Leucine 10 allelic variant in signal peptide of PCSK9 increases the LDL cholesterol-lowering effect of statins in patients with familial hypercholesterolaemia.

BACKGROUND AND AIMS: In the normal population, carriers of an additional leucine residue in a stretch of nine leucines in the signal peptide of PCSK9 (L10) have lower total (TC) and low-density lipoprotein cholesterol (LDL-C) than homozygotes for the wild-type allele (L9/L9). A similar effect was detected in familial hypercholesterolaemia (FH) patients with the p.C681X mutation of LDL-receptor (LDLR). We investigated the effect of L10 variant on basal lipid profile and response to statins in molecularly characterised FH patients. METHODS AND RESULTS: Plasma lipids were determined in 322 FH patients screened for the L9/L10/L11 polymorphism and in a subgroup of 54 patients carrying the same LDLR mutation (p.Q474HfsX63). Plasma lipids were also determined in 42 FH patients carrying the L10 variant and in a parallel group of 42 FH patients, L9/L9 homozygotes, matched for gender, age, type of LDLR gene mutation, as well as for type, dose and duration of statin treatment. In FH patients, no difference in the basal plasma TC and LDL-C levels was observed between carriers of L10 variant (L9/L10+L10/L10) and L9/L9 homozygotes. The same was true in FH patients carrying the p.Q474HfsX63 LDLR mutation. In the subgroups of statin-treated patients, the reduction of TC and LDL-C was greater in carriers of L10 (-34.0% and -42.5%, respectively) than in L9/L9 homozygotes (-27.5% and -34.3%, respectively) (P<0.001). CONCLUSION: The variant L10 of the leucine repeats in PCSK9 signal peptide is to be considered as a factor capable of modulating the lipid-lowering effects of statins in FH.

Data collection and advanced statistical analysis in phytotoxic activity of aerial parts exudates of Salvia spp

In order to define the phytotoxic potential of Salvia species a database was developed for fast and efficient data collection in screening studies of the inhibitory activity of Salvia exudates on the germination of Papaver rhoeas L. and Avena sativa L.. The structure of the database is associated with the use of algorithms for calculating the usual germination indices reported in the literature, plus the newly defined indices (Weighted Average Damage, Differential Weighted Average Damage, Germination Weighted Average Velocity) and other variables usually recorded in experiments of phytotoxicity (LC50, LC90). Furthermore, other algorithms were designed to calculate the one-way ANOVA followed by Duncan's multiple range test to highlight automatically significant differences between the species. The database model was designed in order to be suitable also for the development of further analysis based on the artificial neural network approach, using Self-Organising Maps (SOM).

Hypertriglyceridaemia and low plasma HDL in a patient with apolipoprotein A-V deficiency due to a novel mutation in the APOA5 gene.

APOA5 encodes a novel apolipoprotein (apo A-V) which appears to be a modulator of plasma triglyceride (TG). In apoA5 knock out mice plasma TG level increases almost fourfold, whereas in human APOA5 transgenic mice it decreases by 70%. Some SNPs in the APOA5 gene have been associated with variations in plasma TG in humans. In addition, hypertriglyceridaemic (HTG) patients have been identified who carried rare nonsense mutations in the APOA5 gene (Q139X and Q148X), predicted to result in apo A-V deficiency. In this study we report a 17-year-old male with high TG and low high density lipoprotein cholesterol (HDL-C), who at the age of two had been found to have severe HTG and eruptive xanthomas suggesting a chylomicronaemia syndrome. Plasma postheparin LPL activity, however, was normal and no mutations were found in LPL and APOC2 genes. The sequence of APOA5 gene revealed that the patient was homozygous for a point mutation (c.289 C>T) in exon 4, converting glutamine codon at position 97 into a termination codon (Q97X). Apo A-V was not detected in patient's plasma, indicating that he had complete apo A-V deficiency. The administration of a low-fat and low-oligosaccharide diet, either alone or supplemented with omega-3 fatty acids, started early in life, reduced plasma TG to a great extent but had a negligible effect on plasma HDL-C. Loss of function mutations of APOA5 gene may be the cause of severe HTG in patients without mutations in LPL and APOC2 genes.

Effect of statins on fibroblasts from human nasal polyps and turbinates.

BACKGROUND: Statins are serum cholesterol-lowering agents used for the prevention and treatment of atherosclerotic vascular disease. There is, however, growing evidence that statins have immunomodulatory and anti-inflammatory activities and may prove invaluable in the treatment of immunological and inflammatory disorders. OBJECTIVE: On these basis we evaluated the effect of statins on the proliferation of fibroblasts derived from human nasal polyps and turbinates and determined their ability to modulate airway remodelling. METHODS: Fluvastatin (0.01-0.1-1 microM), Atorvastatin (0.1-1-10 microM) and Simvastatin (0.1-1-10 microM) were tested on cultured fibroblasts derived from human nasal polyps and turbinates stimulated or not with Fibroblast Growth Factor beta (10 ng/ml). All cultures were treated with 3H-Thymidine (1 microCi/ml) to test cell proliferation. RESULTS: Our results show that proliferation of turbinate-derived fibroblasts is significantly inhibited by the three statins. Fluvastatin is already effective at the lowest dose (0.01 microM), whereas Atorvastatin and Simvastatin act at the plasmatic peak concentration (1 microM). No significant effect was found on fibroblasts derived from nasal polyps, except for Simvastatin which was effective after 144 hours of stimulation. CONCLUSIONS: These drugs show a remarkable antiprolhferative effect and their different outcome depending on the different kind of fibroblasts in vitro is prompting news in the studies about statin use for the treatment of chronic inflammatory diseases.

Beta-sitosterolaemia: a new nonsense mutation in the ABCG5 gene.

BACKGROUND: Sitosterolaemia is a rare autosomal recessive disorder characterised by elevated plasma levels of plant sterols and cholesterol. Sitosterolaemia is caused by gene mutations in either of two ATP-binding cassette (ABC) half transporters, ABCG5 and ABCG8. The plasma sterol profile and genetic analysis of a 10-year-old girl who had tuberous xanthomas is the subject of this report. MATERIALS AND METHODS: Genomic DNA was isolated from white blood cells from the proband, her family and a control group of healthy people. All exons of ABCG5 and ABCG8 were sequenced. Plasma cholesterol and triglycerides were measured by routine methods. All other plasma sterols were measured by Gas Chromatography coupled to Mass Spectrometry. RESULTS: The proband was found to be homozygous for a single nucleotide mutation in exon 10 of the ABCG5 gene, consisting of a C to T transition at nucleotide 1336 of the coding sequence, which results in the premature termination of the ABCG5 protein at amino acid 446 (Arg446X). Her mother and brother were also homozygous for the same mutation and all had elevated plasma beta-sitosterol levels. The father was heterozygous and showed normal beta-sitosterol levels. This mutation was not found in healthy normolipidaemic subjects. CONCLUSIONS: We describe a novel nonsense mutation in exon 10 of the ABCG5 gene in a 10-year-old girl showing clinical and biochemical features of sitosterolaemia. This family study broadens the spectrum of the ABCG5/ABCG8 mutations causing sitosterolaemia and helps highlight the correlations between such gene mutations, biochemical phenotype and the development of cardiovascular disease.

[Treatment of colorectal cancer liver metastases]. / Il trattamento delle metastasi epatiche da cancro del colon-retto.

Liver is the main target for colorectal cancer (CRC) metastases. About 50% of all patients affected by CRC develop liver metastases. Surgery remains the only potentially curative strategy and indications to surgery and resectability criteria are now less restrictive than before so that a more aggressive attitude in the treatment of metastatic lesions is the rule. However surgery is not possible in the majority of patients. For non resectable patients two options are available: local treatment strategies (Radio-frequency ablation and Cryosurgery: alone or in combination with surgery) and chemotherapy. High rates of objective response achieved with Fluoropyrimidines, Oxaliplatin (OHP) and Irinotecan (CPT-11) based chemo-therapy, enable initially unresectable patients to undergo surgery, with a 5-year survival rate comparable to that observed for primary resectable patients. Therefore chemotherapy has not only a palliative aim, but becomes a fundamental moment of a combined medical and surgical treatment with curative purpose. After surgery two-thirds of patients will relapse in first two years, so that adjuvant therapy has been investigated to reduce recurrence rates, mainly testing hepatic arterial infusion (HAI) schedules. So far no randomized trials have been published on the role of systemic intravenous adjuvant chemo-therapy. Finally we report the results of our monoinstitutional experience, suggesting a possible role of systemic adjuvant chemotherapy in reducing recurrence rates after liver metastasectomy. Probably in the next years new targeted drugs and locoregional therapies will contribute to further improve prognosis of such patients, in a neoadjuvant, adjuvant and palliative setting.

Clinical usefulness of serum pepsinogens I and II, gastrin-17 and anti-Helicobacterpylori antibodies in the management of dyspeptic patients in primary care.

BACKGROUND: Several tests have been proposed for evaluating dyspeptic symptoms and their relationship to the underlying gastric disease. Serum pepsinogens and gastrin-17 are known to be useful biomarkers for the detection of gastric pathologies. AIM: To evaluate the capability of screening dyspeptic patients in the primary care by analyses of serum pepsinogens I (sPGI) and II (sPGII), gastrin-17 (sG-17) and the IgG anti-Helicobacter pylori antibodies (IgG-Hp). PATIENTS AND METHODS: Three hundred and sixty-two consecutive patients with dyspeptic symptoms (208 females, mean age 50.6 +/- 16 years, range 18-88 years) referred by general practitioners for upper gastrointestinal endoscopy were enrolled. A blood sample was taken from each subject for IgG-Hp, sPGI, sPGII and sG-17 analyses. RESULTS: Two hundred and eighty-seven patients had a complete screening; of these, 132 resulted positive for Hp infection. Patients with atrophic chronic gastritis showed significantly lower serum pepsinogen I levels and sPGI/sPGII ratio than patients with non-atrophic chronic gastritis. Moreover, by calculating the values of sPGI by sG-17 and sG-17 by sPGII/sPGI, subjects with atrophic chronic gastritis could be distinguished from those with non-atrophic chronic gastritis and from those with normal mucosa, respectively. sG-17 levels were found to be a useful biomarker for the detection of antral atrophic gastritis, while the combination of sPGI, the sPGI/sPGII ratio and sG-17 was found effective in identifying corpus atrophy. CONCLUSION: A panel composed of PGI, PGII, G-17 and IgG-Hp could be used as a first approach in the 'test and scope' and/or 'test and treat' strategy in the primary care management of dyspeptic patients.

Physical activity modulates effects of some genetic polymorphisms affecting cardiovascular risk in men aged over 40 years.

BACKGROUND AND AIM: Several genetic polymorphisms have been found to be involved in cardiovascular risk, and many studies have documented the beneficial effect of systematic physical activity (PA) on the cardiovascular system. Our aim was to investigate the interactive effects of PA and genetic background on plasma lipids and homocysteine (tHcy) levels. METHODS AND RESULTS: Clinical and metabolic parameters, dietary intakes and some polymorphisms of the genes involved in cardiovascular risk (Apo E, fatty acid binding protein-2, Apo AII, hepatic lipase and methylene tetrahydrofolate reductase) were determined in 100 men aged over 40 years who cycle 120-150 Km/week and 100 age-matched sedentary controls. The physically active subjects had lower concentrations of plasma LDL cholesterol (LDL-C), triglyceride (TG), Apo B, glucose and tHcy, and higher concentrations of plasma HDL cholesterol (HDL-C) and Apo AI than the sedentary men; they also had larger LDL particle sizes (LDLs). The LDL-C and Apo B raising effect of the Apo E epsilon 4 allele detectable in the sedentary subjects was totally absent in the cyclists, in whom the LDL-C and Apo B lowering effect of the epsilon 2 allele was observed. PA blunted the TG-raising effect of the Apo AII-265TT genotype, and amplified the HDL-C raising effect of the HL-250AA genotype. PA had a small but significant lowering effect on plasma tHcy adjusted for folate levels in subjects with the 677TT genotype of the MTHFR gene. CONCLUSIONS: Extended high-intensity PA in men aged over 40 years may modify their metabolic cardiovascular risk factors even in the presence of some unfavourable genotypes.

Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia.

This study evaluated the applicability of microchip electrophoresis to the sizing of microsatellites suitable to genetic, clinical and forensic applications. The evaluation was performed with the D19S394 tetranucleotide (AAAG) repeat characterized by a wide variation in the repeat number (1-17) and a short recombination distance from the low-density lipoprotein (LDL)-receptor gene that makes it suitable to cosegregation analysis of familial hypercholesterolemia (FH). The study was performed with 70 carriers of two LDL-receptor mutations common in northern Italy (i.e., the 4 bp insertion in exon 10 known as FH-Savona and the D200G missense mutation in the exon 4, known as FH-Padova 1) and 100 healthy controls. The polymerase chain reaction (PCR) amplification products prepared with a cosolvent PCR protocol and an antibody-protected polymerase were directly analyzed with an apparatus for high-voltage capillary electrophoresis on microchips and laser-induced fluorescence detection equipped with chips for the analysis of 25-500 bp dsDNA fragments. The test could not be extended to dinucleotide repeats due to the resolution characteristics of the available microchip. This novel approach was able to distinguish 17 microsatellite alleles varying from 0 to 17 repeats. Many of these alleles were quite rare, but the seven more abundant accounted for over the 70% of allele distribution in control population. The standard deviation in the sizing of the most abundant alleles ranged from +0.60 to +/- 0.75 bp. This indicated that the size attribution to a conventional allele using the +/- 1 bp range around it allowed a confidence limit above the 80 %. The sizing of D19S394 obtained this way allowed the cosegregation analysis with both the FH mutations tested. Therefore, this innovative approach to microsatellite sizing was much simpler, but equally effective as traditional capillary electrophoresis, at least with tetranucleotide repeats.

Low-density lipoprotein apheresis in a patient aged 3.5 years.

UNLABELLED: A 3.5 y-old girl carrying a severe mutation of the LDL-receptor gene known as "FH Pavia", affected by homozygous familial hypercholesterolaemia (FH), and at high risk of developing coronary artery atherosclerosis was treated with selective dextran sulphate cellulose (DSC) column low-density lipoprotein apheresis (LDL-a). This is the youngest patient ever treated with LDL-a. Plasma total cholesterol (982 mg/dl) and LDL-cholesterol (939 mg/dl) (T-Chol, LDL-Chol) levels at baseline showed a transient decrease: -13.4%, and -16.8%, respectively, after 9 mo of combined treatment with a diet, cholestyramine (max. 12 g/d) and atorvastatin (max. 30 mg/d). However, the drugs were discontinued because of intolerance and an increase in aminotransferases and creatine phosphokinase in the plasma. Moreover, after 9 mo of this therapy, the mean plasma T-Chol and LDL-Chol levels were still high (930 mg/dl and 869.5 mg/dl, respectively). Therefore, 9 consecutive treatments with LDL-a were carried out every 15 d (plasma volumes treated: 1000-1700 ml). Mean plasma T-Chol, LDL-Chol, triglycerides (TG), and Lp(a) decreased significantly: -75.5%, -77.2%, -67.5% and -50.8%, respectively. HDL-cholesterol (HDL-Chol) concentration was considerably decreased immediately after apheresis because of haemodilution (X: -45.1%). CONCLUSION: LDL-a treatment improved the plasma apo B 100-containing lipoproteins--LDL, Lp(a)--profile in a homozygote with a severe inherited disorder in which coronary artery atherosclerosis frequently has its clinical onset before 10 y of age. At the time of this report, no significant side effects had been observed.

Hypocomplementemic type II membranoproliferative glomerulonephritis in a male patient with familial lecithin-cholesterol acyltransferase deficiency due to two different allelic mutations.

Patients with familial lecithin-cholesterol acyltransferase (LCAT) deficiency very often show progressive glomerulosclerosis with evolution to end-stage disease. High levels of an abnormal lipoprotein (lipoprotein X) cause glomerular capillary endothelial damage. The ultrastructural study of renal biopsy specimens shows characteristic glomerular deposits of membrane-like, cross-striated structures and vacuole structures. The gene encoding for LCAT has been mapped to chromosome 16q22.1, and several mutations of this gene cause LCAT deficiency which is inherited as an autosomal recessive trait and which is characterized by corneal opacities, normochromic normocytic anemia, and renal dysfunction. Herein we report clinical features and renal histological findings concerning a 24-year-old male patient with classical familial LCAT deficiency due to two different allelic mutations: a nonsense mutation inherited from the father and a missense mutation inherited from the mother. Moreover, the patient showed glomerular histological lesions and an immunofluorescent glomerular pattern typical of hypocomplementemic membranoproliferative type II glomerulonephritis (dense-deposit disease). The nature of electron-dense material that characterizes dense-deposit disease is still unknown, but there are suggestions that some chemical modifications might occur in the renal basement membranes. Therefore, this clinical case might induce to consider possible relations between disorders of the lipoprotein metabolism and renal dense-deposit disease.

Identification of an alternative transcript of ABCA1 gene in different human cell types.

We have observed two ABCA1 gene transcripts in human skin fibroblasts. The RT-PCR amplification of the exon 3-exon 8 region generated a normal fragment (740 bp) and an abnormal fragment (600 bp) in a ratio ranging from 3:1 to 8/9:1. These two transcripts were present in other cells such as leukemia T-cells, endothelial and smooth muscle cells as well human hepatoma cells (HepG2). Restriction enzyme analysis and sequencing indicated that in the abnormal fragment exon 3 was followed by exon 5. The complete skipping of exon 4 leads to a premature stop and a predicted translation product of 74 amino acids. The ratio between the normal and alternative transcript is not affected by variation in ABCA1 gene expression induced by incubating cells in serum-free medium and in the presence of cholesterol. It is possible that this alternative splicing represents as mechanism that regulates the ABCA1 content in tissues.

A point mutation in ABC1 gene in a patient with severe premature coronary heart disease and mild clinical phenotype of Tangier disease.

The proband is a 50 year-old woman born from a consanguineous marriage. She has been suffering from angina pectoris since the age of 38 and underwent coronary bypass surgery for three-vessel disease at 48. The presence of low plasma levels of total cholesterol and high density lipoprotein (HDL) cholesterol (2.4 and 0.1 mmol/l) and apo AI (<15 mg/dl), associated with corneal lesions and a mild splenomegaly suggested the diagnosis of Tangier disease. However, none of the other features of Tangier disease, including hepatomegaly, anemia and peripheral neuropathy, were present. The analysis of the dinucleotide microsatellites located in chromosome 9q31 region demonstrated that the proband was homozygous for the alleles of D9S53, D9S1784 and D9S1832. The mother and son of the proband, both with low levels of HDL cholesterol, shared one of the proband's haplotypes, whereas neither of these haplotypes was present in the normolipidemic proband's sister. The sequence of ATP-binding cassette transporter 1 (ABC1-1) cDNA obtained by reverse transcription-PCR (RT-PCR) of total RNA isolated from cultured fibroblasts showed that the proband was homozygous for a C>T transition in exon 13, which caused a tryptophane for arginine substitution (R527W). This mutation was confirmed by direct sequencing of exon 13 amplified from genomic DNA. It can be easily screened, as the nucleotide change introduces a restriction site for the enzyme Afl III. R527W substitution occurs in a highly conserved region of the NH2 cytoplasmic domain of ABC1 protein. R527W co-segregates with the low HDL phenotype in the family and was not found in 200 chromosomes from normolipidemic individuals.

Polymorphisms of drug-metabolizing enzymes in healthy nonagenarians and centenarians: difference at GSTT1 locus.

Drug metabolizing enzymes are involved in the detoxification of several drugs, environmental substances, and carcinogenic compounds, and their polymorphisms have been associated with risk for a variety of cancer. In this paper, we compared the frequency of polymorphisms in cytochrome P450-1A1 gene (CYP1A1), a phase 1 gene (oxidation, activation), and of two polymorphisms of glutathione S-transferase enzymes (GSTM1, GSTT1), two phase 2 genes (conjugation, detoxification). Two groups were studied and compared, i.e., 94 nonagenarians and centenarians and 418 control subjects of younger age. A significant difference in the proportion of nonagenarians and centenarians homozygotes for a GSTT1 deletion (28%) was observed in comparison to control subjects (19%, P = 0.03). The distribution of the other gene polymorphisms did not differ in the two groups. These findings on phase 2 drug-metabolizing enzyme gene polymorphisms may help in disentangling gene-environmental interactions which can have a role in successful aging and longevity, as well as in cancer incidence in the oldest old.

Italian familial defective apolipoprotein B patients share a unique haplotype with other Caucasian patients.

Familial defective apolipoprotein (apo) B-100 together with familial hypercholesterolemia are the two common genetic conditions that cause hypercholesterolemia. Familial defective apolipoprotein B-100 is due to mutations around codon 3500 of the apo B gene. The most-characterized mutation is a G>A transition at nucleotide 10,708 that results in the substitution of arginine by glutamine at codon 3500 (Apo B Arg3500Gln). Two other mutations are caused by a C>T transition, one at nucleotide 10,800 (Apo B Arg3531Cys) and the other at nucleotide 10,707 (apo B Arg3500Trp). In the present study we describe three new Italian cases of familial defective apolipoprotein B-100 (Apo B Arg3500Gln), one from the Liguria region and two from Sicily, and the haplotype of the apo B gene co-segregating with the mutation. By screening two groups of probands, clinically diagnosed as having Familial Hypercholesterolemia (700 from mainland Italy and 305 from Sicily), the prevalence of familial defective apolipoprotein B-100 due to Arg3500Gln was found to be very low (0.28% and 0.65%, respectively). The Arg3531Cys mutation was not detected in any proband. In the three new families with Arg3500Gln mutation in the present study and in one previously described in Italy, the mutation was associated with a unique apo B haplotype, which is consistent with data previously reported for Caucasian patients [XbaI-, MspI+, EcoRI-, presence of the 5' signal peptide insertion (Ins) allele, and the 49-repeat allele of the 3'-VNTR].

Effectiveness of screening for known mutations in Sicilian patients with "probable" familial hypercholesterolemia.

BACKGROUND AND AIM: More than 750 mutations in the low-density lipoprotein (LDL) receptor gene are currently known to cause familial hypercholesterolemia (FH), but the array of mutations varies considerably in different populations. The definition of essentially all the LDL receptor gene mutations in a population is therefore a prerequisite for the implementation of nation-wide genetic testing for FH. METHODS AND RESULTS: In this study, a screening strategy based on PCR-enzymatic digestion and PCR-allele specific hybridisation procedures was used to evaluate the frequency distributions of 11 known mutations in a cohort of 214 unrelated subjects meeting the diagnostic criteria of "probable" FH. We identified 20 mutation carriers (9.3%). One mutation (FH Palermo-1) occurred with a relatively high frequency, accounting for 7% of the entire study cohort. We also report the first observation of the receptor-negative mutation V408M (Afrikaner-2) in Italy. CONCLUSIONS: Our screening approach is not effective and, at least in our area, it is not a suitable alternative to the more expensive and time-consuming sequencing approach. However, our data suggest that it is possible to identify the molecular defect in about 10% of Sicilian patients with a clinical diagnosis of "probable FH" using a rapid laboratory diagnostic mutation panel. Four mutations were responsible for all of the diagnosed cases, and it could be reasonable to use this 4-mutation panel as a preliminary step before adopting a more complex laboratory approach.

Clinical expression of familial hypercholesterolemia in clusters of mutations of the LDL receptor gene that cause a receptor-defective or receptor-negative phenotype.

Seventy-one mutations of the low density lipoprotein (LDL) receptor gene were identified in 282 unrelated Italian familial hypercholesterolemia (FH) heterozygotes. By extending genotype analysis to families of the index cases, we identified 12 mutation clusters and localized them in specific areas of Italy. To evaluate the impact of these mutations on the clinical expression of FH, the clusters were separated into 2 groups: receptor-defective and receptor-negative, according to the LDL receptor defect caused by each mutation. These 2 groups were comparable in terms of the patients' age, sex distribution, body mass index, arterial hypertension, and smoking status. In receptor-negative subjects, LDL cholesterol was higher (+18%) and high density lipoprotein cholesterol lower (-5%) than the values found in receptor-defective subjects. The prevalence of tendon xanthomas and coronary artery disease (CAD) was 2-fold higher in receptor-negative subjects. In patients >30 years of age in both groups, the presence of CAD was related to age, arterial hypertension, previous smoking, and LDL cholesterol level. Independent contributors to CAD in the receptor-defective subjects were male sex, arterial hypertension, and LDL cholesterol level; in the receptor-negative subjects, the first 2 variables were strong predictors of CAD, whereas the LDL cholesterol level had a lower impact than in receptor-defective subjects. Overall, in receptor-negative subjects, the risk of CAD was 2.6-fold that of receptor-defective subjects. Wide interindividual variability in LDL cholesterol levels was found in each cluster. Apolipoprotein E genotype analysis showed a lowering effect of the epsilon2 allele and a raising effect of the epsilon4 allele on the LDL cholesterol level in both groups; however, the apolipoprotein E genotype accounted for only 4% of the variation in LDL cholesterol. Haplotype analysis showed that all families of the major clusters shared the same intragenic haplotype cosegregating with the mutation, thus suggesting the presence of common ancestors.

Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia : a study of patients with familial hypercholesterolemia from Sardinia.

One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the alpha-globin gene (alpha-thalassemia trait) and that 6% to 17% are beta-thalassemia carriers. In this population, a single mutation of beta-globin gene (Q39X, beta(0) 39) accounts for >95% of beta-thalassemia cases. Because previous studies have shown that Sardinian beta-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the beta-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76+/-1.08 mmol/L in subjects with beta(0)-thalassemia trait and 8.25+/-1.66 mmol/L in subjects without this trait (P<0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had beta(0)-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of beta(0)-thalassemia trait emerged also when we pooled the data from all FH subjects with and without beta(0)-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of beta(0)-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B-containing lipoproteins. The observation that our FH subjects with beta(0)-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of beta(0)-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.

Rapid screening of the LDL receptor point mutation FH-Genoa/Palermo. Mutation in brief no. 238. Online.

The LDL-receptor gene point mutation FH-Genoa/Palermo is the most frequent mutation responsible for Familial Hypercholesterolemia in Sicily. The mutation does not introduce or abolish any useful restriction site. We establish a GeneComb-based strategy to identify this mutation in a population of Sicilian unrelated clinically diagnosed FH probands. The method was very sensitive and specific; 12 out of 90 (13.3%) unrelated FH probands were found to carry the FH-Genoa/Palermo mutation. According to these results, the FH-Genoa/Palermo is the more frequent LDL-receptor gene mutation among the Sicilian FH patients. Moreover FH-Genoa/Palermo is the mutation cluster to date more represented in Southern Italy.

A third major locus for autosomal dominant hypercholesterolemia maps to 1p34.1-p32.

Autosomal dominant hypercholesterolemia (ADH), one of the most frequent hereditary disorders, is characterized by an isolated elevation of LDL particles that leads to premature mortality from cardiovascular complications. It is generally assumed that mutations in the LDLR and APOB genes account for ADH. We identified one large French pedigree (HC2) and 12 additional white families with ADH in which we excluded linkage to the LDLR and APOB, implicating a new locus we named "FH3." A LOD score of 3.13 at a recombination fraction of 0 was obtained at markers D1S2892 and D1S2722. We localized the FH3 locus to a 9-cM interval at 1p34.1-p32. We tested four regional markers in another set of 12 ADH families. Positive LOD scores were obtained in three pedigrees, whereas linkage was excluded in the others. Heterogeneity tests indicated linkage to FH3 in approximately 27% of these non-LDLR/non-APOB ADH families and implied a fourth locus. Radiation hybrid mapping located four candidate genes at 1p34.1-p32, outside the critical region, showing no identity with FH3. Our results show that ADH is genetically more heterogeneous than conventionally accepted.