Fh-Souassi: a founder frameshift mutation in exon 10 of the LDL-receptor gene, associated with a mild phenotype in Tunisian families

Familial hypercholesterolemia (FH) has a higher prevalence in central Tunisia together with a milder clinical expression than in western countries. The molecular basis of FH in Tunisia remains unknown. Our aim was to identify FH-causing mutations in three unrelated families (21 subjects) from the area of Souassi (central Tunisia). In probands with a presentation of homozygous FH, the promoter and 18 exons of the low density lipoprotein (LDL)-receptor gene were sequenced in both orientations. A novel complex frameshift mutation was identified in exon 10, nucleotides 1477-1479 (TCT) at Serine 472 were replaced by an insertion of seven nucleotides (AGAGACA), producing a premature termination codon 43 amino acids downstream. Binding of 125I-labelled LDL at 4 degrees C to cultured fibroblasts from two probands showed <2% normal LDL-receptor activity. AvaII digestion of PCR amplified genomic DNA identified this unique mutation in all families; homozygotes n=11, heterozygotes n=10. All mutation carriers shared the same haplotype (7 RFLPs), suggesting that they had a common ancestor. Despite high plasma LDL levels (m=16.0+/-3.0 mmol/l) and extravascular cholesterol deposits, most homozygotes were diagnosed after puberty and had a delayed onset of cardiovascular complications. Moreover, most heterozygotes were free of clinical signs and had plasma LDL cholesterol in the normal range (4.7+/-1.3 mmol/l) without taking any lipid-lowering medication. This mild clinical phenotype which contrasted with the severity of the mutation, could not be explained by specific apolipoprotein E or lipoprotein lipase alleles.     

Molecular genetics of familial hypercholesterolaemia in Norway

Objectives. To characterize mutations in the low density lipoprotein (LDL) receptor gene causing familial hypercholesterolaemia (FH) amongst Norwegian patients.
Design. Molecular genetic analyses of the LDL receptor gene have been performed in patients with a clinical diagnosis of FH.
Subjects. A total of 742 probands have been studied. Of these, 476 had a diagnosis of definite FH. The rest had a diagnosis of possible FH.
Results. Twenty-three different mutations in the LDL receptor gene as well as the apolipoprotein B-3500 mutation have been found. Six of the mutations in the LDL receptor gene are novel mutations. A molecular genetic diagnosis was achieved in 295 of the probands with definite FH (62%) and in 317 probands total. Of the 317 probands, 3% carried the apolipoprotein B-3500 mutation. When family members were included, a total of 624 persons carried a mutation in the LDL receptor gene and 20 carried the apolipoprotein B-3500 mutation.
Conclusions. Approximately 5% of Norwegian FH patients have been provided with a molecular genetic diagnosis. Our data suggest that molecular diagnosis of FH in Norway is feasible and should be implemented in clinical medicine.     

Effects of atorvastatin on electrophoretic characteristics of LDL particles among subjects with heterozygous familial hypercholesterolemia

The effects of the HMG CoA reductase inhibitor atorvastatin on electrophoretic characteristics of LDL particles were evaluated in 46 patients (28 males and 18 females) with heterozygous familial hypercholesterolemia (FH) aged 20-61 carrying either a negative or a defective LDL receptor gene mutation. Following a 6 week drug-free baseline period, FH heterozygotes were treated with atorvastatin (median dose: 20 mg/day, range 10-80 mg/day)) for 6 months to maintain their plasma LDL-cholesterol concentrations between 4.0 and 5.0 mmol/l. Atorvastatin treatment significantly reduced plasma total cholesterol, LDL-cholesterol and triglyceride levels and increased plasma HDL-cholesterol. Furthermore, atorvastatin treatment significantly increased LDL peak particle diameter (LDL-PPD) by 0.5% (from 255.0+/-6.2 to 256.4+/-5.5 A, P=0.004) and reduced the absolute concentration of cholesterol among small (<255 A) and large (>260 A) LDL particles by 35% (P<0.001). Changes in LDL-PPD and plasma triglyceride levels were inversely correlated (R=-0.34; P=0.02). Stepwise multiple linear regression analyses showed that 41.6% of the variation in the LDL-PPD response to atorvastatin was attributable to the initial LDL-PPD (14.4%, P=0.003), the apo E polymorphism (12.4%, P=0.02), the nature of the LDL receptor gene mutation (9.6%, P=0.01) and change in triglyceride levels (5.2%, P=0.04). Moreover, the reduction in the cholesterol content of LDL <255 A was directly correlated with the daily dosage of atorvastatin (P=0.05). Results of the present study showed that atorvastatin alters significantly LDL heterogeneity in patients at high risk of coronary heart disease (CHD) such as FH heterozygotes. These results also suggest that genetic and metabolic factors may be important determinants of atorvastatin-induced changes of LDL particle size and distribution among FH heterozygotes.     

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.     

Autosomal recessive hypercholesterolaemia: normalization of plasma LDL cholesterol by ezetimibe in combination with statin treatment

BACKGROUND: Severe hereditary hypercholesterolaemia is most frequently due to familial hypercholesterolaemia (FH), caused by mutations in the LDL receptor (LDLR) gene. However, a phenotype very similar to FH may also be caused by defects in other genes like the genes for apolipoprotein (apo) B-100 or autosomal recessive hypercholesterolaemia (ARH). SUBJECT: An 8-year-old male of Lebanese origin was diagnosed with severe hypercholesterolaemia and extensive cutaneous and tendon xanthomas. Plasma LDL cholesterol before treatment was 17 mmol L(-1), whilst parents and both siblings had normal levels. DIAGNOSIS: Degradation of (125)I-labelled LDL in blood lymphocytes was reduced, but not abolished. Sequencing analysis of the LDLR and apoB-100 genes were negative, whilst a splice acceptor mutation in intron 1 (IVS 1 -1G>C) was detected in the ARH gene. The patient was homozygous for the mutation, whilst the parents were heterozygous. These findings were in agreement with a diagnosis of ARH. TREATMENT AND CLINICAL COURSE: Monthly LDL apheresis and atorvastatin 120 mg daily reduced LDL cholesterol preapheresis level to 4.8 mmol L(-1). When ezetimibe was given 10 mg day(-1) in combination with rosuvastatin 80 mg day(-1), LDL cholesterol was further lowered to 1.6 mmol L(-1), which made apheresis unnecessary. Cutaneous and tendon xanthomas disappeared completely and the intima-media thickness of the common carotid arteries decreased. At age 23 he developed a small myocardial infarction. CONCLUSION: ARH should be considered in cases of severe hypercholesterolaemia with a pattern of recessive inheritance. Combination therapy with high-dose statin and ezetimibe seems to be the treatment of choice in ARH and may reduce or eliminate the need for LDL apheresis treatment.     

Molecular genetic analysis of familial hypercholesterolemia: spectrum and regional difference of LDL receptor gene mutations in Japanese population

To determine the molecular basis of familial hypercholesterolemia (FH) in Japan, 200 unrelated patients with clinically diagnosed heterozygous FH were screened for mutations in coding and promoter region of the low density lipoprotein (LDL) receptor gene using denaturing gradient-gel electrophoresis (DGGE), DNA sequencing and Southern blotting analysis. About 37 different mutations in the LDL receptor gene were identified in 125 (62.5%) of the patients, 22 of these mutations have not been described before. The most common mutations were K790X (19.5%), P664L (6.0%), FH-Tonami-1 (6.0%), IVS15-3C>A (5.5%) and FH-Tonami-2 (4.5%), whereas the other mutations were rare. No apolipoprotein B (apoB) mutations responsible for familial ligand-defective apoB-100 (FDB) were identified. Polymorphisms of apolipoprotein E (apoE) and scavenger receptor class B type I (SR-BI) were observed to have minor effects on the lipid and lipoprotein profile. In 75 (32.5%) of the FH patients, LDL receptor gene mutations could not be identified. These patients had significantly lower total cholesterol (7.71+/-1.64 vs. 8.68+/-1.47 mmol/l, P<0.001) and LDL-cholesterol (6.02+/-1.51 vs. 6.87+/-1.47 mmol/l, P<0.001) in plasma, also a lower incidence of coronary heart disease (CHD) (22 vs. 29%, P=0.05) compared with patients with a LDL receptor gene mutation, suggesting that besides LDL receptor, defect of other genes involved in LDL metabolism may be a cause of FH with a milder phenotypic expression in Japanese population.     

Recommendations for the use of LDL apheresis

Plasma exchange has been shown to increase life-expectancy in homozygous familial hypercholesterolaemia (FH) but increasingly is being replaced by LDL apheresis. Several methods are now available for undertaking this procedure, which lowers LDL cholesterol and Lp(a) efficiently and safely when performed weekly or bi-weekly and causes only slight decreases in HDL cholesterol. Hitherto the main clinical indication has been homozygous FH, including children and pregnant women, but there are limited data showing that LDL apheresis has effects on the progression of cardiovascular disease in FH heterozygotes which are similar to those of maximal lipid-lowering drug therapy. Hence it has the potential to be beneficial in hypercholesterolaemic patients with overt coronary disease who are refractory to or intolerant of drugs. It is therefore recommended that LDL apheresis should be the treatment of choice for: (1) all FH homozygotes from the age of seven onwards unless their serum cholesterol can be reduced by >50% and/or decreased to 5.0mmol/l or is decreased by <40% with maximal drug therapy. Apheresis may also occasionally be indicated on a case-by-case basis for patients with lower levels of LDL. (3) LDL apheresis should also be considered for patients with aggressive progressing coronary disease and Lp(a)>60mg/l whose LDL cholesterol remains >3.2mmol/l despite maximal drug therapy.     

Long-term outcome after living donor liver transplantation for two cases of homozygous familial hypercholesterolemia from a heterozygous donor

AIM: We experienced two pediatric siblings with homozygous familial hypercholesterolemia (FH) who received living donor liver transplantation (LDLT) from their parents who were heterozygous for FH. METHODS: The elder brother presented orange cutaneous xanthomas and was diagnosed as homozygous FH at the age of one. The plasma lipid profile showed that his total cholesterol level was 898 mg/dL (23.2 mmol/L), LDL cholesterol level was 756 mg/dL (19.6 mmol/L) and triglyceride level was 60 mg/dL (0.7 mmol/L). There were no living donors with a normal LDL receptor in their family, and it was difficult to find a deceased donor in Japan; thus he underwent LDLT with his father as the donor. His sister was born 2 years after his LDLT. She underwent ABO-incompatible LDLT at the age of 2 with her mother as the donor. RESULTS: The boy's liver function tests normalized immediately after transplantation, and his cholesterol has remained controlled at around 280 mg/dL (7.2 mmol/L), with HMG-CoA reductase inhibitor for 6 years after LDLT. The girl's cholesterol remained stable at around 280 mg/dL (7.2 mmol/L) under treatment with HMG-CoA reductase inhibitor two years after LDLT. At present, the four patients, including the two donors, are leading normal daily lives. CONCLUSION: Living-donor liver transplantation from a donor with heterozygous FH is a feasible indication for the treatment of homozygous FH.     

The type of mutation in the low density lipoprotein receptor gene influences the cholesterol-lowering response of the HMG-CoA reductase inhibitor simvastatin in patients with heterozygous familial hypercholesterolaemia

In a genetically heterogeneous group of 109 patients with a clinical diagnosis of heterozygous familial hypercholesterolaemia (FH), the influence of gender, apolipoprotein (apo) E genotype and the type of molecular defect in the LDL-receptor (LDLR) gene on the reduction of plasma LDL-cholesterol levels to treatment with a HMG-CoA reductase inhibitor (simvastatin) were studied. Response was determined as the percentage fall in LDL-cholesterol from untreated levels and as the proportion of patients where levels fell below 4.9 or 4.1 mmol/l. Of the patients, 86 individuals had tendon xanthomata (TX+) and a diagnosis of 'definite' FH and these individuals presented with a significantly higher untreated LDL-cholesterol compared to the 23 individuals who did not have xanthomas (TX-) and a diagnosis of 'probable' FH (8.14+/-0.19 vs. 6.81+/-0.25, P= 0.001). Overall, HMG-CoA reductase inhibitor doses of 10, 20 or 40 mg/day resulted in a significant fall of LDL-cholesterol levels of 29, 39 and 49%, but at all doses those with TX had significantly higher levels than those without, and significantly fewer TX + patients achieved LDL-cholesterol levels below 4.9 or 4.1 mmol/l than the TX - group (P < 0.05 at each dose). In the TX+ group the response to treatment was of similar magnitude in men and women and in patients with different apoE genotype. In the 'probable' FH probands only three mutations were identified (detection rate 13%), one in the LDLR gene and two in the APOB gene, a detection rate significantly lower (P= 0.02) than in the 'definite' FH probands where 28 mutations were detected (detection rate 37%). In the TX + patients where no mutation was detected, treatment resulted in a greater proportion achieving LDL-cholesterol levels below 4.9 and 4.1 mmol/l compared to those with any LDLR mutation, this difference was close to statistical significance at the 4.9 mmol/l threshold at 10 mg/day (41 vs. 13%, P = 0.058). For the 14 patients with an LDLR mutation that was predicted to be 'severe', fewer achieved LDL-cholesterol levels below 4.9 or 4.1 mmol/l at each dosage compared to the 16 individuals with 'mild' mutations, and this difference was statistically significant at the maximal dosage of 40 mg/day (P = 0.018). Thus although characterisation of the molecular defect in FH patients may not be relevant to their immediate clinical management, those with a particular mutation may need more aggressive lipid-lowering treatment to reach LDL-cholesterol levels recommended to reduce the risk of coronary heart disease (CHD).     

Blood rheology and fibrinogen in children with familial hypercholesterolaemia.

Whole blood and plasma viscosity, red cell aggregability and deformability, and plasma fibrinogen have been compared between 16 children aged between 6 and 18 years with heterozygous familial hypercholesterolaemia (FH) and 16 controls individually matched for age and sex. Mean (SD) plasma cholesterol was 7.19 (1.23) mmol/l in the patients and 4.31 (0.84) mmol/l in the controls. This was due to a similar difference in low density lipoprotein (LDL) cholesterol, while triglycerides and high density lipoprotein (HDL) cholesterol were similar between groups. No differences were seen in any of the rheological parameters between the two groups. This suggests that the rheological abnormalities seen in adults with FH are not a direct consequence of their hyperlipidaemia, and may instead be a reflection of their more extensive atherosclerosis.     

Autosomal recessive hypercholesterolemia in a Sicilian kindred harboring the 432insA mutation of the ARH gene

We describe a Sicilian family presenting a recessive form of hypercholesterolemia harboring a mutation of the autosomal recessive hypercholesterolemia (ARH) gene. In two of the three sibs, a 26-year-old male and a 22-year-old female, a severe hypercholesterolemia was diagnosed with very high levels of plasma cholesterol (15.9 and 12.2 mmol/l, respectively); tendon xanthomatas and xanthelasms were present and in the male proband was documented a diffuse coronary atherosclerotic disease with a rapid and fatal progression. Both the parents had normal or slightly increased levels of plasma cholesterol. All causes of secondary hypercholesterolemia were ruled out as well as an involvement of the LDL receptor or apoB genes. Beta-Sitosterol plasma levels were in the normal range. Cultured fibroblasts from skin biopsy from parents and the two probands displayed a normal ability to bind and degrade 125I-LDL. Direct sequencing of ARH gene demonstrated the presence of a 432insA mutation in homozygosis in the two probands; parents were heterozygotes for the same mutation. This mutation is the first report of a mutation of the ARH gene responsible for recessive forms of hypercholesterolemia in Sicily.     

Mutations in the LDL receptor gene in four Chinese homozygous familial hypercholesterolemia phenotype patients

Background and aimsFamilial hypercholesterolemia (FH) is an autosomal dominant disorder of lipoprotein metabolism caused by mutations in the low-density lipoprotein receptor (LDL-R) gene, leading to elevated levels of cholesterol and an increased risk of coronary heart disease. In this article, from four homozygous FH phenotype probands we identified disease causing mutations and analyzed the relationship between genotype and phenotype.Methods and resultsDNA sequencing identified five LDL-R point mutations in four unrelated families. We found a novel homozygous mutation (C210R), a homozygous mutation at W462X, a compound heterozygous mutation of C122Y and T383I, and a G>A intron 3 splice site homozygous mutation. The functional alteration caused by the novel C210R mutation was confirmed by FACS analysis. Four probands have high low-density lipoprotein cholesterol (LDL-C) levels, ranging from 14.65 to 27.66 mmol/L. Their heterozygous parents had relatively low levels. B-mode ultrasound supplemented by Doppler was used to examine aortic/mitral valve structural alterations and carotid intima-media thickness (ITM) in all probands. The ITM values were between 1.2 and 2.3 mm, much higher than the normal value of <0.8 mm.ConclusionOur data demonstrated that all the probands were associated with severe hypercholesterolemia, thick carotid IMT and a low CFVR (coronary flow velocity reserve) value. The novel mutation (C120Y) is a disease causing mutation.     

The extended abnormalities in lipoprotein metabolism in familial hypercholesterolemia: Developing a new framework for future therapies

Familial hypercholesterolemia (FH) is a dominantly inherited disorder characterized by marked elevation of plasma low-density lipoprotein (LDL) cholesterol concentrations and premature coronary artery disease (CHD). In addition to impaired LDL receptor-mediated clearance of LDL particles, in vitro and in vivo studies suggest that hepatic oversecretion of apolipoprotein (apo) B may contribute to the hypercholesterolemia in FH. This may be due to an effect of the expanded hepatic pool of cholesterol (a consequence of increased receptor-independent uptake of LDL) and/or a direct effect of the LDL receptor on apoB secretion. Hepatic oversecretion of apoB may depend on the type and severity of the genetic mutation causing FH. FH can also increase plasma Lp(a) concentration by an undefined mechanism that may not directly involve the LDL receptor pathway. Decreased catabolism of triglyceride-rich lipoproteins could also be due to deficient LDL receptor function, accounting for postprandial dyslipidemia in FH. The metabolism of high-density lipoprotein (HDL) in FH is poorly understood, but preliminary data suggest abnormal HDL composition and functionality, as well as altered transport of apoA-I. Beyond effects related to specific genetic defects in the LDL pathway, co-existing secondary causes, particularly obesity and insulin resistance, and other genetic variants may also perturb lipoprotein metabolism in individuals with FH. Furthermore, residual risk remains high in statin-treated FH. Knowledge of an extended metabolic framework will, therefore, provide the basis for judiciously selecting new pharmacotherapies to treat FH, including apoB antisense oligonucleotides, microsomal transfer protein (MTP) inhibitors and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors.     

Beta-thalassemia is a modifying factor of the clinical expression of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a codominant disorder due to a variety of mutations of the low-density lipoprotein (LDL) receptor gene that result in an elevation of plasma LDL-cholesterol (LDL-C). Plasma levels of LDL-C show large interindividual variation even in subjects carrying the same mutation of the LDL receptor gene. This variability may be due to genetic factors (modifier genes). Several surveys indicate that the overall contribution of common polymorphisms of modifier genes (such as the genes encoding apolipoproteins E and B) to this variability is less than 10%. In contrast, beta-thalassemia has a profound LDL-lowering effect. This was documented in FH patients identified on the island of Sardinia, in Italy, where 12% of the inhabitants are carriers of beta-thalassemia due to a single mutation (Q39X) of the beta-globin gene that abolishes the synthesis of beta-globin chain of hemoglobin (beta(o)-thalassemia). Plasma LDL-C in FH heterozygotes carrying the beta(o)-thalassemia trait is 25% lower than in noncarriers, regardless of the LDL receptor gene mutation. It is likely that this effect is due to two main mechanisms: (1) increased uptake of LDL by the bone marrow to provide cholesterol for the increased proliferation of erythroid progenitor cells and (2) increased production of inflammatory cytokines that reduce the hepatic secretion and increase the catabolism of LDL. In view of its LDL-C-lowering effect, beta-thalassemia trait may protect FH heterozygotes against premature coronary atherosclerosis.     

Mutations in the low-density-lipoprotein receptor gene in German patients with familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) is an autosomal dominant disorder of lipid metabolism characterized by elevated low-density lipoproteins (LDL), the formation of tendon and skin xanthomata and the development of premature coronary atherosclerosis. It is caused by a defect in the receptor-mediated hepatic uptake of LDL due to mutations in the LDL receptor. In 25 FH families with a total of 160 members and in two individuals without available relatives, all of German origin, we identified LDL receptor mutations by a multiplex-PCR-based single-strand conformation polymorphism method followed by direct sequencing. Of the 24 mutations found, 15 are missense mutations, 2 are nonsense mutations, 4 are small deletions or insertions leading to frameshifts, 2 are an in-frame insertion and deletion, respectively, and one is a splice site mutation. Propositi carrying mutations that are known to completely abolish receptor function (nonsense and frameshift mutations, missense mutation V480M) had significantly higher untreated total and LDL-cholesterol levels compared to those patients carrying missense and in-frame insertion mutations of unknown functional consequence, which may lead to either reduced or completely abolished receptor function (11.30±1.64 vs 9.76±1.50 mmol/L, and 9.39±1.23 vs 7.99±1.45 mmol/L, respectively). These results confirm the clinical and molecular heterogeneity of FH and the influence of different functional classes of mutations on lipid values.     

The association of LDL receptor activity, LDL cholesterol level, and clinical course in homozygous familial hypercholesterolemia

Patients with homozygous familial hypercholesterolemia (FH), reveal a marked heterogeneity in plasma cholesterol levels, response to diet as well as drug treatment, and clinical course. Low-density lipoprotein (LDL) receptor activities were assessed by the rate of 14C-oleate cholesteryl ester biosynthesis in fibroblasts from 13 FH homozygotes in tissue culture. The receptor activity of the individual patients was highly correlated with initial pretreatment plasma cholesterol and LDL cholesterol levels (P less than .001, r = -0.89). In addition, the LDL receptor activity was positively correlated with the age of onset of angina based on the Cox model (P less than .035, likelihood ratio = 6.71). An association was also noted between LDL receptor activity and cholesterol reduction with drugs. These data provide direct evidence for the correlation between the heterogeneity of the LDL receptor and the expression of the clinical manifestations of homozygous FH. The determination of pretreatment plasma cholesterol level and LDL receptor activity in patients with homozygous FH provide useful parameters on which to base predictions of the clinical progression of cardiovascular disease. These parameters may also influence the selection of a program for diet and drug therapy. Patients with markedly elevated plasma cholesterol levels and very low LDL receptor activity should be considered to be candidates for multiple drug therapy, and portacaval shunt, and/or periodic plasma exchanges.     

Genetic screening of patients with familial hypercholesterolaemia (FH): a New Zealand perspective

Using denaturing high performance liquid chromatography (DHPLC) to screen the LDL receptor gene of people with familial hypercholesterolaemia (FH) in Christchurch, New Zealand, we have identified mutations in 65 patients (44 different mutations, of which 15 are novel). We also test family members of probands for the mutation identified in their relative, allowing diagnosis of affected children and those without classical FH symptoms. This screening programme is helpful to clinicians and benefits FH patients and their families, and has provided us with a pool of LDL receptor variants on which to base research into this disease.     

Familial hypercholesterolemia in Utah kindred with novel R103W mutations in exon 4 of the LDL receptor gene

Heterozygous familial hypercholesterolemia (FH) is a serious disorder causing twice normal low-density lipoprotein cholesterol levels early in childhood and very early coronary disease in both men and women. Previously published blood cholesterol criteria greatly under-diagnosed new cases of FH among members of known families with FH and over-diagnosed FH among participants of general population screening. Thus, there is a need for accurate and genetically validated criteria for the early diagnosis of heterozygous FH. In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To carry out molecular genetic diagnosis of the disease, we screened DNA samples for mutations in all 18 exons and the exon- intron boundaries of the low-density lipoprotein (LDL) receptor gene. Novel point mutations were identified in the proband: a C-to-T transversion at nucleotide position 369, causing substitution of Tryptophan for Arginine at codon 103 in exon 4 of the LDL receptor gene. The SSCP method was used to examine seven members of the family recruited for the diagnosis. This method helped to unequivocally diagnose only the proband as heterozygous for this particular LDL receptor mutation while excluding the remaining six individuals from carrier status with FH.     

Spectrum of LDL receptor gene mutations in Denmark: implications for molecular diagnostic strategy in heterozygous familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (FH) is one of the most common potentially fatal single-gene diseases leading to premature coronary artery disease, but the majority of heterozygous FH patients have not been diagnosed. FH is due to mutations in the gene coding for the low-density lipoprotein (LDL) receptor, and molecular genetic diagnosis may facilitate identification of more FH subjects. The Danish spectrum of 29 different mutations, five of which account for almost half of heterozygous FH, is intermediate between that of countries such as South Africa, where three mutations cause 95% of heterozygous FH in the Afrikaners, and Germany or England, where there are many more mutations. In clinical practice, a strategy for the genetic diagnosis of heterozygous FH, tailored to the mutational spectrum of patients likely to be seen at the particular hospital/region of the country, will be more efficient than screening of the whole LDL receptor gene by techniques such as single-strand conformation polymorphism (SSCP) analysis in every heterozygous FH candidate. In Aarhus, Denmark, we have chosen to examine all heterozygous FH candidates for the five most common LDL receptor gene mutations (W23X, W66G, W556S, 313 + 1G --> A, 1846 - 1G --> A) and the apoB-3500 mutation by rapid restriction fragment analysis. Negative samples are examined for other mutations by SSCP analysis followed by DNA sequencing of the exon indicated by SSCP to contain a mutation. If no point mutation or small insertion/deletion is detected, Southern blot or Long PCR analysis is performed to look for the presence of large gene rearrangements. In conclusion, our data suggest that an efficient molecular diagnostic strategy depends on the composition of common and rare mutations in a population.     

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.