusion: For the ADH phenotype, pedigree information and mapping data are essential to identify shared haplotypes, phenocopies and nonpenetrance, to be able to select the most distantly related and informative individuals before exome sequencing is applied. Additionally, TC and LDL cut-points need refinement to potentially increase success rates in novel-gen-finding related to ADH. 40 - Inherited dyslipidemias EAS-0826. DYSLIPIDEMIA AND SUSTAINED ELEVATIONS IN TRANSAMINASES FROM EARLY CHILDHOOD ARE COMMON IN LYSOSOMAL ACID LIPASE DEFICIENCY P. Deegana, B. Burtonb, M. Di Roccoc, G. Ennsd, O. Guardmagnae, S. Horslenf, G. Hovinghg, S. Lobrittoh, V. Malinovai, V. McLinj, J. Raimank, S.