Duplication of seven exons in LDL receptor gene caused by Alu-Alu recombination in a subject with familial hypercholesterolemia

A defective LDL receptor gene in a child with familial hypercholesterolemia produces a receptor precursor that is 50,000 daltons larger than normal (apparent Mr 170,000 vs. 120,000). The elongated protein resulted from a 14 kilobase duplication that encompasses exons 2 through 8. The duplication arose from an unequal crossing-over between homologous repetitive elements (Alu sequences) in intron 1 and intron 8. The mutant receptor has 18 contiguous cysteine-rich repeat sequences instead of the normal nine. Seven of these duplicated repeats are derived from the ligand-binding domain, and two repeats are part of the epidermal growth factor precursor homology region. The elongated receptor undergoes normal carbohydrate processing, its apparent molecular weight increases to 210,000, and the receptor reaches the cell surface where it binds reduced amounts of LDL but undergoes efficient internalization and recycling. The current findings support an evolutionary model in which homologous recombination between repetitive elements in introns leads to exon duplication during evolution of proteins.     

Four DNA polymorphisms in the LDL-receptor gene and their use in diagnosis of familial hypercholesterolemia

Summary To examine the potential usefulness of restriction fragment length polymorphisms (RFLPs) for diagnosis of familial hypercholesterolemia (FH), we determined the genotype of FH patients and their relatives for the ApalI, NcoI, PvuII and StuI RFLP of the LDL-receptor gene in a sample of German patients attending the Lipid Clinic in Munich. There was no significant difference in the relative allele frequency between the group of FH patients and controls for any of the four polymorphisms. Using linkage analysis, we could determine the four-RFLP haplotypes of 39 defective and 90 normal LDL-receptor genes in 38 FH families. In our sample, defective LDL-receptor genes occur on 6 different chromosomes determined by the four RFLPs. This suggests that at least 6 different genetic defects may cause FH in this sample. RFLPs of the LDL-receptor gene cannot be used to detect FH in individuals; however, appropriate diagnosis can be carried out in more than 90% of families using linkage analysis and these RFLPs.     

A "de novo" mutation of the LDL-receptor gene as the cause of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a common genetic disorder caused by mutations of the LDL-receptor gene and transmitted as a co-dominant trait. However, there are some forms of hypercholesterolemia which have a recessive type of transmission. We have identified a subject with the clinical phenotype of heterozygous FH whose parents had normal plasma lipid values, suggesting a recessive type of transmission. The analysis of the LDL-receptor gene revealed that the patient was heterozygous for a G>C transversion in exon 4, which results in a serine for cysteine substitution at position 88 (C88S) of the receptor protein. Since this novel mutation was not found in the proband's parents and non-paternity was excluded, we concluded that the patient was a carrier of a "de novo" mutation. Haplotype analysis of LDL-receptor locus indicated that this "de novo" mutation occurred in the paternal germ line. The C88S mutation is the likely cause of LDL-receptor defect as it was present in the proband's hypercholesterolemic son and was not found in 200 chromosomes of control subjects.     

Recurrent and novel LDL receptor gene mutations causing heterozygous familial hypercholesterolemia in La Habana

The molecular basis of familial hypercholesterolemia (FH) in three families of Spanish descent from La Habana was investigated by the candidate gene approach. The Arg3500Gln mutation of apolipoprotein B-100 was not found. Identification of low density lipoprotein receptor (LDLR) gene haplotypes segregating with FH guided the characterisation of three point mutations by automated sequencing. One, a Val408Met missense mutation, a founder mutation in Afrikaner FH patients, was recurrent, being associated with a distinct DNA haplotype. The other two, Glu256Lys and Val776Met missense mutations, were novel and modified highly conserved residues. These mutations were absent in normolipidemic subjects and were associated in heterozygous carriers with twice the cholesterol levels observed in noncarriers. Noticeably, cardiovascular complications were rarely observed in older heterozygotes, even in those with the Afrikaner FH-2 mutation. These findings confirm the molecular heterogeneity of LDLR gene mutations causing FH and the variability of their expression across different populations.     

A double mutant LDL receptor allele in a Cypriot family with heterozygous familial hypercholesterolemia

Two novel mutations Q363X and D365E were identified in the low-density lipoprotein receptor gene in a Cypriot patient with heterozygous familial hypercholesterolemia. Restriction enzyme analysis of the index case and seven of her family members, by using AvaII and PvuII respectively, demonstrated that the two exon 8 mutations are transmitted in cis within the family. The disease phenotype is probably caused by the stop-363 mutation; this would result in a truncated protein that would probably be rapidly degraded in the extracellular space. Received: 15 August 1996 / Accepted: 10 February 1997     

Macrophage lipoprotein lipase expression is increased in patients with heterozygous familial hypercholesterolemia

FH is associated with accelerated atherosclerosis. Based on the crucial role of macrophage LPL in atherogenesis, we determined in the present study macrophage LPL expression in patients with FH. Monocytes isolated from 13 FH patients and 13 control subjects were differentiated into macrophages by culturing the cells for 9 days in 20% autologous or heterologous serum. Macrophages of patients with FH cultured in their own sera showed a significant increase in LPL mRNA levels, extracellular LPL mass, and activity compared with macrophages of control subjects. Although these alterations positively correlated with the levels of serum platelet-derived growth factor-BB (PDGF-BB) in FH subjects, increased LPL secretion by cultured FH macrophages was reduced neither by immunoneutralizing FH serum with an anti-PDGF-BB antibody, nor by culturing these cells in sera from control subjects. With the exception of LPL, levels of other cytokines and 8-isoprostane were not increased in the supernatants of macrophages of FH patients. Serum from FH patients also enhances the levels of LPL secreted by macrophages from control subjects. Immunoneutralization of FH serum with an anti-PDGF-BB antibody totally reversed this alteration. Overall, this study demonstrates that macrophages from FH subjects overproduce LPL and that PDGF present in the serum from FH patients stimulates LPL secretion by control macrophages. These findings suggest that macrophage LPL induction in patients with FH might be related to the increased atherogenesis observed in these subjects.     

Identification of two new LDL-receptor mutations causing homozygous familial hypercholesterolemia in a South African of Indian origin

South Africans of Indian origin have a high frequency of Familial Hypercholesterolemia (FH). Fibroblasts from a South African Indian FH homozygote, D, expressed about 30% of the normal number of LDL receptors. These receptors showed defective LDL binding. Sequence and haplotype analysis revealed that D had two different mutant LDL receptor alleles: FH Durban-1 is a point mutation [asp₆₉(GAT) to tyr(TAT)] in ligand-binding repeat 2 and FH Durban-2 is a point mutation [glu₁₁₉GAG) to lys(AAG)] in ligand-binding repeat three of the LDL receptor. Single-strand conformational polymorphism analysis, which was used in the initial detection of these mutations, was also employed for subsequent population screening assays. These mutations were not detected in amy of the South African Indian FH of hypercholesterolemic patients that were screened.     

Apolipoprotein A-I kinetics in heterozygous familial hypercholesterolemia: a stable isotope study.

Heterozygous familial hypercholesterolemia (FH) is associated with a moderate decrease of plasma apoA-I and HDL-cholesterol levels. The aim of the study was to test the hypothesis that these abnormalities were related to an increase of HDL-apoA-I fractional catabolic rate (FCR). We performed a 14-h infusion of [5,5,5-(2)H(3)]leucine in seven control subjects and seven heterozygous FH patients (plasma total cholesterol 422 +/- 27 vs. 186 +/- 42 mg/dL, P < 0.001, respectively). Plasma apoA-I concentration was not changed in FH compared to controls (respectively 115 +/- 18 vs. 122 +/- 15 mg/dL, NS), and HDL-cholesterol level was decreased (37 +/- 7 vs. 46 +/- 19 mg/dL, NS). Kinetics of HDL metabolism were modeled as a single compartment as no differences were observed between HDL(2) and HDL(3) subclasses. Both mean apoA-I FCR and absolute production rate (APR) were increased in FH (respectively, 0.36 +/- 0.14 vs. 0.22 +/- 0.05 pool/d, P < 0.05, and 18.0 +/- 7.7 and 11.2 +/- 2.3 mg/kg/d, P < 0.05). Higher HDL-triglyceride and HDL-apoE levels were observed in patients with heterozygous FH. (Respectively 19 +/- 8 vs. 8 +/- 3 mg/dL, P < 0.05, and 5.3 +/- 0.8 vs. 3.7 +/- 0.9 mg/dL, P < 0.05). We conclude that the catabolism of HDL-apoA-I is increased in heterozygous FH patients. However, plasma apoA-I concentration was maintained because of an increased HDL-apoA-I production rate.     

Partial recovery of the endothelial glycocalyx upon rosuvastatin therapy in patients with heterozygous familial hypercholesterolemia

The endothelial glycocalyx has been shown to serve as a protective barrier between the flowing blood and the vessel wall in experimental models. The aim of this study was to evaluate whether hypercholesterolemia is associated with glycocalyx perturbation in humans, and if so, whether statin treatment can restore this. We measured systemic glycocalyx volume (V(G)) in 13 patients with heterozygous familial hypercholesterolemia (FH) after cessation of lipid-lowering therapy for a minimum of 4 weeks and 8 weeks after initiating rosuvastatin therapy. Normocholesterolemic subjects were used as controls. V(G) was estimated by subtracting the intravascular distribution volume of a glycocalyx permeable tracer (dextran 40) from that of a glycocalyx impermeable tracer (labeled erythrocytes). V(G) in untreated FH patients [LDL 225 +/- 57 mg/dl (mean +/- SD)] was significantly reduced compared with controls (LDL 93 +/- 24 mg/dl) (V(G) 0.8 +/- 0.3 vs. 1.7 +/- 0.6, respectively, P < 0.001). After normalization of LDL levels (95 +/- 33 mg/dl) upon 8 weeks of statin treatment, V(G) recovered only partially (V(G) 1.1 +/- 0.4 L, P = 0.04). The endothelial glycocalyx is profoundly reduced in FH patients, which may contribute to increased atherogenic vulnerability. This perturbation is partially restored upon short-term statin therapy.     

Evidence for a cholesterol-lowering gene in a French-Canadian kindred with familial hypercholesterolemia

We describe a four-generation kindred with familial hypercholesterolemia (FH) in which two of the eight heterozygotes for a 5-kb deletion (exons 2 and 3) in the low density lipoprotein (LDL) receptor gene were found to have normal LDL-cholesterol levels. In our search for a gene responsible for the cholesterol-lowering effect in this family, we have studied variation in the genes encoding the LDL receptor, apolipoprotein (apo) B, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, apoAI-CIII-AIV, and lipoprotein lipase. The analysis showed that it was unlikely that variation in any of these genes was responsible for the cholesterol-lowering effect. Expression of the LDL receptor, as assessed in vitro with measurements of activity and mRNA levels, was similar in normo and hyperlipidemic subjects carrying the deletion. Analysis of the apo E isoforms revealed that most of the e2 allele carriers in this family, including the two normolipidemic 5-kb deletion carriers, were found to have LDL-cholesterol levels substantially lower than subjects with the other apo E isoforms. Thus, this kindred provides evidence for the existence of a gene or genes, including the apo e2 allele, with profound effects on LDL-cholesterol levels.C. S. and M. G. contributed equally to this work.     

家族性高胆固醇血症低密度脂蛋白受体第13外显子突变分析

目的:探讨本课题组收集家族性高胆固醇血症(FH)患者中存在低密度脂蛋白受体(LDLR)第13外显子(E13)基因突变患者临床生化和心血管系统损害特点.方法:对9例临床诊断为FH、基因检测到LDLR基因E13突变的患者进行回顾性分析.结果:(1)临床诊断FH纯合子患者7名,其总胆固醇(TC)水平15.12～26.14 mmol/L,杂合子患者2名,TC水平11.30～11.75 mrnol/L.(2)均可见不同程度黄色瘤;(3)FH纯合子3例心电图出现ST-T改变;4例儿童和1例青年患者出现瓣膜损害,冠脉血流储备(CVFR)减低;杂合子心电图检查均正常,1例出现瓣膜损害,CVFR均正常.(4)核苷酸序列分析证实:9例E13突变患者中,A606T纯舍突变3名;D601Y纯合突变2名;A606T+、W462X和A606T+D601Y复合杂合突变各1名;A606T和D601Y杂合突变各1名.结论:FH严重损害患儿心血管系统和皮肤,LDLR基因E13出现的A606T和D601Y突变可能成为中国FH人群的高频突变位点.     

Biliary lipid secretion in patients with heterozygous familial hypercholesterolemia and combined hyperlipidemia. Influence of bezafibrate and fenofibrate

Serum and biliary lipid metabolism were examined in 13 patients with different types of hyperlipoproteinemia before and after 4 weeks of treatment with either bezafibrate or fenofibrate. In patients with heterozygous familial hypercholesterolemia (FH), bezafibrate (n = 5) and fenofibrate (n = 7) produced a similar significant reduction of total cholesterol, LDL-cholesterol, and triglycerides by 21, 23, and 32%, respectively. In patients with familial combined hyperlipidemia (CHL), only triglycerides decreased markedly. Biliary lipid secretion rates in patients with heterozygous FH were not different from those of young male volunteers, indicating that a reduction of hepatic LDL receptors did not affect hepatic elimination of cholesterol or bile acids. Biliary cholesterol secretion increased significantly from 57 to 75 mg/hr during bezafibrate therapy (n = 8) and from 62 to 71 mg/hr during fenofibrate therapy (n = 9). No consistent change in bile acid or phospholipid secretion was observed. The elevated output of biliary cholesterol increased cholesterol saturation significantly from 147 to 185% and from 152 to 173% during administration of bezafibrate and fenofibrate, respectively. The present study indicates that treatment with bezafibrate or fenofibrate is effective in lowering LDL cholesterol in patients with heterozygous FH, but both drugs increase cholesterol saturation of bile, which might enhance the risk of cholesterol gallstone formation.     

Metabolic pathways of apolipoprotein B in heterozygous familial hypercholesterolemia: studies with a [3H]leucine tracer.

The kinetics of apolipoprotein B (apoB) were measured in seven studies in heterozygous, familial hypercholesterolemic subjects (FH) and in five studies in normal subjects, using in vivo tracer kinetic methodology with a [3H]leucine tracer. Very low density (VLDL) and low density lipoproteins (LDL) were isolated ultracentrifugally and LDL was fractionated into high and low molecular weight subspecies. ApoB was isolated, its specific radioactivity was measured, and the kinetic data were analyzed by compartmental modeling using the SAAM computer program. The pathways of apoB metabolism differ in FH and normal subjects in two major respects. Normals secrete greater than 90% of apoB as VLDL, while one-third of apoB is secreted as intermediate density lipoprotein IDL/LDL in FH. Normals lose 40-50% of apoB from plasma as VLDL/IDL, while FH subjects lose none, metabolizing all of apoB to LDL. In FH, there is also the known prolongation of LDL residence time. The leucine tracer, biosynthetically incorporated into plasma apoB, permits distinguishing the separate pathways by which the metabolism of apoB is channeled. ApoB synthesis and secretion require 1.3 h. ApoB is secreted by three routes: 1) as large VLDL where it is metabolized by a delipidation chain; 2) as a rapidly metabolized VLDL fraction converted to LDL; and 3) as IDL or LDL. ApoB is metabolized along two pathways. The delipidation chain processes large VLDL to small VLDL, IDL, and LDL. The IDL pathway channels nascent, rapidly metabolized VLDL and IDL particles into LDL. It thus provides a fast pathway for the entrance of apoB tracer into LDL, while the delipidation pathway is a slower route for channeling apoB through VLDL into LDL. LDL apoB is derived in almost equal amounts from both pathways, which feed predominantly into large LDL. Small LDL is a product of large LDL, and the major loss of LDL-apoB is from small LDL. Two features of apoB metabolism in FH, the major secretory pathway through IDL and the absence of a catabolic loss of apoB from VLDL/IDL, greatly facilitate measuring the metabolic channeling of apoB into LDL.     

TaqI polymorphism in the LDL receptor gene and a TaqI 1.5-kb band associated with familial hypercholesterolemia

Summary The low density lipoprotein (LDL) receptor gene was analyzed in 67 unrelated healthy Japanese and 38 members of six consecutive families with familial hypercholesterolemia (FH) by Southern blot hybridization with TaqI, an LDL receptor cDNA fragment containing exons 1 to 8 being used as a probe. A new TaqI RFLP at the LDL receptor locus was detected with allele frequencies of 0.67 and 0.33. The data obtained with smaller cDNA subfragment probes revealed that the TaqI RFLP site is located within 1.1 kb of the 5 side of the EcoRI site of exon 5. The TaqI RFLP was in linkage disequilibrium with the PstI RFLP but showed no significant linkage disequilibrium with the RFLPs for AvaII, ApaLI/I15, PvuII, NcoI, and ApaLI/3. Among the seven RFLPs at the LDL receptor locus, the TaqI RFLP was the only useful genetic marker in one of the six families with FH. Furthermore, the association of an additional TaqI 1.5-kb band with a mutant LDL receptor gene was observed in another family with FH in which the proband was homozygous for all of the seven RFLPs. The data obtained with various restriction enzymes and smaller cDNA subfragments probes suggested that a minor change in nucleotide sequences in the region including exons 5 to 8 is present in the mutant gene. These data suggest that the TaqI RFLP is a useful genetic marker at the LDL receptor locus and that TaqI serves for the analysis of some mutant LDL receptor genes, when used with small LDL receptor cDNA probes.     

Clinical course of homozygous familial hypercholesterolemia during childhood: report on 4 unrelated patients with homozygous or compound heterozygous mutations in theLDLR gene

Natural history of the disease in 4 unrelated Polish children with homozygous familial hypercholesterolemia (FH) is described. Their phenotypic homozygosity was established by identification of known LDLR gene mutations on both alleles, respectively: p.G592E & p.G592E in Patient 1; p.G592E & p.C667Y in Patient 2; p.S177L & p.R350X in Patient 3; and p.G592E & deletion in the promoter region, exons 1 and 2 in Patient 4. Heterozygosity of the mutations was revealed in all patients' mothers and fathers (obligatory heterozygotes) and in 1 out of 4 siblings studied. FH was diagnosed at the age of 4 months to 9 years by cholesterol screening among family members of patients with early cardiovascular disease episodes. At the time of FH detection, the children were asymptomatic. Only in 2, some skin eruptions were found. Antihyperlipidemic therapy was started, including a lipid-lowering diet, cholestyramine, and HMG-CoA inhibitors if necessary. No cardiovascular symptoms appeared during the observation up to the age of 18, 20, 19, and 17 years, respectively. An increase in external carotid artery diameter was found in a patient at the age of 9 years, and LDL-apheresis was introduced in his therapy. We conclude that the analysis of LDLR gene mutations in the studied FH children made it possible to identify 4 presymptomatic FH homozygotes and to introduce early appropriate treatment. Multicenter analysis of such persons would finally determine if the early lipid-lowering procedures can significantly reduce the risk of premature cardiovascular disease in homozygous FH.     

Novel and recurrent mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia

We have identified 16 different mutations of the low-density lipoprotein receptor (LDLR) gene in 25 unrelated Korean patients with heterozygous familial hypercholesterolemia (FH), including five novel mutations, C83Y, 661del17, 1705insCTAG, C675X, and 941-1G>A. The 1705insCTAG mutation in which the four 3 cent -terminal nucleotides of exon 11 are duplicated was found to prevent splicing of exon 11 and would therefore generate a truncated polypeptide. The in-frame 36-bp deletion (1591del36) in exon 11, which had been reported only in one Korean FH patient, was also found. We showed that this change affects transport of the LDL receptor from the endoplasmic reticulum to the cell surface. In addition, we found 8 mutations (-136C>T, E119K, E207K, E207X, F382L, R574Q, 1846-1G>A, and P664L) that had been described in other ethnic groups but not in Koreans, and 2 mutations (R94H and D200N) that had been described in Koreans as well as other ethnic groups. 5 mutations (1591del36, E119K, E207X, E207K, and P664L) were found more than once in the Korean FH samples. Identification of the novel and recurring LDLR mutations in Korean FH patients should facilitate prenatal and early diagnosis in families at high risk of FH.     

Duplication within chromosome region 15q11-q13 in a patient with similarities to Prader-Willi syndrome confirmed by region-specific and band-specific fish.

We report on a patient presenting with mental retardation and obesity and a proximal duplication of chromosome 15. The patient shared some clinical signs with Prader-Willi syndrome. With a region-specific paint, generated by microdissection, a duplication in region 15q11.2-q13 was shown to be present. Subsequently, FISH with probes localized to chromosome region 15q11.2-q12 and microsatellite analysis was used to characterize this chromosome aberration further and an insertion duplication within the region frequently deleted in Prader-Willi and Angelman syndrome was demonstrated.     

Screening of APOB gene mutations in subjects with clinical diagnosis of familial hypercholesterolemia

Monogenic hypercholesterolemia is a group of lipid disorders, most of which have autosomal dominant transmission. Familial defective apoB (FDB) resulting from mutations in the APOB gene is a well-recognized cause of autosomal dominant monogenic hypercholesterolemia (ADMH). However, the frequency of FDB among patients with ADMH is not well established. The aim of our research was to screen for mutations responsible for FDB in subjects with a clinical diagnosis of familial hypercholesterolemia. We studied 408 patients from the Spanish Register of Familial Hypercholesterolemia, proportionally distributed among all Spanish regions. Abnormal SSCP patterns of the APOB gene were checked by DNA sequencing and restriction analysis. Three out of the 408 patients were carriers of the R3500Q mutation, and 2 subjects were carriers of the silent T3552T mutation; in both of these patients functional mutations in the LDL receptor gene were found. We conclude that FDB is not a common cause of ADMH in Spain; the R3500Q mutation is the only mutation in APOB causing FDB, and the LDL receptor binding domain of APOB is highly conserved in the studied sample.     

Influence of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, on corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia.

The present study examined whether hypolipidemic therapy with a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, influences corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia (FH). Urinary excretion of tetrahydrocortisone, tetrahydrocortisol, 6 beta-hydroxycortisol and free cortisol were determined in 22 patients with heterozygous FH before and after pravastatin administration (10 mg/day for 2 months). Pravastatin induced a statistically significant decrease in serum total cholesterol in patients with heterozygous FH from 6.9 +/- 0.1 to 5.9 +/- 0.1 mmol/l (p less than 0.05). No significant changes were seen in the urinary tetrahydrocortisone, tetrahydrocortisol and free cortisol levels before and after pravastatin therapy. Urinary excretion of 6 beta-hydroxycortisol was significantly (p less than 0.05) increased after pravastatin administration. These results suggest that the hypolipidemic effect of pravastatin in patients with heterozygous FH does not influence the corticosteroid metabolism. The increase in urinary 6 beta-hydroxycortisol may be caused by pravastatin-induced hepatic microsomal 6 beta-hydroxylase induction.