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Backgrounds: Familial hypercholesterolemia (FH) is an autosomal dominant disease characterized by hypercholesterolemia, tendon xanthomas, and premature coronary heart disease. FH is caused by mutations of "FH genes," which include the LDL-receptor (LDLR), apolipoprotein B-100 (APOB) or proprotein convertase subtilisin/kexin type 9 (PCSK9). We evaluated the usefulness of FH gene analysis for diagnosing homozygous FH (homo-FH), particularly in cases caused by gain-of-function (g-o-f) mutations in PCSK9 (PCSK9 E32K)., Objectives: To evaluate the frequency of homo-FH caused by PCSK9 E32K compared with FH due to other genetic causes and to report the phenotypic features of homo-FH caused by PCSK9 E32K., Methods: Genomic DNA was prepared from white blood cells, and LDLR and PCSK9 mutations were identified using the Invader assay method., Results: Of the 1055 hetero-FH patients, 62 patients (5.9%) carried the PCSK9 E32K mutation, while in the 82 alleles of 41 homo-FH patients, 13 (15.9%) had double mutations of LDLR allele and PCSK9 E32K mutation. Mean plasma total cholesterol (TC) (9.93 ± 2.95 mmol/L, mean ± SD) in true homo-FH cases with PCSK9 E32K or double hetero-FH cases with PCSK9 E32K and LDLR mutations were significantly lower than those in true homo-FH (18.06 ± 4.96 mmol/L) and compound heterozygous cases with LDLR mutations (14.84 ± 1.62 mmol/L). Mean plasma TC concentrations in the 59 hetero-FH cases with PCSK9 E32K (7.21 ± 1.55 mmol/L) were significantly lower than those (8.94 ± 1.53 mmol/L) in the hetero-FH by LDLR mutations., Conclusions: FH caused by PCSK9 g-o-f mutations is relatively common in Japan and causes a mild type of homo- and hetero-FH compared with FH caused by LDLR mutations., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Aim: Familial hypercholesterolemia (FH) is caused by mutations of FH genes, i.e. LDL-receptor (LDLR), PCSK9 and apolipoprotein B (ApoB) gene. We evaluated the usefulness of DNA analysis for the diagnosis of homozygous FH (homo-FH), and studied the frequency of FH in the Hokuriku district of Japan., Methods: Twenty-five homo-FH patients were recruited. LDLR mutations were identified using the Invader assay method. Mutations in PCSK9 were detected by PCR-SSCP followed by direct sequence analysis., Results: We confirmed 15 true homozygotes and 10 compound heterozygotes for LDLR mutations. Three types of double heterozygotes for LDLR and PCSK9 were found. No FH patients due to ApoB mutations were found. The incidences of homo-FH and hetero-FH in the Hokuriku district were 1/171,167 and 1/208, respectively., Conclusions: Our observations underlined the value of FH gene analysis in diagnosing homo-FH and confirmed extraordinarily high frequency of FH in the Hokuriku district of Japan., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The long-term efficacy and safety of HMG-CoA reductase inhibitors (statins) have been established in large multicenter trials. Inhibition of this enzyme, however, results in decreased synthesis of cholesterol and other products downstream of mevalonate, such as CoQ10 or dolichol. This was a randomized double-blind, placebo-controlled study that examined the effects of CoQ10 and placebo in hypercholesterolemic patients treated by atorvastatin. Eligible patients were given 10mg/day of atorvastatin for 16 weeks. Half of the patients (n=24) were supplemented with 100mg/day of CoQ10, while the other half (n=25) were given the placebo. Serum LDL-C levels in the CoQ10 group decreased by 43%, while in the placebo group by 49%. The HDL-C increment was more striking in the CoQ10 group than in the placebo group. All patients showed definite reductions of plasma CoQ10 levels in the placebo group, by 42%. All patients supplemented with CoQ10 showed striking increases in plasma CoQ10 by 127%. In conclusion atorvastatin definitely decreased plasma CoQ10 levels and supplementation with CoQ10 increased their levels. These changes in plasma CoQ10 levels showed no relation to the changes in serum AST, ALT and CK levels. Further studies are needed, however, for the evaluation of CoQ10 supplementation in statin therapy.

The clinical efficacy and safety of pitavastatin (NK-104), a novel HMG-CoA reductase inhibitor, during long-term treatment, were examined in 25 patients (male/female=11/14, mean age=53+/-13 (mean+/-SD) years) with heterozygous familial hypercholesterolemia (FH). After a period on placebo of >4 weeks, 2 mg/day of pitavastatin was administered for 8 weeks, and the dose was increased to 4 mg/day for up to 104 weeks. Total cholesterol (TC) decreased by 31% from the initial value of 340+/-57 to 237+/-40 mg/dl (P<0.0001) at week 8. During treatment with the higher dose, TC decreased even further to 212+/-35 mg/dl at week 12; it decreased by 37% from the initial value (P<0.0001). Similarly, the baseline low-density lipoprotein (LDL)-cholesterol (LDL-C) decreased by 41% at week 8, and by 49% at week 12, from 267+/-61 mg/dl at baseline. These findings indicate a dose-dependent effect of the drug on TC and LDL-C concentrations. To examine whether the levels of circulating matrix metalloproteinases (MMPs) and their endogenous inhibitors (tissue inhibitors of metalloproteinases: TIMPs) are altered during lipid-lowering therapy, we also measured their plasma levels. The mean levels of MMP-2 and -3 were significantly increased. No significant alteration was found in MMP-9, TIMP-1 and -2 levels. As for the safety of pitavastatin, adverse reactions were observed in one case (4%) of subjective and objective symptoms. The effects of pitavastatin on TC and LDL-C were stable during long treatment of patients with heterozygous FH.

Long-term effects of pravastatin and probucol on serum cholesteryl ester transfer protein (CETP) and xanthoma/xanthelasma size were compared. Twenty-three patients with primary hypercholesterolemia and xanthoma/xanthelasma, including 11 patients with heterozygous familial hypercholesterolemia, were treated with pravastatin (20 mg/day) or probucol (1000 mg/day) for 24 months. Serum CETP levels were measured by sandwich ELISA. In 11 patients (six men and five women, 55 +/- 2 [SE] yr) treated with pravastatin, serum cholesterol levels decreased from 262 +/- 13 to 229 +/- 13 mg/dl during the 24-month treatment period (P = 0.05). Serum HDL cholesterol levels were not changed. Serum CETP levels decreased from 2.5 +/- 0.2 to 2.0 +/- 0.2 microg/ml (-21%, P = 0.002). By contrast, in 12 patients (four men and eight women, 57 +/- 4 year) treated with probucol, serum cholesterol levels did not significantly decrease from 236 +/- 11 to 207 +/- 13 mg/dl. Serum HDL cholesterol levels decreased from 44 +/- 2 to 30 +/- 2 mg/dl (P = 0.009). Serum CETP levels increased from 2.3 +/- 0.1 to 2.8 +/- 0.2 microg/ml (+23%, P = 0.02). Xanthelasma regression was found in two of four patients (50%) each treated with pravastatin and probucol, respectively. In contrast, Achilles' tendon xanthoma regressed in four of five patients (80%) treated with pravastatin, but only in two of five patients (40%) treated with probucol. Patients with xanthoma/xanthelasma regression after 2 years treatment had higher baseline levels of serum CETP than those without regression (2.7 +/- 0.2 microg/ml [n = 9] versus 2.1 +/- 0.2 microg/ml [n = 7], P = 0.05). Serial changes in serum CETP levels during treatment with pravastatin and probucol were discordant, but not related to the degree of xanthoma regression. However, higher level of serum HDL3 cholesterol was an independent factor in the smaller size of Achilles' tendon xanthoma at baseline. In addition, higher levels of serum HDL3 triglyceride on lipid-lowering therapy (6 months) appear to be a common predictor of regression of Achilles' tendon xanthoma in the treatment with either pravastatin or probucol.

Coronary heart disease (CHD) in familial hypercholesterolemia (FH) may be modified by genetic and/or environmental factors. We described the effect of the cholesteryl ester transfer protein (CETP) gene on CHD in heterozygous FH caused by low density lipoprotein receptor (LDL-R) gene mutation. In 288 unrelated Japanese subjects with heterozygous FH, the allele frequency of an intron 14 G(+1)-to-A mutation (Int14 A) and a missense mutation in exon 15 (Asp442 to Gly, D442G) was 0.3 and 3.0%, respectively. HDL-C levels (1.55 +/- 0.08 mmol/l) in FH patients with heterozygous CETP deficiency were higher than those (1.19 +/- 0.08 mmol/l) in FH without CETP deficiency (P < 0.03), while LDL-C levels in FH with CETP deficiency were moderately reduced. However, two FH patients with CETP deficiency suffered myocardial infarction, and six patients had effort angina pectoris and/or coronary atherosclerosis. No difference in the score of coronary stenosis index (CSI) was found in FH with/without CETP deficiency, although CSI was inversely correlated with HDL-C levels (P < 0.05). Thus, the effect of increased HDL-C levels caused by partial deficiency of CETP is insufficient to prevent CHD in FH.

A 66-year-old male heterozygous familial hypercholesterolemia (FH) patient with significant coronary atherosclerosis has been treated by us with probucol (1000 mg daily) for eight years. This treatment has produced significant reductions in the cholesterol levels of his serum, low density lipoprotein (LDL), and high density lipoprotein (HDL) from 237 +/- 20 mg/dl (mean +/- S.D.) to 156 +/- 15, from 175 +/- 8 to 111 +/- 16 mg/dl, and from 23 +/- 4 to 19 +/- 2 mg/dl, respectively. These reductions have been maintained for eight years. Serum triglyceride levels also decreased, from 220 +/- 54 to 146 +/- 36 md/dl. During this period, marked regression of xanthomas on the eyelids and finger extensor tendons was observed, while thickness of the Achilles tendons was reduced from 21.0 mm to 13.0 mm. On other hand, effort-induced anginal symptoms requiring additional antianginal medication have been noticed, and angiographically-demonstrated coronary atherosclerosis has progressed significantly during these eight years. These observations lead us to suggest that maintaining low levels of HDL cholesterol with probucol, even though resulting in satisfactory reduction of LDL cholesterol and marked regression of xanthomas, appears to be associated with the progression of atherosclerosis in the coronary arteries.

Lipoprotein(a) (Lp(a)) was eliminated by LDL-apheresis using a dextran sulfate cellulose column in 3 homozygous and 10 heterozygous familial hypercholesterolemic patients. Immediately after LDL-apheresis by the LA-15 system (continuous LDL apheresis), there were significant reductions in Lp(a) concentrations (28.6 +/- 11.8 mg/dl (mean +/- S.E.) to 9.6 +/- 5.6 mg/dl (P < 0.01)), and in LDL-cholesterol concentrations (156 +/- 32 mg/dl to 48 +/- 18 mg/dl (P < 0.01)). Immediately following LDL-apheresis, Lp(a) and LDL-cholesterol were reduced by 67.4% +/- 11.6% and 68.3% +/- 11.8%, respectively. The removal of Lp(a) paralleled that of LDL-cholesterol. The reduced levels of Lp(a) nearly returned to baseline within 7 days. In 6 of the heterozygous FH patients the rates of recovery of LDL cholesterol and Lp(a) were calculated, according to Apstein's equation after discontinuing lipid altering drug treatment for 4 weeks. Mean constant k values of LDL cholesterol and Lp(a) were 0.354 (range: 0.136-0.752) and 0.427 (range 0.112-0.933), respectively. The average concentration during the 7 days following LDL-apheresis was calculated. Average reductions were 28% in LDL cholesterol and 18% in Lp(a). Pravastatin treatment, which continued for 4 weeks, significantly decreased LDL cholesterol (P < 0.01); however, before LDL-apheresis pravastatin treatment significantly increased Lp(a) levels (P < 0.05) in a small number (n = 6) of the FH patients, who had been regularly treated with LDL-apheresis. These results suggest that LDL-apheresis using the dextran sulfate cellulose column is an effective treatment to reduce levels of serum Lp(a) and LDL proportionally. This therapy may be of value in the prevention and regression of coronary artery disease in FH patients.

Six homozygous, 10 heterozygous and 8 unaffected subjects in a CETP deficient family confirmed by CETP gene analysis were studied to characterize serum lipoproteins separated by ultracentrifugation, and to examine the relations between CETP levels and lipoprotein lipid concentration and composition. The serum CETP levels were measured by radioimmunoassay using 125I-labeled monoclonal antibodies (TP2). The serum CETP levels in the homozygotes were undetectable and those in the heterozygotes were significantly lower than those in the unaffected subjects (1.5 +/- 0.1 vs. 2.2 +/- 0.5 microgram/ml, P less than 0.01). In the HDL fraction, esterified cholesterol (EC) levels in the homozygotes were significantly increased (P less than 0.01), and those in the heterozygotes were slightly increased (n.s.), in comparison with those in the unaffected and the normolipidemic controls. The EC levels in the IDL fractions were lower in the homozygotes than in the normolipidemic controls. The EC/triglyceride (TG) molar ratios in IDL, the fraction obtained from the homo- and heterozygotes, were lower than those from the unaffected subjects (P less than 0.01 and less than 0.01, respectively), and the EC/TG ratios in the HDL fraction obtained from the homo- and heterozygotes were higher than those from the unaffected subjects (P less than 0.01 and n.s., respectively). Linear regression analysis showed that positive correlates of the serum CETP levels in all subjects were: IDL-EC (r = 0.463), HDL-TG (r = 0.603) and VLDL- and IDL-EC/TG ratio (r = 0.698 and 0.843).(ABSTRACT TRUNCATED AT 250 WORDS)

Serum lipids and lipoprotein lipids were studied in 53 patients (21 males and 32 females) with xanthelasma palpebrarum and 40 age-matched normal controls (20 males and 20 females). Patients were subdivided into patients with normolipidemia, hyperlipidemia or familial hypercholesterolemia (FH). In both male and female patients with hyperlipidemia or FH, the serum cholesterol (Chol) levels were significantly higher than in normal controls. In both male and female patients with normolipidemia or hyperlipidemia, the VLDL-Chol levels were significantly higher than in normal controls. Male patients with FH showed significantly higher levels of VLDL-Chol than normal controls. Both male and female patients with normolipidemia, hyperlipidemia or FH showed significantly higher levels of LDL-Chol, lower HDL-Chol levels and lower HDL-Chol/LDL-Chol ratios than normal controls. In both male and female patients with hyperlipidemia and in male patients with FH, the serum triglyceride (TG) levels were significantly higher than in normal controls. Both male and female hyperlipidemic patients showed significantly higher levels of VLDL-TG than normal controls. In male patients with FH, the VLDL-TG levels were significantly above the control levels. In male patients with normolipidermia, the LDL-TG levels were significantly higher than in normal controls. In both male and female patients with hyperlipidemia or FH, the LDL-TG levels were significantly higher than in normal controls. The HDL-TG levels in patients with normolipidemia (males) or FH (females) were significantly lower than in normal controls. The prevalence of coronary heart disease in patients with normolipidemia, hyperlipidemia or FH was 29.4%, 24.0% and 45.4%, respectively.

Lipoprotein patterns and cholesteryl ester transfer activity (CETA) were examined in 2 patients with familial hyperalphalipoproteinaemia (FHALP). The proband was a healthy 58-year-old Japanese male who had an HDL cholesterol of 7.83 mmol/l (301 mg/dl). His sister's HDL cholesterol was 4.52 mmol/l (174 mg/dl), which suggested that both were homozygous carriers of FHALP. In both subjects HDL showed a high cholesterol/apo A-I ratio and appeared to be a larger-sized particle than normal HDL on agarose gel chromatography. Two of the proband's children showed higher HDL cholesterol levels (1.74 mmol/l, 2.16 mmol/l) than normal, but another 2 children showed normal levels (1.48 mmol/l, 1.40 mmol/l). However, the ratios of HDL cholesterol to total cholesterol and to apo A-I in all children were higher than normal. These data suggest, but do not prove, that all his children were heterozygotes. Apo B levels in all of the family members studied were lower than normal (47-80 mg/dl). Deceased members of the same family had not died from cardiovascular disease. Cholesteryl-ester transfer activity was studied in both patients. When serum or lipoprotein deficient serum (d greater than 1.21) and [3H]cholesteryl ester labelled HDL3 were incubated in the presence of an LCAT inhibitor, there was no evidence of cholesteryl ester transfer from HDL to VLDL and/or LDL, unlike normal subjects. The deficiency of CETA in these patients with FHALP presumably accounted for the increase in particle size and cholesterol enrichment of HDL.

We describe a new low density lipoprotein (LDL) apheresis system using dextran sulfate cellulose column in an automated column regenerating unit (LDL continuous apheresis). Two columns containing 150 ml of dextran sulfate cellulose were used, and the whole extracorporeal circulation was about 400 ml in volume. After 600 ml of plasma was adsorbed into the first column, the second column was used as an adsorbent and meanwhile the first column was regenerated. Thus, the 2 columns were used alternately without losing the potency of the columns. As the apparatus was automatically controlled by a computerized unit, no extra manipulation is necessary compared with the conventional single-column method. By treating 4-5 liters of plasma, non-high density lipoprotein (HDL)-cholesterol levels decreased by 63-71%, and HDL-cholesterol levels remained unchanged. Thus, this new method of LDL apheresis can safely reduce LDL-cholesterol to any desired level and will be applicable for the treatment of child and adult family hypercholesterolemic patients with severe coronary heart disease.

Nine heterozygous patients with familial hypercholesterolemia (FH) were treated by low density lipoprotein (LDL)-apheresis using dextran sulfate cellulose columns. After more than 3 procedures of LDL-apheresis without drug therapy, combination therapy with LDL-apheresis and CS-514 (eptastatin), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase, at a dose of 10 mg twice daily was started. Pre- and post-apheresis serum cholesterol levels were decreased significantly by CS-514, from 289 +/- 24 mg/dl (mean +/- SEM) to 247 +/- 25 mg/dl and from 118 +/- 7 mg/dl to 106 +/- 9 mg/dl, respectively. Pre- and post-apheresis apolipoprotein B levels decreased significantly on CS-514 from 160 +/- 9 mg/dl to 138 +/- 8 mg/dl and from 58 +/- 6 mg/dl to 45 +/- 6 mg/dl, respectively. No adverse effects were observed during the combination therapy. Thus, the addition of an inhibitor of HMG-CoA reductase to LDL-apheresis is a useful method for further reducing serum cholesterol and apolipoprotein B levels in FH heterozygotes.

The presence of apo E-containing HDL in familial hypercholesterolaemia was investigated and its removal by LDL-apheresis using a dextran sulphate cellulose column was demonstrated by measurement of the apo E/apo A-I molar ratio of serum and by nondenaturing polyacrylamide gel electrophoresis followed by immunoblotting. The molar ratios of apo E/apo A-I in the density greater than 1.063 kg/l fraction of serum obtained from two homozygous patients with familial hypercholesterolaemia were higher (0.021 and 0.030) than that from normal subjects (mean +/- SE 0.011 +/- 0.002) (P less than 0.05). Polyacrylamide gel electrophoresis and immunoblotting showed an increase in apo E-containing HDL similar to HDL2, in the plasma obtained from the homozygous patient with familial hypercholesterolaemia. The increased amounts of apo E-enriched HDL were removed from plasma by adsorption with a dextran-sulphate cellulose column. These results suggested that LDL-apheresis using the dextran-sulphate cellulose column, may cause an increase in the turnover rate of the apo E-containing HDL and thus facilitate cholesterol removal from the peripheral tissues.

Coronary angiographic findings were analyzed in 51 consecutive patients (36 males and 15 females) with heterozygous familial hypercholesterolemia (FH) and 279 consecutive patients (216 males and 63 females) without FH (non-FH). The coronary stenosis index and over 75% stenosis vessel subset were almost three times as high in the FH group. The incidence of myocardial infarction was almost twice as high in the FH group. Levels of total cholesterol and its lipoprotein fractions, except HDL-cholesterol, were almost twice as high in the FH group. In the FH group aged under 50 years, the two parameters of coronary angiogram and the incidence of myocardial infarction were significantly higher in males than in females. However, in the group aged over 50 years, all three parameters were not significantly different between those in males and females. The level of HDL-cholesterol was significantly lower in males than in females. A significantly higher incidence (18%) of coronary ectasia was observed in the FH group compared with the incidence (2%) in non-FH. All patients with coronary ectasia were males, except one female with FH. On comparison of the males among the FH patients with those among the non-FH patients matched for total cholesterol, age and other risk factors, the FH patients were associated with a significantly higher degree of coronary atherosclerosis and lower level of HDL-cholesterol. Seven FH patients with a normal coronary angiogram were observed. However, any factors as regards age, total cholesterol, HDL-cholesterol and Achilles tendon thickness failed to distinguish between the FH patients with a normal coronary angiogram and those without.(ABSTRACT TRUNCATED AT 250 WORDS)

The serum cholesterol and triglyceride levels and the incidence of ischemic heart disease were studied in 122 (55 men and 67 women) consecutive heterozygous familial hypercholesterolemic patients in the Hokuriku district of Japan. (1) The mean +/-SD of serum cholesterol level was 354.0 +/- 71.0 mg/100 ml, which was lower than those of the Western countries by about 60--70 mg/100 ml. (2) The mean +/-SD of serum triglyceride level was 116.5 +/- 54.0 mg/100 ml. (3) The average serum cholesterol values in the 20--50-year-old group showed no differences from those of the Western countries. However, in the above 50 years of age group the serum cholesterol levels were much lower than those in the United States. (4) The occurrence of ischemic heart disease in 83 heterozygous familial hypercholesterolemic patients was 43.3%. The incidence of myocardial infarction was 20.5%. Thus, familial hypercholesterolemia is as highly atherogenic as that of the Western countries even in Japan where the low incidence of coronary heart disease in the general population has been attributed to the low level of serum cholesterol.