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594 NOVEL MUTATIONS IN SAR1B AND MTP GENES IN CHYLOMICRON RETENTION DISEASE AND ABETALIPOPROTEINEMIA
79th EAS Congress
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
Conclusions: These data suggest that some of these novel missense mutations of PCSK9 found only in hypocholesterolemic individuals are to be considered loss of function mutations. 591 FAMILIAL HYPERCHOLESTEROLEMIA IN MEXICO. AN APPROXIMATION TO THE MUTATIONAL DIVERSITY 1 1 G. Vaca1 , A. Vazquez ` , T. Magana ˜ 1 , L. Ram`ırez1 , I. Davalos ` , B. Mar`ın2 , ` Biomedica ` de Occidente, E. Mart`ınez3 , G. Carrillo3 . 1 Centro de Investigacion Instituto Mexicano del Seguro Social, 2 Hospital de Especialidades, Centro ` Medico Nacional de Occidente, Instituto Mexicano del Seguro Social, 3 Hospital ` de Especialidades, Centro Medico Nacional de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Mexico Objective: To define the genetic cause of autosomal dominant hypercholesterolemia (ADH) in probands with a clinical and/or biochemical diagnosis and to perform family genetic cascade screening. Patients and Methods: Sequencing analysis of the LDL receptor (LDLR) and the APOB genes in 62 index cases. Results: In 37 (60%) index cases the genetic cause of ADH was defined.36 probands had ADH due to a loss-of-function mutations at the LDLR and 1 at the APOB genes respectively. The clinical and biochemical phenotypes were more severe in the group of mutation positive indiiduals than in in the mutation negative group. Sequencing analysis and/or PCR-restriction enzyme analysis performed in 269 index cases’ relatives lead to the identification of 162 individuals with ADH (49 subjects aged <18 years). Among the 24 LDLR mutation identified: (a) four are novel c.695 −1G>T; c.1034_1035insA; c.1586 G>A; and c.2264_2273del; and the four fulfill the criteria to assign pathogenicity to a DNA sequence change; (b) five were observed in 3 or more apparently unrelated index cases; and (c) the more common were c.682 G>A (FH-Mexico); c.1055 G>A (FH-Mexico 2); and c.1090 T>C (FH Mexico 3). Conclusions: These results suggest that in Mexico ADH by mutations at the LDLR gene shows allelic heterogeneity with 5 mutations relatively common and that mutations at the APOB gene are not a common cause of ADH.It is expected that the early detection and the long term treatment will produce a reduction in the high cardiovascular risk and an increment in the life expectancy of the patients. 592 MUTATION SPECTRUM IN THE LDL RECEPTOR GENE IN PATIENTS FROM THE SOUTH-EASTERN PART OF POLAND M. Walus-Miarka1 , B. Idzior-Walus1 , M. Sanak2 , B. Katra1 , M. Malecki1 , J. Starzyk3 , M. Wojcik3 , S. Dziedzina2 . 1 Department of Metabolic Diseases, 2 Department of Medicine, 3 Department of Pediatric and Adolescent Endocrinology, Jagiellonian University School of Medicine, Krakow, Poland The purpose of this investigation was to identify a frequency and spectrum of mutations in the LDL-receptor (LDLR) gene in 48 families with clinically diagnosed familial hypercholesterolemia (FH) from the South-Eastern part of Poland. Mutational screening with exon by exon sequencing analysis was performed in all patients. DNA of patients, whose exons sequences did not have any mutations were screened using MLPA assay to detect major rearrangements of LDLR gene. In probands without mutations in the LDLR gene, direct sequencing of the apo-B gene was performed. In 29 probands we detected altogether 13 mutations of LDLR, including 10 missense (one novel recurrent mutation), 1 frameshift, 1 nonsense and 1 splice site. The novel mutation 1079 G>T (Cys308Phe) in the exon 7 o LDLR gene was found in three unrelated probands with FH. Analysis of the receptor activity of peripheral blood lymphocytes by binding and uptake of DiL-LDL showed a significant reduction (by 24% vs. healthy control) of the fluorescent label in the cells of patients carrying this mutation. Major rearrangements of LDLR were found in two patients. In 4 remaining patients mutation in apo-B gene was found. A one fifth of patients studied for LDLR mutations had an intron 7 variant IVS7+10GC, which could potentially interfere with mRNA processing. Our results indicate a high prevalence of missense mutations of LDLR, in line with other reports. Elevated frequency of the common intronic variant in our of FH patients suggest need for functional studies of mRNA splicing in FH. 593 NOVEL RARE VARIANTS OF APOA5 GENE IN PATIENTS WITH SEVERE HYPERTRIGLYCERIDEMIA L. Pisciotta1 , R. Fresa1 , V. Guido1 , A. Bellocchio1 , S. Calandra2 , S. Bertolini1 . 1 Department of Internal Medicine, University of Genoa, Genoa, 2 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy Apolipoprotein A-V encoded by APOA5 gene affects plasma triglycerides (TG) by increasing lipoprotein Lipase (LPL) activity. Hypertriglyceridemia (HTG) was observed in Apoa5 knockout mice, in patients with truncation causing mutations of apo A-V and in individuals carrying rare APOA5 gene SNPs. Aim of this study was to ascertain the presence of APOA5 variants in a group of 101 HTG patients without mutations in LPL, APOC2, LMF1 and GPIHBP1. Two kindred with novel apo A-V variants were identified. Proband E.C. was a 32 year-old overweight male (BMI > 30.2 kg/m2 ) with plasma TG ranging from 7.17
125
to 11.73 mmol/L and no history of pancreatitis, diabetes or alcohol consumption. However, his diet was rich in fat and simple sugars. He was a compound heterozygous for a novel variant (c.758 T>C, p.L253P) and the common TGraising c.56 C>G (p.S19W) (5*3 haplotype). In silico analysis predicted the p.L253P to be probably damaging. The proband’s father and brother carriers of p.L253P variant only, had mildly elevated plasma TG (7.85 and 2.10 mmol/L). Proband Z.G. was a 41 year-old male (BMI > 24.4 kg/m2 ) with plasma TG ranging from 7.26 to 13.56 mmol/L and no history of pancreatitis, diabetes or alcohol abuse. He was heterozygous for a minute deletion (c.293–295 del AGG) in exon 4, which eliminates a glutamic acid residue at position 98 or 99 of ApoAV, and carried in trans the TG-raising 5*2 haplotype. These novel APOA5 variants, not found in 200 controls, predispose to severe HTG in the presence of other genetic or nutritional factors 594 NOVEL MUTATIONS IN SAR1B AND MTP GENES IN CHYLOMICRON RETENTION DISEASE AND ABETALIPOPROTEINEMIA T. Fancello1 , M. Najah2 , A.L. Magnolo1 , A. Jelassi2 , E. Di Leo1 , N. Slimene2 , P. Tarugi1 . 1 Dept. of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy, 2 Research Unit of Genetic and Biological Factors of Atherosclerosis, University of Monastir, Monastir, Tunisia Object of the study: Primary Hypobetalipoproteinemias include three genetic conditions: Abetalipoproteinemia (ABL) and Chylomicron Retention Disease (CMRD) with recessive transmission and severe clinical manifestations related to intestinal lipid malabsoption and Familial Hypobetalipoproteinemia (FHBL) with dominant transmission often associated with fatty liver and mild intestinal fat malabsorption. Methods and Results: We investigated two Tunisian children with a similar clinical phenotype characterized by diarrhea, retarded growth, and the presence of lipid droplets in enterocytes. Proband #805, a 7-year old girl, had a lipid profile and mode of transmission of the lipid trait consistent with heterozygous FHBL. Genetic analysis showed that she had no mutations in APOB and MTP genes. The analysis of SAR1B gene showed that she was homozygous for a novel mutation (c.184 G>A) in exon 4 which causes a non conservative amino acid substitution (p.Glu62Lys). This change involves a highly conserved amino acid and “in silico” is predicted to be possibly damaging. Proband #AB5, a 8-month old girl, had very low plasma cholesterol level and undetectable LDL-C and apoB, suggesting the diagnosis of ABL. MTP gene sequencing showed that she was homozygous for a single nucleotide substitution in intron 16 involving the donor splice site (c.2342+1G>A). This novel mutation is predicted to abolish the function of the canonical donor splice site causing a splicing defect on MTP mRNA. Conclusion: This study demonstrates that patients with ABL and CRD sometime show a similar clinical phenotype despite a different lipid profile. 595 CORRELATION OF GENETIC VARIATIONS OF GENES INVOLVED IN LOW HIGH DENSITY LIPOPROTEINS CHOLESTEROL SYNTHESIS IN INDIANS T. Ashavaid1 , A. Sawant2 , R. Mankeshwar3 . 1 Biochemistry, 2 Research Laboratories, 3 Research Department, P. D. Hinduja National Hospital and Medical Research Centre, Mumbai, India Background: The excess burden of Coronary Artery Disease (CAD) amongst Asians is primarily due to dyslipidemia mainly characterized by low levels of High density lipoprotein-cholesterol (HDL-C). Literature states that only 14% of Asian Indian men and 5% of women have optimal HDL-C level. Therefore the study objective was to identify the genetic variants responsible for low levels of HDL-C in Indian population. Materials and Methods: A case-control study was conducted on 130 subjects attending Health-Check program at P. D. Hinduja Hospital. 65 subjects with low HDL-C (<5th percentile) formed the study group and 65 healthy subjects with high HDL-C (>95th percentile) formed the control group. Blood samples were collected and DNA was extracted. The entire APOA1, ATP-Binding Cassette A-1 (ABCA1) and Lecithin cholesterol acyltransferase (LCAT) genes were amplified by PCR. SSCP analysis was carried out individually for each region to determine genetic variations. Regions showing abnormal electrophoretic mobility on SSCP gels were sequenced. Results: Mutation analysis of entire APO A1 gene revealed 6 genetic variations. Of 50 exons analyzed of ABCA1, we identified 23 genetic variations. The variations E868G, R1082C, R639Q, V825I and E1172G were the novel mutations that were observed. Of the total 6 exons of LCAT, 2 intronic variations were observed. A significant (p = 0.0001) correlation was observed between HDL-C and APOA1 levels. Conclusion: Multiple variants in three genes responsible for low HDL-C may lead to rise in severity of CAD and can emerge as an important tool in identification of patients predisposed to developing atherosclerosis.
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
Conclusions: These data suggest that some of these novel missense mutations of PCSK9 found only in hypocholesterolemic individuals are to be considered loss of function mutations. 591 FAMILIAL HYPERCHOLESTEROLEMIA IN MEXICO. AN APPROXIMATION TO THE MUTATIONAL DIVERSITY 1 1 G. Vaca1 , A. Vazquez ` , T. Magana ˜ 1 , L. Ram`ırez1 , I. Davalos ` , B. Mar`ın2 , ` Biomedica ` de Occidente, E. Mart`ınez3 , G. Carrillo3 . 1 Centro de Investigacion Instituto Mexicano del Seguro Social, 2 Hospital de Especialidades, Centro ` Medico Nacional de Occidente, Instituto Mexicano del Seguro Social, 3 Hospital ` de Especialidades, Centro Medico Nacional de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Mexico Objective: To define the genetic cause of autosomal dominant hypercholesterolemia (ADH) in probands with a clinical and/or biochemical diagnosis and to perform family genetic cascade screening. Patients and Methods: Sequencing analysis of the LDL receptor (LDLR) and the APOB genes in 62 index cases. Results: In 37 (60%) index cases the genetic cause of ADH was defined.36 probands had ADH due to a loss-of-function mutations at the LDLR and 1 at the APOB genes respectively. The clinical and biochemical phenotypes were more severe in the group of mutation positive indiiduals than in in the mutation negative group. Sequencing analysis and/or PCR-restriction enzyme analysis performed in 269 index cases’ relatives lead to the identification of 162 individuals with ADH (49 subjects aged <18 years). Among the 24 LDLR mutation identified: (a) four are novel c.695 −1G>T; c.1034_1035insA; c.1586 G>A; and c.2264_2273del; and the four fulfill the criteria to assign pathogenicity to a DNA sequence change; (b) five were observed in 3 or more apparently unrelated index cases; and (c) the more common were c.682 G>A (FH-Mexico); c.1055 G>A (FH-Mexico 2); and c.1090 T>C (FH Mexico 3). Conclusions: These results suggest that in Mexico ADH by mutations at the LDLR gene shows allelic heterogeneity with 5 mutations relatively common and that mutations at the APOB gene are not a common cause of ADH.It is expected that the early detection and the long term treatment will produce a reduction in the high cardiovascular risk and an increment in the life expectancy of the patients. 592 MUTATION SPECTRUM IN THE LDL RECEPTOR GENE IN PATIENTS FROM THE SOUTH-EASTERN PART OF POLAND M. Walus-Miarka1 , B. Idzior-Walus1 , M. Sanak2 , B. Katra1 , M. Malecki1 , J. Starzyk3 , M. Wojcik3 , S. Dziedzina2 . 1 Department of Metabolic Diseases, 2 Department of Medicine, 3 Department of Pediatric and Adolescent Endocrinology, Jagiellonian University School of Medicine, Krakow, Poland The purpose of this investigation was to identify a frequency and spectrum of mutations in the LDL-receptor (LDLR) gene in 48 families with clinically diagnosed familial hypercholesterolemia (FH) from the South-Eastern part of Poland. Mutational screening with exon by exon sequencing analysis was performed in all patients. DNA of patients, whose exons sequences did not have any mutations were screened using MLPA assay to detect major rearrangements of LDLR gene. In probands without mutations in the LDLR gene, direct sequencing of the apo-B gene was performed. In 29 probands we detected altogether 13 mutations of LDLR, including 10 missense (one novel recurrent mutation), 1 frameshift, 1 nonsense and 1 splice site. The novel mutation 1079 G>T (Cys308Phe) in the exon 7 o LDLR gene was found in three unrelated probands with FH. Analysis of the receptor activity of peripheral blood lymphocytes by binding and uptake of DiL-LDL showed a significant reduction (by 24% vs. healthy control) of the fluorescent label in the cells of patients carrying this mutation. Major rearrangements of LDLR were found in two patients. In 4 remaining patients mutation in apo-B gene was found. A one fifth of patients studied for LDLR mutations had an intron 7 variant IVS7+10GC, which could potentially interfere with mRNA processing. Our results indicate a high prevalence of missense mutations of LDLR, in line with other reports. Elevated frequency of the common intronic variant in our of FH patients suggest need for functional studies of mRNA splicing in FH. 593 NOVEL RARE VARIANTS OF APOA5 GENE IN PATIENTS WITH SEVERE HYPERTRIGLYCERIDEMIA L. Pisciotta1 , R. Fresa1 , V. Guido1 , A. Bellocchio1 , S. Calandra2 , S. Bertolini1 . 1 Department of Internal Medicine, University of Genoa, Genoa, 2 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy Apolipoprotein A-V encoded by APOA5 gene affects plasma triglycerides (TG) by increasing lipoprotein Lipase (LPL) activity. Hypertriglyceridemia (HTG) was observed in Apoa5 knockout mice, in patients with truncation causing mutations of apo A-V and in individuals carrying rare APOA5 gene SNPs. Aim of this study was to ascertain the presence of APOA5 variants in a group of 101 HTG patients without mutations in LPL, APOC2, LMF1 and GPIHBP1. Two kindred with novel apo A-V variants were identified. Proband E.C. was a 32 year-old overweight male (BMI > 30.2 kg/m2 ) with plasma TG ranging from 7.17
125
to 11.73 mmol/L and no history of pancreatitis, diabetes or alcohol consumption. However, his diet was rich in fat and simple sugars. He was a compound heterozygous for a novel variant (c.758 T>C, p.L253P) and the common TGraising c.56 C>G (p.S19W) (5*3 haplotype). In silico analysis predicted the p.L253P to be probably damaging. The proband’s father and brother carriers of p.L253P variant only, had mildly elevated plasma TG (7.85 and 2.10 mmol/L). Proband Z.G. was a 41 year-old male (BMI > 24.4 kg/m2 ) with plasma TG ranging from 7.26 to 13.56 mmol/L and no history of pancreatitis, diabetes or alcohol abuse. He was heterozygous for a minute deletion (c.293–295 del AGG) in exon 4, which eliminates a glutamic acid residue at position 98 or 99 of ApoAV, and carried in trans the TG-raising 5*2 haplotype. These novel APOA5 variants, not found in 200 controls, predispose to severe HTG in the presence of other genetic or nutritional factors 594 NOVEL MUTATIONS IN SAR1B AND MTP GENES IN CHYLOMICRON RETENTION DISEASE AND ABETALIPOPROTEINEMIA T. Fancello1 , M. Najah2 , A.L. Magnolo1 , A. Jelassi2 , E. Di Leo1 , N. Slimene2 , P. Tarugi1 . 1 Dept. of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy, 2 Research Unit of Genetic and Biological Factors of Atherosclerosis, University of Monastir, Monastir, Tunisia Object of the study: Primary Hypobetalipoproteinemias include three genetic conditions: Abetalipoproteinemia (ABL) and Chylomicron Retention Disease (CMRD) with recessive transmission and severe clinical manifestations related to intestinal lipid malabsoption and Familial Hypobetalipoproteinemia (FHBL) with dominant transmission often associated with fatty liver and mild intestinal fat malabsorption. Methods and Results: We investigated two Tunisian children with a similar clinical phenotype characterized by diarrhea, retarded growth, and the presence of lipid droplets in enterocytes. Proband #805, a 7-year old girl, had a lipid profile and mode of transmission of the lipid trait consistent with heterozygous FHBL. Genetic analysis showed that she had no mutations in APOB and MTP genes. The analysis of SAR1B gene showed that she was homozygous for a novel mutation (c.184 G>A) in exon 4 which causes a non conservative amino acid substitution (p.Glu62Lys). This change involves a highly conserved amino acid and “in silico” is predicted to be possibly damaging. Proband #AB5, a 8-month old girl, had very low plasma cholesterol level and undetectable LDL-C and apoB, suggesting the diagnosis of ABL. MTP gene sequencing showed that she was homozygous for a single nucleotide substitution in intron 16 involving the donor splice site (c.2342+1G>A). This novel mutation is predicted to abolish the function of the canonical donor splice site causing a splicing defect on MTP mRNA. Conclusion: This study demonstrates that patients with ABL and CRD sometime show a similar clinical phenotype despite a different lipid profile. 595 CORRELATION OF GENETIC VARIATIONS OF GENES INVOLVED IN LOW HIGH DENSITY LIPOPROTEINS CHOLESTEROL SYNTHESIS IN INDIANS T. Ashavaid1 , A. Sawant2 , R. Mankeshwar3 . 1 Biochemistry, 2 Research Laboratories, 3 Research Department, P. D. Hinduja National Hospital and Medical Research Centre, Mumbai, India Background: The excess burden of Coronary Artery Disease (CAD) amongst Asians is primarily due to dyslipidemia mainly characterized by low levels of High density lipoprotein-cholesterol (HDL-C). Literature states that only 14% of Asian Indian men and 5% of women have optimal HDL-C level. Therefore the study objective was to identify the genetic variants responsible for low levels of HDL-C in Indian population. Materials and Methods: A case-control study was conducted on 130 subjects attending Health-Check program at P. D. Hinduja Hospital. 65 subjects with low HDL-C (<5th percentile) formed the study group and 65 healthy subjects with high HDL-C (>95th percentile) formed the control group. Blood samples were collected and DNA was extracted. The entire APOA1, ATP-Binding Cassette A-1 (ABCA1) and Lecithin cholesterol acyltransferase (LCAT) genes were amplified by PCR. SSCP analysis was carried out individually for each region to determine genetic variations. Regions showing abnormal electrophoretic mobility on SSCP gels were sequenced. Results: Mutation analysis of entire APO A1 gene revealed 6 genetic variations. Of 50 exons analyzed of ABCA1, we identified 23 genetic variations. The variations E868G, R1082C, R639Q, V825I and E1172G were the novel mutations that were observed. Of the total 6 exons of LCAT, 2 intronic variations were observed. A significant (p = 0.0001) correlation was observed between HDL-C and APOA1 levels. Conclusion: Multiple variants in three genes responsible for low HDL-C may lead to rise in severity of CAD and can emerge as an important tool in identification of patients predisposed to developing atherosclerosis.