Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels.

Plasma high density lipoprotein (HDL) levels are strongly genetically determined and show a general inverse relationship with coronary heart disease (CHD). The cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and is a key participant in the reverse transport of cholesterol from the periphery to the liver. A high prevalence of two different CETP gene mutations (D442G, 5.1%; intron 14G:A, 0.5%), was found in 3,469 men of Japanese ancestry in the Honolulu Heart Program and mutations were associated with decreased CETP (-35%) and increased HDL chol levels (+10% for D442G). However, the overall prevalence of definite CHD was 21% in men with mutations and 16% in men without mutations. The relative risk (RR) of CHD was 1.43 in men with mutations (P < .05); after adjustment for CHD risk factors, the RR was 1.55 (P = .02); after additional adjustment for HDL levels, the RR was 1.68 (P = .008). Similar RR values were obtained for the D442G mutation alone. Increased CHD in men with mutations was primarily observed for HDL chol 41-60 mg/dl; for HDL chol > 60 mg/dl men with and without mutations had low CHD prevalence. Thus, genetic CETP deficiency appears to be an independent risk factor for CHD, primarily due to increased CHD prevalence in men with the D442G mutation and HDL cholesterol between 41 and 60 mg/dl. The findings suggest that both HDL concentration and the dynamics of cholesterol transport through HDL (i.e., reverse cholesterol transport) determine the anti-atherogenicity of the HDL fraction.     

心脑血管疾病患者胆固醇酯转运蛋白及某些基因缺陷

目的 分析心脑血管疾病患者和健康人血清胆固醇酯转运蛋白（CETP）水平、CETPD442G和114A突变频率和基因突变者血脂异常的特点。方法 94例心肌梗死、110例脑卒中和335名健康人,应用ELISA测定GETP浓度;合成^14C胆固醇酯标记的含载脂蛋白AI的双盘状、双层颗粒作基质测定GETP活性;并采用聚合酶链反应－限制性片断长度多态性分析CETP基因突变。结果 心梗组、脑卒中组CETP水平较健康人明显增高。心梗组、脑卒 中组和健康人D442G检出率分别为3.5%、3.6%和5％;114A检出率分别为1.2%、0.9%和1％,其中1名为健康人D442G纯合子。心梗、脑卒中患者两种CETP基因突变频率与健康人无差别。 基因突变者ETP浓度和活性仅为非基因突变者1／3,并伴有血清高密度脂蛋白胆固醇升高,甘油三酯降低。结论 心脑血管疾病患者CETP水平升高;CETP基因突变者有显著的脂蛋白异常。     

CETP gene mutation (D442G) increases low-density lipoprotein particle size in patients with coronary heart disease

BACKGROUND: Small, dense low-density lipoprotein (LDL) in subjects with the atherogenic pattern B has been established as a risk factor of atherosclerosis. Cholesteryl ester transfer protein (CETP) plays an important role in the transfer and exchange of cholesteryl esters and triglycerides between the lipoprotein classes of human plasma. It has been shown that CETP can also change the particle size of high-density lipoprotein (HDL) and LDL subfractions in vitro. Previous clinical studies about CETP gene mutations mainly focused on abnormalities in HDL, few involved those in LDL. OBJECTIVES: To investigate the effect of the D442G mutation in the CETP gene on major peak size of LDL particles in patients with coronary heart diseases (CHD). METHODS: D442G mutation in the CETP gene was detected using the PCR-RFLP. LDL particles sizes were analyzed by 2-16% nondenaturing polyacrylamide gradient gels in CHD patients with D442G mutation in the CETP gene. RESULTS: Six heterozygotes and one homozygote were found to have the D442G mutation among 200 CHD patients. The frequency of this mutation was 3.5%. The major peak size of LDL in patients with gene mutation (n=7) was significantly larger than that in patients without the mutation (n=40) (26.92 +/- 0.79 nm vs. 25.71 +/- 0.66 nm, respectively; P<0.01). All the patients with the gene mutation expressed pattern A, whereas only about half of the patients without the mutation expressed this pattern. The patients with gene mutation had decreased plasma CETP concentration, while increased concentration of HDL-C and apolipoprotein A-I compared with controls. CONCLUSIONS: CETP gene mutation (D442G) increases LDL particle size. This suggests that CETP play an antiatherogenic role.     

Higher frequency of abnormal serum angiopoietin-like protein 3 than abnormal cholesteryl ester transfer protein in Japanese hyperalphalipoproteinemic subjects

BACKGROUND: Either a decrease of cholesteryl ester transfer protein (CETP) or an increase of angiopoietin-like protein 3 (ANGPTL3) in plasma has been shown to increase HDL-cholesterol (HDL-C) levels. However, as yet, it is not known which protein is more strongly associated with the modulation of HDL in the Japanese hyperalphalipoproteinemic (HALT) subjects. METHODS: The serum concentration of ANGPTL3 and CETP, together with total cholesterol (TC), triglycerides (TG), adiponectin and ApoE phenotypes were determined in three groups with different HDL-C concentrations: low, <40 mg/dl (n=51); normal, 40-90 mg/dl (n=126) and high, >90 mg/dl (n=89) in the average Japanese population. RESULTS: The normal range (mean+/-2SD) of serum ANGPTL3 (218+/-144 ng/ml) and CETP (1.29+/-0.90 microg/ml) were determined in cases with 40-90 mg/dl HDL-C concentration. The frequency of abnormally high ANGPTL3 cases (>362 ng/ml) were found to be significantly greater (44%) compared with those of low CETP cases (<0.39 microg/ml, 4.5%) in HALT cases (>90 mg/dl). ANGPTL3 showed a high correlation with HDL-C (r=0.67, P<0.0001) and adiponectin (r=0.57, P<0.0001), but not with CETP. CONCLUSION: In average Japanese population, abnormally higher frequency of increased ANGPTL3 prevail in HALT cases as compared with cases with low CETP. These findings suggest that ANGPTL3, the inhibitor of endothelial lipase, may be more strongly associated with increased HDL-C rather than CETP in plasma. Accordingly, ANGPTL3 seems to be a better target for the modulation of HDL-C.     

A novel missense mutation C127R (FH Zagreb) in the LDL-receptor gene.

We employed the analysis of single-strand conformation polymorphisms to identify mutations in exon 4 of the low density lipoprotein receptor gene causing familial hypercholesterolemia. Three familial hypercholesterolemia heterozygotes had abnormal single-strand conformation polymorphism patterns. DNA sequencing revealed that the abnormal pattern of exon 4A was due to heterozygosity (T/C) at nucleotide 442. Nucleotide 442 is the first base of codon 127, and the T-->C mutation (C127R) changes this codon from CysTGT to ArgCGT. Abnormal patterns of exon 4B were due to heterozygosity (A/G) at nucleotide 662: nucleotide 662 is the second base of codon 200, and the A-->G mutation (D200G) changes this codon from AspGAC to GlyGGC. Mutation D200G was previously described as FH Padova, but mutation C127R (FH Zagreb) has not been reported previously. This novel mutation was confirmed by restriction endonuclease analysis with Dsa I. The screening of 420 familial hypercholesterolemia heterozygotes suggests that C127R and D200G account for about 0.7% of mutations causing familial hypercholesterolemia in Croatia.     

Cholesteryl ester transfer protein inhibitor (JTT-705) and the development of atherosclerosis in rabbits with severe hypercholesterolaemia

Cholesteryl ester transfer protein (CETP) is a major determinant of plasma levels of high-density lipoprotein-cholesterol (HDL-C) in humans. The anti-atherogenic effect of lowering CETP levels is dependent not only on HDL-C levels but also on a metabolic background of increased low-density lipoprotein or very-low-density lipoprotein. Here we investigated the effects of JTT-705, a chemical inhibitor of CETP, on the development of atherosclerosis in Japanese white rabbits fed on a high cholesterol diet. After 4 weeks on a diet of 0.25% cholesterol-containing chow, 100 mg/kg (low dose) or 300 mg/kg (high dose) JTT-705 was given, and the animals were monitored at weeks 0, 4, 8 and 12. Aortic atherosclerotic lesions were determined at the end of this period. JTT-705 induced a significant increase in HDL-C in the high-dose group [from 21+/-3 to 50+/-7 mg/dl (mean+/-S.E.M.); P <0.0001] compared with the control group (from 21+/-2 to 27+/-2 mg/dl). The atheromatous area was 60+/-9% in the high-dose group and 58+/-9% in the control group. Moreover, correlation analysis showed that triacylglycerol and non-HDL-C levels had a direct relationship with the development of atherosclerosis, but CETP activity and HDL-C levels did not. Thus the CETP inhibitor JTT-705 alone did not have an anti-atherogenic effect in our rabbit model, of severe hypercholesterolaemia suggesting a relatively minor effect of HDL-elevating therapy as compared with decreases in non-HDL-C (or triacylglycerol) levels in patients with severe hypercholesterolaemia, such as familial hypercholesterolaemia.     

A missense mutation in the cholesteryl ester transfer protein gene with possible dominant effects on plasma high density lipoproteins.

Plasma HDL are a negative risk factor for atherosclerosis. Cholesteryl ester transfer protein (CETP; 476 amino acids) transfers cholesteryl ester from HDL to other lipoproteins. Subjects with homozygous CETP deficiency caused by a gene splicing defect have markedly elevated HDL; however, heterozygotes have only mild increases in HDL. We describe two probands with a CETP missense mutation (442 D:G). Although heterozygous, they have threefold increases in HDL concentration and markedly decreased plasma CETP mass and activity, suggesting that the mutation has dominant effects on CETP and HDL in vivo. Cellular expression of mutant cDNA results in secretion of only 30% of wild type CETP activity. Moreover, coexpression of wild type and mutant cDNAs leads to inhibition of wild type secretion and activity. The dominant effects of the CETP missense mutation during cellular expression probably explains why the probands have markedly increased HDL in the heterozygous state, and suggests that the active molecular species of CETP may be multimeric.     

A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans. We identified a novel allele, designated CYP2A6*17 , which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8). We developed a genotyping method by polymerase chain reaction-restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation. The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval [CI], 5.3%-13.5%). The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209). To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli . For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 +/- 0.11 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/L). The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 +/- 0.06 and 0.64 +/- 0.07 pmol . min -1 . pmol -1 CYP, respectively. For nicotine C -oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 +/- 2.9 micromol/L and 31.3 +/- 3.1 micromol/L, respectively. The maximum velocity value of variant CYP2A6 (0.72 +/- 0.21 pmol . min -1 . pmol -1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 +/- 0.42 pmol . min -1 . pmol -1 CYP). Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C -oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type. Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 +/- 2.7, n = 12) compared with homozygotes of the wild type (11.5 +/- 10.5, n = 37). A subject with CYP2A6*17 / CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8). We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo.     

Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.

Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by single-strand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-->Cys mutation, caused by a C-->T transition at nucleotide 10800. One unrelated 59-yr-old man of Italian ancestry was found with the same mutation after screening 1,560 individuals. He had coronary heart disease, a TC of 310 mg/dl, and an LDL-C of 212 mg/dl. A total of eight individuals were found with the defect in the families of the two patients. They had an age- and sex-adjusted TC of 240 +/- 14 mg/dl and LDL-C of 169 +/- 10 mg/dl. This compares with eight unaffected family members with age- and sex-adjusted TC of 185 +/- 12 mg/dl and LDL-C of 124 +/- 12 mg/dl. In a dual-label fibroblast binding assay, LDL from the eight subjects with the mutation had an affinity for the LDL receptor that was 63% that of control LDL. LDL from eight unaffected family members had an affinity of 91%. By way of comparison, LDL from six patients heterozygous for the Arg3500-->Gln mutation had an affinity of 36%. The percentage mass ratio of the defective Cys3531 LDL to normal LDL was 59:41, as determined using the mAb MB19 and dynamic laser light scattering. Thus, the defective LDL had accumulated in the plasma of these patients. Using this mass ratio, it was calculated that the defective Cys3531 LDL particles bound with 27% of normal affinity. Deduced haplotypes using 10 apoB gene markers showed the Arg3531-->Cys alleles to be different in the two kindreds and indicates that the mutations arose independently. The Arg3531-->Cys mutation is the second reported cause of familial ligand-defective apoB.     

Low-dose effect of simvastatin (MK-733) on serum lipids, lipoproteins, and apolipoproteins in patients with hypercholesterolemia

The effects of simvastatin (MK-733), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on serum lipids, lipoproteins, and apolipoproteins were investigated in 29 patients (12 men, 17 women, aged 37 to 73) with moderate to severe hypercholesterolemia. It was given in doses of 2.5 mg/day for four months and 5 mg/day for the succeeding four months. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and apolipoprotein (apo) B decreased by 18% (263 +/- 7 mg/dl to 216 +/- 7 mg/dl, P less than 0.01), 24% (180 +/- 7 mg/dl to 136 +/- 7 mg/dl, P less than 0.01), and 21% (133 +/- 4 mg/dl to 104 +/- 3 mg/dl, P less than 0.01), respectively, four months after treatment. Similar reductions (17%, 24%, and 23%, respectively, P less than 0.01) were observed at eight months. A significant reduction in triglyceride (TG) was observed (173 +/- 15 mg/dl to 136 +/- 11 mg/dl at eight months, P less than 0.01), as was a significant increase in serum high-density lipoprotein cholesterol (HDL-C) (48 +/- 2 mg/dl to 52 +/- 2 mg/dl at eight months, P less than 0.01). However, apo AI and apo AII remained unchanged. Atherogenic indices of (TC--HDL-C)/ HDL-C, LDL-C/HDL-C, and apo B/Apo AI ratios were significantly (P less than 0.01) reduced after treatment. No significant changes were observed in lipoprotein lipase, hepatic TG lipase, and lecithin: cholesterol acyltransferase (LCAT) activities. Simvastatin was well tolerated and no critical side effects were noted in the eight-month study period. These data indicate that simvastatin, even at a low dose of 2.5 to 5 mg daily, causes consistent reductions in serum TC, LDL-C, apo B, and TG, and a rise in HDL-C and antiatherogenic lipoproteins.     

CD14C（-260）T启动子多态性对冠心病患者C反应蛋白水平的影响

目的探讨位于CD14基因启动子区域的C260T多态等位基因变异体CD14启动子区域-260位点C、T等位基因[CD14C(-260)T]启动子多态性对冠心病患者C反应蛋白水平的影响。方法通过研究82例稳定性冠心病患者的高敏C反应蛋白(hs-CRP)水平检测组织炎症。结果CD14基因中C260T多态性基因型分布如下:CC18例(22%)、TC48例(58.5%)、TT16例(19.5%)。相比于其它等位基因携带者TT型个体具有较高的hs-CRP(P=0.04)。具有较高百分比的T等位基因纯合子其hs-CRP>0.3mg/dl(P=0.01)。结论功能多态性的T纯合子在缺血性危险中是增高的,与hs-CRP>0.3mg/dl是独立相关的(P=0.004)。     

Common genetic variants of microsomal epoxide hydrolase affect warfarin dose requirements beyond the effect of cytochrome P450 2C9

BACKGROUND: Warfarin dose response is partially explained by the polymorphisms in the cytochrome P450 (CYP) 2C9 gene, affecting S -warfarin clearance, as well as by age and body weight. We examined the influence on warfarin dose requirements of candidate genes encoding microsomal epoxide hydrolase (mEH), as well as glutathione S -transferase A1 (GSTA1) components of vitamin K epoxide reductase and the gamma-glutamylcarboxylase (GGCX) gene. METHODS: We studied the effects of CYP2C9, mEH, GSTA1, and GGCX genotypes on warfarin maintenance doses, accounting for age, weight, vitamin K plasma concentrations and concurrent medications, in 100 patients undergoing therapeutic anticoagulation. RESULTS: Allele frequencies were 76.5%, 12.5%, and 11% for CYP2C9*1 , *2 , and *3 , respectively; 75% and 25% for mEH T 612 C; 75.8% and 24.2% for mEH A 691 G; 73.5% and 26.5% for GSTA1 T 631 G; and 70.5% and 29.5% for GGCX G 8762 A. Warfarin doses differed among the CYP2C9 ( 2C9*1 , 2C9*2 , and 2C9*3 ) genotype groups: 6.3 +/- 1.9 mg/d, 5.3 +/- 1.8 mg/d, and 3.8 +/- 1.7 mg/d, respectively (F = 4.83, P < .01). There were no differences in any of the other genotype groups. Among the 62 wild-type CYP2C9 patients, variant mEH T 612 C homozygotes required higher doses than heterozygotes and wild-type patients (7.5 +/- 2.9 mg/d, 6.5 +/- 4.2 mg/d, and 6.0 +/- 2.6 mg/d, respectively [F = 3.57, P = .03]). The odds ratio for requiring greater than 7 mg/d in variant mEH T 612 C patients versus wild-type patients was 3.14 (95% confidence interval, 1.47-6.67), accounting for CYP2C9. CONCLUSIONS: Variant mEH T 612 C genotypes are associated with warfarin doses of greater than 50 mg/wk beyond the effect of CYP2C9.     

两种新的F8内含子突变导致剪接异常的机制研究

目的：分析从2个血友病A家系中检出的2个未报道的内含子突变对剪接的影响,明确这2种突变的致病性,为遗传咨询提供依据。方法：通过检测相关的凝血指标,明确血友病A的诊断。进行F8相关检测,包括PCR扩增及测序分析F8的26个外显子及其侧翼序列、拷贝数检测、内含子1和内含子22倒位检测,明确致病突变。采用巢式PCR扩增外周血中的mRNA,从异位转录水平分析内含子突变对剪接的影响。针对6个短串联重复序列（short tandem repeats, STR）位点F8Up226、F8Up146、F8Int13、F8Int25、F8Down48和DXS1073进行家系遗传连锁分析,用SNaPshot SNP分型技术检测外周血、口腔黏膜细胞和毛囊细胞嵌合情况,分析突变来源。结果：家系1中血友病A先证者1的 FⅧ：C为0.9%,检测到F8的9号内含子c.1444-2dupA突变,mRNA分析显示该突变导致F8的10号和11号外显子缺失;家系2中血友病A先证者2的 FⅧ：C为5.1%,检测到F8的18号内含子c.5999-29T>G突变,mRNA分析显示该突变产生2种转录本,即缺失19号外显子的异常转录本和少量正常转录本。家系1无血友病A家族史,遗传分析显示突变来源于先证者的外公,但外公的血液、毛发和口腔样本嵌合检测均未检出突变。结论：c.1444-2dupA和c.5999-29T>G突变均为国际首次报道,突变导致了不同程度的剪接异常,分别引起重型和轻型血友病A。家系1的c.1444-2dupA为新发突变,可能是在先证者外公的精子形成过程中发生。     

HFE mutations and hemochromatosis in Danish patients admitted for HFE genotyping

Analysis of the common C282Y and H63D mutations in the HFE gene is widely used to diagnose hereditary hemochromatosis (HH). The aim of this study was to evaluate the efficiency with which different hospitals and general practitioners select patients for HH genotype and to determine the distribution of HFE mutations in such patients. Nine hundred unrelated patients from Danish hospitals and general practitioners (group A) and 69 consecutive patients from a specialized liver unit (group B) were examined for HFE substitutions using multiplex real-time polymerase chain reaction. In group A we found 13.0% (0%) C282Y homozygotes, 5.8% (2.6%) H63D/C282Y compound heterozygotes and 1.9% (3.1%) S65C heterozygotes. The values for 420 Danish blood donors are shown in parentheses. The distribution of genotypes in group B was similar to that of the blood donors. Serum ferritin, transferrin iron saturation and pathological data were collected from 38 randomly selected C282Y homozygotes, 36 H63D/C282Y compound heterozygotes, 19 H63D heterozygotes, 17 S65C heterozygotes and 144 wild-types. All of the C282Y homozygotes and 28% of the compound heterozygotes were diagnosed as HH patients. There was no evidence of HH in the H63D homozygotes or S65C heterozygotes. Moreover, 7 wild-type patients, 2 C282Y heterozygote patients and one H63D heterozygote patient fulfilled the criteria for HH. The significant enrichment of HH among associated genotype samples submitted for HFE testing indicates that the clinical selection is generally adequate. However, the study showed substantial deviation in the selection efficiency among the various hospitals and general practitioners.     

HFE mutations and hemochromatosis in Danish patients admitted for HFE genotyping

Analysis of the common C282Y and H63D mutations in the HFE gene is widely used to diagnose hereditary hemochromatosis (HH). The aim of this study was to evaluate the efficiency with which different hospitals and general practitioners select patients for HH genotype and to determine the distribution of HFE mutations in such patients. Nine hundred unrelated patients from Danish hospitals and general practitioners (group A) and 69 consecutive patients from a specialized liver unit (group B) were examined for HFE substitutions using multiplex real-time polymerase chain reaction. In group A we found 13.0% (0%) C282Y homozygotes, 5.8% (2.6%) H63D/C282Y compound heterozygotes and 1.9% (3.1%) S65C heterozygotes. The values for 420 Danish blood donors are shown in parentheses. The distribution of genotypes in group B was similar to that of the blood donors. Serum ferritin, transferrin iron saturation and pathological data were collected from 38 randomly selected C282Y homozygotes, 36 H63D/C282Y compound heterozygotes, 19 H63D heterozygotes, 17 S65C heterozygotes and 144 wild-types. All of the C282Y homozygotes and 28% of the compound heterozygotes were diagnosed as HH patients. There was no evidence of HH in the H63D homozygotes or S65C heterozygotes. Moreover, 7 wild-type patients, 2 C282Y heterozygote patients and one H63D heterozygote patient fulfilled the criteria for HH. The significant enrichment of HH among associated genotype samples submitted for HFE testing indicates that the clinical selection is generally adequate. However, the study showed substantial deviation in the selection efficiency among the various hospitals and general practitioners.     

A family-based study of hyperinsulinemia and hypertriglyceridemia in heterozygous lipoprotein lipase deficiency.

CASE REPORT: A case is presented of predisposing a patient's father with obligate heterozygous lipoprotein lipase (LPL) deficiency to mild hypertriglyceridemia in Japanese I-family members (n=8) with patient DI, who was a compound heterozygote for a novel missense mutation of G154V (GG(716)C-->GTC/Gly(154) Val) in exon 5 and a novel splice mutation (Int8/5'-dss/t(+2)c; a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (dss)) in intron 8 of the LPL gene. RESULTS: The patient's father and paternal grandmother were heterozygotes for the Int8/5'-dss/t(+2)c allele, while the patient's mother and maternal grandmother were heterozygotes for the G154V allele. These four heterozygous carriers with one defective LPL allele showed 45-57% of the mean LPL activity and mass in the post-heparin plasma (PHP) observed in normal individuals. Among the four heterozygous carriers, the patient's father, who was <40 years old, nonobese and hyperinsulinemia, manifested mild hypertriglyceridemia (type IV hyperlipoproteinemia). The remaining three healthy heterozygous carriers (two were >40 years old and the other was <40 years old) were all normolipidemic state. CONCLUSION: In this family, hyperinsulinemia as a marker of insulin resistance may be a strong determinant of hypertriglyceridemia in the carrier with heterozygous LPL deficiency.     

D抗原阳性个体及无效等位基因携带者RHD基因数目测定

目的　测定Rh血型D抗原阳性个体的RHD基因数目。方法　采用聚合酶链式反应 限制性片段长度多态性 (PCR RFLP)技术 ,扩增 10名D阳性、5名弱D、2 5名Del表型和 12名携带无效等位基因的Rh阴性个体的RH基因座位下游盒子和融合盒子序列 ,产物经DNA限制性内切酶PstI消化后电泳 ,以 5份经血清学和序列分析基因分型方法确认的D阴性样本作为RHD-/RHD-纯合子对照 ,鉴定RHD+ /RHD+ 或RHD+ /RHD-基因型。结果　 10名D阳性个体均为RHD+ /RHD+ 纯合子、5份弱D样本及 12名携带无效等位基因的Rh阴性个体全部为RHD+ /RHD-杂合型。 2 5名Del表型个体中 9人为RHD+ /RHD+ 纯合型 (36 % ) ,其中 6人Rh表型为DCCee、3人为DCcee ;另 16名Del个体为RHD+ /RHD-杂合型 ,Rh表型均为DCcee。结论　D阳性个体RHD+ /RHD+ 纯合型多见 ,弱D型个体以RHD+ /RHD-杂合型为主 ,在Del表型个体存在高比率的RHD+ /RHD+ 纯合型 ;携带无效等位基因的个体多为RHD+ /RHD-杂合型     

Genetic modulation of the FVLeiden/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes

Summary. Background and Objectives: The factor (F) V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC‐mediated inactivation and by impairing the APC‐cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. Patients/methods: APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase‐based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single‐nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. Results: In FV Leiden heterozygotes, VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2–80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G (K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FV_Leiden/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo‐homozygotes, i.e. they carried a deleterious mutation on the non‐Leiden allele. Conclusions: In FV Leiden heterozygotes, the prothrombinase‐based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FV_Leiden/normal FV ratio in plasma.     

Porphobilmogen deaminase gene mutations in Brazilian acute intermittent porphyria patients

Acute intermittent porphyria (AIP) is an autosomal dominant disorder resulting from porphobilmogen deaminase (PBGD) deficiency. Seven unrelated Brazilian patients were investigated regarding PBGD gene mutations by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing. The PBG gene screening disclosed abnormal SSCP patterns in exons 7, 9, 12, 13, and 15, as well as in introns 3 and 10. Direct DNA sequencing revealed the occurrence of three nonsense mutations (R149X, R225X, and R325X) in exons 9, 12, and 15, respectively, and one missense mutation G111R in exon 7. The G111R mutation was detected in two unrelated patients. Intragenic polymorphisms (3119G/T in intron 2, 3581G/A in intron 3, 7052A/G and 7064C/A in intron 10, and -65C/T in exon 1) were also observed. In addition, two silent mutations (V202V in exon 10 and A266A in exon 13) were found. The latter has not heretofore been reported. Thus, this study revealed the mutations involved in Brazilian symptomatic AIP patients, as well as the intragenic polymorphisms found in the patients.     

Pharmacogenetic study of apolipoprotein E, cholesteryl ester transfer protein and hepatic lipase genes and simvastatin therapy in Brazilian subjects

BACKGROUND: In recent years, one of the focuses of genetic investigation in cardiology has been to identify the genetic factors associated with variable response to statin treatment. Polymorphisms in apolipoprotein E (APOE), cholesteryl ester transfer protein (CETP) and hepatic lipase (LIPC), proteins with major roles in lipid metabolism and homeostasis have been shown associated with lipid-lowering drugs response. METHODS: One hundred forty-six hypercholesterolemic patients of European descent were prospectively enrolled and treated with simvastatin 20 mg per day for over 6 months. Ninety-nine subjects completed the 6-month follow-up. Plasma lipids and lipoproteins were measured before and throughout the study. APOE (E*2, E*3 and E*4), LIPC-250A > G and CETP TaqIB genotypes were determined by PCR and restriction mapping. RESULTS: After a 6-month follow-up, no differences among genotypes in the percentage variation in lipid and lipoprotein concentrations for APOE and LIPC SNPs were observed. After adjustment for covariates, CETP B2B2 homozygotes showed a greater HDL-cholesterol increase compared to B1B2 and B1B1 subjects (14.1% vs. 1.7% and 1.3%, P < 0.05, respectively). CONCLUSION: Our study demonstrates that individual plasma HDL-cholesterol response to simvastatin is mediated, in part, by the CETP gene locus, with the B2 homozygotes having more benefit in HDL-C improvement than carriers of B1 allele.