Genetic variants in PCSK9 in the Japanese population: rare genetic variants in PCSK9 might collectively contribute to plasma LDL cholesterol levels in the general population

The aim of this study was to investigate whether plasma low-density lipoprotein cholesterol (LDL-C) levels in the general population are influenced by rare sequence variations in the PCSK9 gene. We sequenced the promoter and coding regions of the PCSK9 gene in individuals from the general population (n=3655) with the lowest (n=78) and highest (n=96) LDL-C levels and in individuals taking antihypercholesterolemia medication (n=96). We identified 33 sequence variants in the PCSK9 gene among which 24 were specific for Japanese. Statistical analysis showed that one missense mutation, R93C, was associated with low LDL-C levels. The other variants had no association with LDL-C levels or the numbers of individuals with the variants were too small for statistical analysis. A comparison of the numbers of individuals with nonsynonymous mutations between the low LDL-C and high LDL-C/treatment groups found that four missense mutations and one nonsense mutation were identified only in the low LDL-C group and six missense mutations were identified only in the high LDL-C/treatment group. As we have analyzed groups at opposite ends of the LDL-C spectrum, it is likely that some of these nonsynonymous mutations may be associated with either low or high LDL-C in the Japanese population. Based on the extremely high frequencies of the nonsynonymous mutations in PCSK9 compared with those of LDLR or apoB-100, PCSK9 mutations could be important factors that cumulatively influence plasma LDL-C levels in the general population.     

Mutations in low-density lipoprotein receptor gene as a cause of hypercholesterolemia in Taiwan

Familial hypercholesterolemia (FH) is inherited as an autosomal dominant trait that has been associated with more than 920 different mutations in the low-density lipoprotein receptor (LDLR) gene. To characterize LDLR gene mutations in the Chinese of Han descent with FH, we isolated genomic DNA from peripheral blood samples of 20 affected subjects and 50 healthy subjects with no family history of hypercholesterolemia. We used polymerase chain reaction and long polymerase chain reaction to amplify the 18 coding exons and the minimal promoter of the LDLR gene, and subjected amplicons to direct sequence analysis. We identified 6 mutations in LDLR gene, including heterozygous missense mutations I420T (ATC-->ACC), C660W (TGC-->TGG), H562Y (CAC-->TAC), and A606T (GCC-->ACC), and a heterozygous and a homozygous mutation in codon P664L (CCG-->CTG) as well as a homozygous large deletion of exons 6 to 8. The FH homozygotes manifested generalized xanthomatosis. One of the mutations we identified (C660W) was novel. In conclusion, we identified 5 missense mutations and 1 large deletion in LDLR gene, including 1 novel mutation in Han Chinese with FH in Taiwan.     

Effect of ezetimibe coadministered with statins in genotype-confirmed heterozygous FH patients

We investigated the effect of statins and statins plus ezetimibe in 65 FH heterozygotes carrying LDLR-defective or LDLR-negative mutations as well as the effect of ezetimibe monotherapy in 50 hypercholesterolemic (HCH) patients intolerant to statins. PCSK9 and NPC1L1 genes were analysed to assess the role of genetic variants in response to therapy. In FH patients combined therapy reduced LDL-C by 57%, irrespective of the type of LDLR mutation. The additional decrease of plasma LDL-C induced by ezetimibe showed wide inter-individual variability (from -39% to -4.7%) and was negatively correlated with percent LDL-C decrease due to statin alone (r=-0.713, P<0.001). The variable response to statins was not due to PCSK9 gene variants associated with statin hyper-sensitivity. The highest response to ezetimibe was observed in a carrier of R174H substitution in NPC1L1, which had been found to be associated with high cholesterol absorption. In HCH patients, ezetimibe monotherapy induced a variable decrease of plasma LDL-C (from -47.7% to -13.4%). To investigate this variability, we sequenced NPC1L1 gene in patients with the highest and the lowest response to ezetimibe. This analysis showed a higher prevalence of the G allele of the c.816 C>G polymorphism (L272L) in hyper-responders, an observation confirmed also in FH patients hyper-responders to ezetimibe. In both FH and HCH patients, the G allele carriers tended to have a higher LDL-C reduction in response to ezetimibe. These observations suggest that in FH heterozygotes LDL-C reduction following combined therapy reflects a complex interplay between hepatic synthesis and intestinal absorption of cholesterol.     

Effect of mutations in LDLR and PCSK9 genes on phenotypic variability in Tunisian familial hypercholesterolemia patients

BackgroundAutosomal dominant hypercholesterolemia (ADH) is commonly caused by mutations in the low-density lipoprotein (LDL) receptor gene (LDLR), in the apolipoprotein B-100 gene (APOB), or in the proprotein convertase subtilisin kexine 9 gene (PCSK9). ADH subjects carrying a mutation in LDLR present highly variable plasma LDL-cholesterol (LDL-C). This variability might be due to environmental factors or the effect of some modifying genes such as PCSK9 and APOE.AimsWe investigated the molecular basis of thirteen Tunisian ADH families and attempted to determine the impact of PCSK9 and APOE gene variations on LDL-cholesterol levels and on the variable phenotypic expression of the disease.Methods and resultsFifty six subjects were screened for mutations in the LDLR gene through direct sequencing. The causative mutation was found to segregate with the disease in each family and a new frameshift mutation, p.Met767CysfsX21, was identified in one family. The distribution of total- and LDL-cholesterol levels, adjusted for age and gender, among homozygous and heterozygous ADH patients varied widely. Within seven families, nine subjects presented low LDL-cholesterol levels despite carrying a mutation in the LDLR gene. To identify the molecular actors underlying this phenotypic variability, the PCSK9 gene was screened using direct sequencing and/or enzymatic restriction analysis, and the apo E genotypes were determined. A new missense variation (p.Pro174Ser) in the PCSK9 gene was identified and characterized as a new putative loss-of-function mutation.ConclusionGenetic variations in PCSK9 and APOE genes could explain only part of the variability observed in the phenotypic expression in Tunisian ADH patients carrying mutations in the LDLR gene. Other genetic variants and environmental factors very probably act to fully explain this phenotypic variability.     

高胆固醇血症患者枯草溶菌素转化酶9基因突变研究

目的探讨国人单纯性高胆固醇血症患者人类枯草溶菌素转化酶9基因突变情况。方法排除载脂蛋白B100基因Q3500R位点突变及低密度脂蛋白受体基因突变后的100例单纯性高胆固醇血症患者,提取其基因组DNA,采用聚合酶链反应方法扩增枯草溶菌素转化酶9基因的12个外显子,进行DNA测序分析,同时选100例健康人群作为对照。结果 100例高胆固醇血症患者中,6例发现3处错义突变(D320N、V312F、R319E)、1处框移突变(934delGV312S)、3处同义突变(A305A、Q342Q、K125K)和1处剪接点突变(第2内含子5’剪接位点突变)。结论枯草溶菌素转化酶9基因在以上位点发生的突变可引起单纯性高胆固醇血症。枯草溶菌素转化酶9基因突变可能与国人高胆固醇血症有关。     

Molecular basis for LDL receptor recognition by PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally regulates hepatic low-density lipoprotein receptors (LDLRs) by binding to LDLRs on the cell surface, leading to their degradation. The binding site of PCSK9 has been localized to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. Here, we describe the crystal structure of a complex between PCSK9 and the EGF-A domain of the LDLR. The binding site for the LDLR EGF-A domain resides on the surface of PCSK9's subtilisin-like catalytic domain containing Asp-374, a residue for which a gain-of-function mutation (Asp-374–Tyr) increases the affinity of PCSK9 toward LDLR and increases plasma LDL-cholesterol (LDL-C) levels in humans. The binding surface on PCSK9 is distant from its catalytic site, and the EGF-A domain makes no contact with either the C-terminal domain or the prodomain. Point mutations in PCSK9 that altered key residues contributing to EGF-A binding (Arg-194 and Phe-379) greatly diminished binding to the LDLR's extracellular domain. The structure of PCSK9 in complex with the LDLR EGF-A domain defines potential therapeutic target sites for blocking agents that could interfere with this interaction in vivo, thereby increasing LDLR function and reducing plasma LDL-C levels.     

矮妖综合征:胰岛素受体缺陷所致的胰岛素抵抗综合征

目的对1例罕见的17岁矮妖综合征患者进行临床及分子生物学研究,探讨其发病机制及特点。方法对该患者进行病史询问及相关检查。采集患者及其家系成员（父母及妹妹）的血DNA样本,进行胰岛素受体编码基因（INSR）各外显子的PCR扩增测序。结果该患者面容特殊,体型消瘦,体重指数15．5kg／m^2,多毛,全身黑棘皮病。空腹C肽2．92μg／L,胰岛素104mIU／L,空腹血糖15．8mmol／L;糖化血红蛋白（HbA1c）12％。住院后予格列美脲4mg／d治疗4d,空腹血糖降至15．1mmol／L,空腹C肽增至7．4μg／L;后加用胰岛素治疗（约200U／d）,空腹血糖降至12．3mmol／L。基因筛查发现,患者INSR存在W659R及V1054M的复合杂合突变,分别位于第9及17号外显子,此为矮妖综合征一新的突变类型。而其父只存在V1054M突变;其母只存在W659R突变,其妹正常。结论患者为一新的INSR突变引起的矮妖综合征,其生存期长,对其特殊表现尚需做进一步的研究。     

A novel type of familial hypercholesterolemia: double heterozygous mutations in LDL receptor and LDL receptor adaptor protein 1 gene

Background

Autosomal recessive hypercholesterolemia (ARH) is an extremely rare inherited hypercholesterolemia, the cause of which is mutations in low-density lipoprotein (LDL) receptor adaptor protein 1 (LDLRAP1) gene.

Methods

A total of 146 heterozygous familial hypercholesterolemic (FH) patients with a mutation in LDLR gene were screened for genes encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLRAP1.

Results

Among the 146 subjects, we identified a 79-year-old Japanese female with double mutations in LDLR gene (c.2431A > T) and LDLRAP1 gene (c.606dup). Two other relatives with double mutations in those genes in her family were also identified. Although the proband exhibited massive Achilles tendon xanthoma and coronary and aortic valvular disease, serum LDL-C level of subjects with double mutations was similar with that of subjects with single LDLR mutation (284.0 ± 43.5 versus 265.1 ± 57.4 mg/dl).

Conclusion

Additional mutation in LDLRAP1 may account for severer phenotype in terms of xanthoma and atherosclerotic cardiovascular disease in FH patients.     

Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa

We analysed the Proprotein Convertase Subtilisin Kexin type 9 (PCSK9) exons and intronic junctions of 71 patients with familial hypercholesterolemia (FH) in whom LDL receptor (LDLR) or apolipoprotein B100 mutations were excluded. The previously reported S127R and R237W mutations were found in South African families, whereas new missense mutations D129G and A168E were found in families from New Zealand. Only, the S127R and D129G mutations modify a highly conserved residue and segregate with the FH phenotype. We overexpressed those mutants in hepatoma cells and found that both S127R and D129G have reduced autocatalytic activity compared with wild-type PCSK9, whereas the A168E mutant is processed normally. The S127R and D129G mutants were not secreted from cells, unlike the A168E mutant and wild-type PCSK9. By immunoblot, we showed that the expression of the LDLR was reduced by 40% in cells overexpressing wild-type or A168E PCSK9 and further reduced by 30% when the S127R or D129G mutants were used. Paralleling the LDLR levels, LDL cellular binding decreased by 25% upon wild-type PCSK9 or A168E overexpression, and by 45% with both S127R and D129G mutants. Our study therefore indicates that PCSK9 mediated inhibition of the LDLR does not require PCSK9 autocatalytic cleavage or secretion, suggesting that PCSK9 may also function intracellularly.     

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

Background and aimsIn 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 resultsPlasma 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).ConclusionThe 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.     

Limited mutational heterogeneity in the LDLR gene in familial hypercholesterolemia in Tunisia

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In previous studies, we have identified novel mutations in Tunisian FH families. In this study, we have extended our investigation to additional families. Five unrelated probands were screened for mutations in the LDLR and APOB genes, using direct sequencing and enzymatic restriction. We identified two novel LDLR mutations: a missense mutation in exon 7: p.Gly343Cys (c.1027G>T), and a nonsense mutation in exon 17: p.Lys816X (c.2446A>T). Using the PolyPhen and SIFT prediction computer programs the p.Gly343Cys is predicted to have a deleterious effect on LDL receptor activity. The missense mutation we found in exon 3, p.Cys89Trp (c.267C>G), has previously been identified in patients from United Kingdom and Spain, and is reported here for the first time in the Tunisian population. Finally, the framshift mutation in exon 10, p.Ser493ArgfsX44, is reported here for the fourth and fifth time in Tunisian families. The latter is the most frequent FH-causing mutation in Tunisia. These LDLR gene mutations enrich the spectrum of mutations causing FH in the Tunisian population. The framshift mutation, p.Ser493ArgfsX44, seems to be a founder mutation in this population.     

Mutations in MTP gene in abeta- and hypobeta-lipoproteinemia

Familial hypobetalipoproteinemia (FHBL) and abetalipoproteinemia (ABL) are inherited disorders of apolipoprotein B (apo B)-containing lipoproteins that result from mutations in apo B and microsomal triglyceride transfer protein (MTP) genes, respectively. Here we report three patients with severe deficiency of plasma low-density lipoprotein (LDL) and apo B. Two of them (probands F.A. and P.E.) had clinical and biochemical phenotype consistent with ABL. Proband F.A. was homozygous for a minute deletion/insertion (c.1228delCCCinsT) in exon 9 of MTP gene predicted to cause a truncated MTP protein of 412 amino acids. Proband P. E. was heterozygous for a mutation in intron 9 (IVS9-1G>A), previously reported in an ABL patient. We failed to find the second pathogenic mutation in MTP gene of this patient. No mutations were found in apo B gene.

The third proband (D.F.) had a less severe lipoprotein phenotype which was similar to that of heterozygous FHBL and appeared to be inherited as a co-dominant trait. However, he had no mutations in apo B gene. He was found to be a compound heterozygote for two missense mutations (D384A and G661A), involving highly conserved regions of MTP. Since this proband was also homozygous for 2 allele of apolipoprotein E (apo E), it is likely that his hypobetalipoproteinemia derives from a combined effect of a mild MTP deficiency and homozygosity for apo E2 isoform.     

Association between causative mutations and response to PCSK9 inhibitor therapy in subjects with familial hypercholesterolemia: A single center real-world study

Background and aimsFamilial hypercholesterolemia (FH) is an autosomal dominant disease that leads to cardiovascular (CV) disease. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9-I) demonstrated efficacy in low-density lipoprotein cholesterol (LDL-C) reduction and in prevention of CV events. The aim of our study is to evaluate the relationship between LDL receptor (LDLR) mutations and response to PCSK9-I therapy.Methods and resultsWe evaluated total cholesterol (TC), LDL-C, high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) in consecutive patients with FH before PCSK9-I treatment and after 12 (T12w) and 36 (T36w) weeks of treatment. We evaluated LDL-C target achievement according to different mutations in LDLR. Eighty FH subjects (mean age:54 ± 13.3 years), 39 heterozygous (He) with defective LDLR gene mutations, 30 He with null mutations and 11 compound-He or homozygous (Ho) were recruited. At baseline, 69 subjects were under maximal lipid lowering therapy (MLLT) and 11 subjects had statin-intolerance. From baseline to T36w we observed an overall 51% reduction in LDL-C. We found no difference in LDL-C changes between subjects with He-defective mutation and He-null mutations both at T12w (p = 1.00) and T36w (p = 0.538). At T36w, LDL-C target was achieved in 59% of He-defective mutations subjects and in 36% of He-null mutations subgroup (p = 0.069), whereas none of compound-He/Ho-FH achieved LDL-C target.ConclusionsAfter 36 weeks there were no differences in response to PCSK9-I therapy between different groups of He-FH subjects. Response to PCSK9-I was significantly lower in carriers of compound-He/Ho mutations.Registration number for clinical trials: NCT04313270 extension.     

PCSK9 − „missing link“ der familiären Hypercholesterinämie

Lowering plasma low-density lipoprotein cholesterol (LDL-C) levels to individual therapeutic goals is one of the most effective measures for the prevention of cardiovascular disease. Besides dietary measures, this can be achieved pharmaceutically by inhibition of hepatic cholesterol synthesis with statins or inhibition of intestinal cholesterol absorption (e.g., ezetimibe and bile acid sequestrants). Decisive for lowering LDL is an increased hepatic uptake of circulating LDL via an increase in LDL receptors (LDLR) in hepatic cell membranes. The formation of new LDLR and recirculation of existing LDLR play a decisive role in this process. An important modulator of LDLR is proprotein convertase subtilisin/kexin type 9 (PCSK9). In the last years genetic studies have identified several mutations in the PCSK9 gene leading to a gain of function and carriers of these mutations suffer from autosomal dominant hypercholesterolemia. In contrast, carriers of PCSK9 loss of function mutations show very low plasma LDL-C concentrations and a markedly reduced risk for coronary artery disease. These fundamental discoveries have sparked the development of a completely novel therapeutic approach to treating hypercholesterolemia. At present, inhibition of PCSK9 by monoclonal antibodies presents the most promising therapeutic approach. First human antibodies were recently approved as the first immunotherapeutic agents for the treatment of severe hypercholesterolemia and in patients with statin intolerance. An additional PCSK9 antibody is presently being studied in phase III clinical trials.     

A Resuscitated Case of Acute Myocardial Infarction with both Familial Hypercholesterolemia Phenotype Caused by Possibly Oligogenic Variants of the PCSK9 and ABCG5 Genes and Type I CD36 Deficiency

A 56-year-old postmenopausal woman with out-of-hospital cardiac arrest caused by acute myocardial infraction was successfully resuscitated by intensive treatments and recovered without any neurological disability. She was diagnosed as having familial hypercholesterolemia (FH) based on a markedly elevated low-density lipoprotein cholesterol (LDL-C) level and family history of premature coronary artery disease. Genetic testing in her family members showed that a variant of the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene (c.2004C＞A, p.S668R), which had been previously reported as having uncertain significance, was associated with FH, indicating that the variant is a potential candidate for the FH phenotype. Next-generation sequencing analysis for the proband also showed that there was a heterozygous mutation of the ATP-binding cassette sub-family G member 5 ( ABCG5 ) gene (c.1166G＞A, R389H), which has been reported to increase LDL-C level and the risk of cardiovascular disease. She was also diagnosed as having type 1 CD36 deficiency based on a lack of myocardial uptake of ¹²³ I-labeled 15-(p-iodophenyl)-3-R,S-methyl-pentadecanoic acid in scintigraphy and the absence of CD36 antigen in both monocytes and platelets in flow cytometry. She had a homozygous mutation of the CD36 gene (c.1126-5_1127delTTTAGAT), which occurs in a canonical splice site (acceptor) and is predicted to disrupt or distort the normal gene product. To our knowledge, this is the first report of a heterozygous FH phenotype caused by possibly oligogenic variants of the PCSK9 and ABCG5 genes complicated with type I CD36 deficiency caused by a novel homozygous mutation. Both FH phenotype and CD36 deficiency might have caused extensive atherosclerosis, leading to acute myocardial infarction in the present case.     

Novel mutations in the Factor VII gene of Taiwanese Factor VII-deficient patients

The genetic defects of four Taiwanese patients with factor VII (FVII) deficiency were studied. FVII activity and antigen levels were < 1 u/dl and 125.7 u/dl (patient I), < 1 u/dl and < 1 u/dl (patient II), 3.4 u/dl and 5.9 u/dl (patient III), and 1.2 u/dl and 30.4 u/dl (patient IV) respectively. The 5' flanking region, and all exons and junctions were amplified using polymerase chain reaction and sequenced. Patient I was homozygous for a 10824C-->A transversion with Pro303-->Thr mutation in exon 8. In patient II, a heterozygous transversion, 9007+1G-->T at the IVS6, a heterozygous decanucleotide insertion polymorphism at -323 (both mutations present in his father) and a heterozygous deletion, del TC (26-27) in exon 1A (originating from his mother) were identified. Patient III had a homozygous 10961T-->G transversion with His348-->Gln mutation in exon 8. Patient IV had a heterozygous 10902T-->G transversion with Cys329-->Gly mutation in exon 8 (transmitted to her second son) and a heterozygous decanucleotide insertion polymorphism at -323 (transmitted to her third son). All but one of the FVII gene mutations detected in the four patients have not been previously reported. In conclusion, four novel mutations of the FVII gene in Taiwanese, including two missense mutations in exon 8, one point mutation at the exon 6 splice site and one deletion in exon 1A, were identified.     

Venous thromboembolism associated with double heterozygosity for R506Q mutation of factor V and for T298M mutation of protein C in a large family of a previously described homozygous protein C-deficient newborn with massive thrombosis

It is remarkable that certain patients with heterozygous protein C (PC) deficiency manifest venous thromboembolism (VTE), whereas others, particularly those belonging to families with homozygous PC deficiency, remain asymptomatic. The goals of the present study of a family, in which the proband had homozygous PC deficiency, were to identify members with and without VTE, to determine the mutation causing PC deficiency, and to search for the R506Q mutation of factor V (FV) causing activated PC resistance. Heterozygosity for a T298M mutation in exon 9 of the PC gene was found in the father of the homozygous proband who died of massive thrombosis. Based on analysis of a three- dimensional molecular model of PC, we speculate that this mutation causes type I deficiency due to disruption of packing of hydrophobic side chains and loss of an H-bond between Q184 and T298. Forty-six family members were examined for the T298M mutation by polymerase chain reaction (PCR) amplification of exon 9 and restriction analysis using Mae III and for the FV R506Q mutation by PCR amplification of exon 10 and restriction analysis using Mnl I. VTE was observed in five of 11 members who were heterozygous for both PC and FV mutations. In contrast, VTE was not observed for the PC mutation in 13 heterozygotes who had normal FV, including the parents of the deceased proband, 10 heterozygotes for the FV mutation who had normal PC, and 12 individuals bearing neither mutation. These observations extend recent evidence of an increased thrombotic risk conferred by the coexistence of heterozygous PC deficiency and heterozygous activated PC resistance and support the paradigm in which hereditary thrombophilia is often a multigenic disease.     

Portuguese Familial Hypercholesterolemia Study: presentation of the study and preliminary results.

INTRODUCTION: Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder caused, in the majority of cases, by a partial or total lack of functional low density lipoprotein receptors (LDLR). Mutations in the LDLR gene lead to increased plasma cholesterol levels, resulting in cholesterol deposition in the arteries, thereby increasing the risk of premature coronary heart disease. The homozygous form of FH is rare but heterozygous FH is common, although underdiagnosed in many populations, including the Portuguese. In 1999 the Portuguese Familial Hypercholesterolemia Study was begun at the National Institute of Health. OBJECTIVES: The aim of the Portuguese Familial H ypercholesterolemia Study is to perform an epidemiological study to determine the prevalence and distribution of FH in Portugal and to better understand the pathophysiology of coronary heart disease in these patients. The aim of the present work is to present the study's criteria and organization as well as its preliminary results. METHODS: The study population consists of individuals of both sexes and all ages with a clinical diagnosis of FH, with biochemical and molecular characterization being performed. The clinical criteria used for the diagnosis of FH were adapted from those of the Simon Broome Heart Research Trust. The study is organized in five stages: 1. selection of individuals with a clinical diagnosis of FH; 2. completion of a clinical questionnaire and declaration of informed consent; 3. collection of blood samples; 4. biochemical characterization; 5. molecular study of three genes associated with the FH phenotype: LDLR, apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9). RESULTS: Between 1999 and June 2006 the LDLR gene and the APOB gene of 141 index cases (38 children and 103 adults) were studied. In 78 of these index cases (76 heterozygotes and two homozygotes) 50 different mutations in the LDLR gene were identified, and two unrelated individuals were found to have the ApoB3500 mutation. The PCSK9 gene was also studied in individuals in whom a mutation in the LDLR or APOB genes was not found, which identified two index cases with a mutation in this gene. The study of 62 families led to the identification of an additional 117 individuals with FH, 90 adults and 27 children (86 adults and 27 children with mutations in the LDLR gene, two adults with the ApoB3500 mutation, and two adults with a mutation in the PCSK9 gene). CONCLUSIONS: Genetic diagnosis enables correct identification of the disease and provides the basis for more aggressive pharmacologic therapeutic interventions to reduce cardiovascular risk in affected individuals. At present thirteen clinicians are collaborating in the Portuguese FH Study, but it is extremely important to obtain the collaboration of more physicians throughout the country, so that the prevalence and distribution of FH in the Portuguese population can be characterized and a greater number of individuals can benefit from appropriate and early therapy.     

Mutations in the galactose-1-phosphate uridyltransferase gene of two families with mild galactosaemia variants

Summary Classical galactosaemia, deficiency of galactose-1-phosphate uridyltransferase (GALT), is characterized by acute symptoms of hepatomegaly, jaundice, sepsis, cataracts and growth retardation. Treatment with dietary galactose restriction corrects these complications immediately; however, most of these children develop long-term complications of verbal dyspraxia, mental retardation and ovarian failure. Our previous molecular study showed that the most common mutation of the GALT gene is a missense mutation of Q188R (replacement of glutamine-188 by arginine) in approximately 60–65% of the German galactosaemic population. The coding region of GALT was amplified by the polymerase chain reaction from genomic DNA of classical galactosaemic individuals, who are negative or heterozygous for Q188R, and was further characterized by direct sequencing. Three new disease-causing mutations, two missense and a stop codon mutation, were identified in three patients from two families with mild galactosaemic variants: firstly R67C, replacement of arginine-67 by cysteine and W316X, the stop codon at tryptophan-316 in one male; secondly A330V, replacement of alanine-330 by valine in two female siblings. In the first family the patient was also heterozygous for the polymorphism N314D and in the second family both girls were compound heterozygotes for Q188R and A330V. All three galactosaemic individuals have a considerable amount of the residual GALT activity in RBC and the galactose-1-phosphate (GALP) level decreased much faster on treatment than that of other galactosaemic patients with missense mutations such as Q188R. The clinical and biochemical data of these patients were much more favourable in comparison with those of two female galactosaemic individuals, one homozygous for L195P and the other compound heterozygous for Q188R and L195P. These three missense mutations (R67C, L195P and A330V) also occur in highly conserved regions. These observations suggest that the phenotypic variation in galactosaemic individuals may be due to different molecular aetiologies.     

Identification and characterization of new gain-of-function mutations in the PCSK9 gene responsible for autosomal dominant hypercholesterolemia

BackgroundThe identification of mutations in PCSK9 (proprotein convertase subtilisin kexin9) in autosomal dominant hypercholesterolemia (ADH), has revealed the existence of a new player in cholesterol homeostasis. PCSK9 has been shown to enhance the degradation of the LDL receptor (LDLR) at the cell surface. Gain-of-function mutations of PCSK9 induce ADH and are very rare, but their identification is crucial in studying PCSK9's role in hypercholesterolemia, its detailed trafficking pathway and its impact on the LDLR.MethodsIn order to identify new mutations and understand the exact mechanisms of action of mutated PCSK9, PCSK9 was sequenced in 75 ADH patients with no mutations in the LDLR or APOB genes. Functional analyses in cell culture were conducted and the impact of novel PCSK9 mutations on the quantitative and qualitative features of lipoprotein particles and on the HDL-mediated cellular cholesterol efflux was studied.ResultsAmong these 75 ADH probands with no mutations in the LDLR or APOB genes, four gain-of-function mutations of PCSK9 were identified, of which two were novel: the p.Leu108Arg and the p.Asp35Tyr substitutions. In vitro studies of their consequences on the activity of PCSK9 on cell surface levels of LDLR showed that the p.Leu108Arg mutation clearly results in a gain-of-function, while the p.Asp35Tyr mutation created a novel Tyr-sulfation site, which may enhance the intracellular activity of PCSK9.ConclusionThese data further contribute to the characterization of PCSK9 mutations and to better understanding of the impact on cholesterol metabolism of this new therapeutic target.