84 STRATEGY OF GENETIC ANALYSIS IN HYPOALPHALIPOPROTEINEMIA (HPA)

Abstracts, XXII National Congress of the Italian Society for the Study of Arteriosclerosis (SISA) 84 STRATEGY OF GENETIC ANALYSIS IN HYPOALPHALIPOPROTEINEMIA (HPA) L. Pisciotta1 , A. Bellocchio1 , R. Fresa1 , V. Guido1 , R. Sallo1 , C. Danovaro1 , E. Pino1 , A. Cantafora2 , T. Fasano3 , S. Calandra3 , S. Bertolini1 . 1 Dpt. of Internal Med, University of Genoa; 2 Nat. Inst. of Health, Rome; 3 Dpt. of Biomed. Sci., Univ. of Modena and R. Emilia, Italy E-mail: [email protected] HPA is defined by HDL-C conc. <10th perc. of the distribution in population (<34 in M and <40 mg/dl in F). The genetic contribution to HDL-C level variability in human being is about 50%: some cases are due to mutations in HDL structural genes but the majority of them is caused by mutations in genes which alter HDL composition and accelerate HDL catabolism. Our strategy of genetic analysis in HPA includes the following steps: APOE genotype (e2 and e4 may cause HPA); screening for A54T polymorphism of FABP2; screening for common variants of LPL gene (D9N and N291S); screening for common Caucasian mutations of GBA gene (N370S and L444P); screening for the mutation c.894G>A of the LIPA gene and direct sequencing in patients suspected for CESD. In most severe cases of isolated HPA (HDL-C <5th perc., <30 in M, <36 mg/dl in F) and in familial HPA we perform direct sequencing of APOA1 and LCAT genes followed by DHPLC analysis and sequencing of ABCA1 gene. In our lipid clinic we collected 163 probands with HPA. The distribution of APOE genotype was: E3E3: 62.0%, E3E4: 23.3%, E2E3: 8.0%, E2E4: 5.5%, E4E4: 1.8%; E2E2: 0.6%; the prevalence of 54TT genotype of FABP2 was 0.141; we detected LPL D9N in 3.7% and N291S in 4.9% of cases. We identified 2 probands carrying N370S mutation and 1 proband carrying L444P mutation of GBA gene. The screening for the mutation c.894G>A of the LIPA gene was positive in 1 case. We sequenced APOA1 and LCAT genes in 40 subjects and identified 3 carriers of the S208>T polymorphism of LCAT. We analyzed ABCA1 gene in 20 subjects and identified one patient heterozygous for the p.R1897W mutation. 85 A NEW ABCA1 SPLICING MUTATION IN AN ITALIAN PATIENT WITH TANGIER DISEASE L. Pisciotta1 , R. Sallo1 , T. Sampietro2 , F. Bigazzi2 , M. Puntoni2 , F. Sbrana2 , A. Bellocchio1 , R. Fresa1 , L. Bocchi3 , C. Candini3 , S. Calandra3 , S. Bertolini1 . 1 Department of Internal Medicine, University of Genoa; 2 Clinical Physiology Institute CNR, Pisa; 3 Dpt. of Biomed. Sci., Univ. of Modena and R. Emilia, Italy E-mail: [email protected] The proband was a 37 year-old male born in Empoli (PI) from a consanguineous marriage. He had been suffering from effort angina since the age of 34 and underwent PTCA and more recently CABG. The presence of very low plasma levels of HDL-C (1 mg/dl) and undetectable Apo AI level (<4.5 mg/dl), associated with hepato-splenomegaly and orange-yellow tonsils suggested the clinical diagnosis of Tangier Disease. The 62 year-old proband’s mother had low levels of HDL-C and Apo AI (25 mg/dl and 99 mg/dl, respectively). The 58 year-old proband’s father suffered from myocardial infarction at 37 and subsequently underwent PTCA and CABG. The DHPLC analysis of ABCA1 gene in the proband showed an abnormal pattern in flanking region of exon 21. The direct sequence of exon 21 and flanking regions revealed that the proband was homozygous for a A>C transversion at the position 2 of the acceptor splice site of intron 20 (IVS20 2 A>C, c.2961 2 A>C). The mutation introduces a new restriction site for the enzyme Nci I (CC/s_GG) and its screening by restriction analysis of genomic DNA of proband’s father and proband’s daughter confirmed the presence of the mutation in heterozygous form in both subjects. Molecular studies to determine the effects of the mutation on primary mRNA splicing and on ABCA1 protein are in progress. 86 A NEW SPLICING MUTATION OF LPL GENE IN AN HYPERCHYLOMICRONEMIC PATIENT WITH RECURRENT PANCREATITIS L. Pisciotta1 , L. Calabresi2 , A. Bellocchio1 , R. Fresa1 , C. Priore Oliva3 , C.R. Sirtori2 , S. Calandra3 , S. Bertolini1 . 1 University of Genoa; 2 University of Milan; 3 University of Modena and R. Emilia, Italy E-mail: [email protected] The proband was a 41 year-old female (BMI 15.2 kg/m2 ) with hypertriglyceridemia (HTG) since age 20. No satisfactory results were obtained with low-fat diet plus MCT. She had been suffering from recurrent pancreatitis since age 25. Inspection of fundus oculi revealed lipemia retinalis, eruptive xanthomas were absent. Lipid profile was TC 367, HDL-C 24, and TG 1996 mg/dl. Family history was negative for cardiovascular disease. The direct sequence of exons and flanking regions of LPL gene revealed that the proband was heterozygous for a T>G transversion at the position +2 of the donor splice site of intron 1 (c.88 +2T>G). Molecular studies to ascertain the effects of the mutation on pre-mRNA splicing are in progress. The subject was also LPL carrier of two previously reported “silent” substitutions: c.179 G>A in exon 3 (V108V) in heterozygous form and c.1338 C>A in exon 8 (T361T) in homozygous form. APOA5 sequencing did not reveal pathogenic mutations but only common variants in heterozygous form: c.56C>G (S19W); c.132C>A (I144I); c.457G>A (V153M); c.1177C>T in 3 -UTR region. The patient was homozygous (TT) for the common allele of the 1131 T>C polymorphism of APOA5 promoter. In addition, she was found to be heterozygous for the 482 C>T polymorphism of APOC3 gene. APOE genotype was E3E3. The LPL

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mutation, even though in heterozygous form, combined with heterozygosity for c.56C>G (S19W) of APOA5 and 482 C>T of APOC3, which were found to be associated with increased TG levels in several studies, might explain the severe lipoprotein phenotype of this patient. 87 A NOVEL MUTATION OF THE LIPA GENE FOUND IN A PATIENT WITH CHOLESTEROL ESTER STORAGE DISEASE (CESD): UNUSUAL FINDING FOR THE OLD AGE OF THE PROBAND L. Pisciotta1 , R. Fresa1 , A. Bellocchio1 , R. Sallo1 , V. Guido1 , A. Cantafora2 , S. Calandra3 , S. Bertolini1 . 1 Dpt. of Internal Medicine, Univ. of Genoa; 2 Nat. Inst. of Health, Rome; 3 Dpt. of Biomed. Sci., Univ. of Modena and R. Emilia, Italy E-mail: [email protected] CESD is a recessive disorder due to mutations of the LIPA gene encoding LAL (Lysosomal Acid Lipase). LAL hydrolyzes CE and TG that have been internalized via receptor-mediated endocytosis of lipoprotein particles. CESD is present in childhood but frequently unrecognised until adulthood with a broad spectrum of severity. The proband was a 82 year-old female (BMI 17.6 kg/m2 ) with hepato-splenomegaly, myelo-dysplasia, severe carotid atherosclerosis and tendon xanthomatosis. Mean lipid values before any treatment were: TC 393±26, TG 295±40, HDL-C 21±4, Apo AI 87.2, Apo B 197.8 mg/dl, AST 42.6, ALT 45 UI/L. The proband’s DNA was collected several years ago in order to study candidate genes for hypercholesterolemia. The sequencing of LDLR, Apo B and PCSK9 genes did not show any pathogenic abnormality. More recently, in view of the low level of HDL-C and Apo AI found in the patient over the years we searched for mutations in the major candidate gene, with negative findings. So, supported by the presence of hepato-splenomegaly, we screened the LIPA gene for the common mutation (c.894 G>A in exon 8) associated with CESD in 60% of the cases reported in literature and found that the patient carried this mutation. Then all exons and flanking regions of the LIPA gene were sequenced to find the expected second mutation and found a complex frameshift mutation in exon 4 of the other allele: c.230 ins 35nt, del 14 nt >Fs after p.G77>X82. The proband’s daughter, who had a mild hypercholesterolemia (TC 258, LDL-C 169, TG 55, HDL-C 78, Apo AI 198, Apo B 118 mg/dl), was a carrier of the c.894 G>A mutation in exon 8. 88 RENAL FUNCTION AND HYPOLIPIDEMIC TREATMENT IN OUTPATIENT OF A LIPID CLINIC S. Postorivo, L. Testoni, F. Bonetti, R. Fontan, A. Masieri, E. Menegatti, G.B. Vigna, R. Fellin. Department of Clinical and Experimental Medicine, University of Ferrara, Italy E-mail: [email protected] About 5 10% of the adult population in Italy is affected by chronic kidney disease (CKD). Because of their atherogenic lipid profile, CKD patients are at increased cardiovascular risk; at the same time dyslipidemia may favour CKD emergence. We evaluated the prevalence and characteristics of renal function in hyperlipidemic subjects addressed to our Metabolic Unit in a 30month period. All consecutive patients with creatinine determination were subdivided into functional classes based on their Glomerular Filtration Rate (GFR) evaluated through Cockroft formula. About two thirds of 2034 hyperlipidemic subjects showed at least some degree of renal damage: 47% were class I or mild CKD (GFR 60 89 ml/min), 18% class II or moderate CKD (GFR 30 59 ml/min) and less than 1% class III or severe CKD (<30 ml/min); hypertensive patients were overrepresented, while diabetics and pre-diabetics did not disclosed any significant trend. Basal plasma lipid levels increased slightly but significantly according to the severity of renal impairment. Before index visit, 65.5% of CKD patients (any class) received lipid-lowering treatment; 61% of subjects showed a lipid profile not requiring treatment modification (32% received only behavioural prescriptions). After index visit, statins were the most prescribed drugs (49% of patients, either lipophilic or hydrophilic) followed by omega-3 fatty acids (4.5%), fibrates (2.8%), bile acid sequestrants (1.1%) and ezetimibe (0.5%). All drugs were well tolerated: a similar rate of adverse drug reaction (in particular muscular, enzymatic and allergic ones) was reported (9.8%, 8.8%, 9.6% and 15.4% in normal and CKD class I III respectively, p = n.s.). In conclusion, light to moderate degree CKD is a common occurrence in dyslipidemic patients, but it does not modify lipid-lowering treatment attitude. 89 IMPACT OF APOLIPOPROTEIN E (apoE) DEFICIENCY ON REVERSE CHOLESTEROL TRANSPORT IN VIVO F. Pot` ı, M. Pedrelli, S. Costa, G. Stomeo, F. Bernini, I. Zanotti. Dept. of Pharmacological and Biological Sciences and Appl. Chemistries, University of Parma, Italy E-mail: [email protected] ApoE is a 34kDa glicoprotein contained in all lipoproteins other than LDL. The physiological role consists in promoting the hepatic clearance of HDL, VLDL, IDL and chylomicrons. Both in humans and animal models, lack or mutations on apoE gene can modify lipoprotein plasma profile. Mice lacking apoE exhibit abnormal deposition of lipids in the proximal aorta and liver even at 3 months of age, indicating that the lack of this apolipoprotein is sufficient to initiate the atherogenic process. This is possibly related to a severe alteration of plasma lipoprotein profile that impairs plasma efflux potential and consequently the atheroprotective process of