Haplotype analysis at the low density lipoprotein receptor locus: application to the study of familial hypercholesterolemia in Israel

Summary Familial hypercholesterolemia (FH) results from mutations in the low density lipoprotein (LDL) receptor gene. It has been shown that restriction fragment length polymorphisms (RFLPs) associated with this gene may be used for family and population studies. The present investigation is a population-based study of 19 Jewish families with hypercholesterolemia representing 9 different countries of origin. Ten RFLP sites were used to construct 24 different haplotypes from 112 chromosomes. These haplotypes vary in frequency from 0.9% to 28.6%. Five previously undescribed haplotypes, which comprise 8.1% of the sample, are reported here. The six most common haplotypes account for 70% of the sample. Segregation analysis reveals that, in Israel, distinct LDL receptor haplotypes are associated with hypercholesterolemia in 12 (63%) out of the 19 Jewish families. Five LDL receptor haplotypes co-segregate with hypercholesterolemia. Two of these haplotypes seem to be unique to specific population groups in Israel and may therefore represent founder mutations.     

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.     

Use of homozygosity mapping to identify a region on chromosome 1 bearing a defective gene that causes autosomal recessive homozygous hypercholesterolemia in two unrelated families

Familial hypercholesterolemia (FH) is a common inherited disorder of metabolism characterized clinically by high levels of low-density lipoprotein (LDL) in plasma owing to reduced catabolism. This leads to accelerated atherosclerosis and thus to an increased risk of coronary heart disease. FH is usually caused by defects in the gene for either the LDL receptor or apolipoprotein B (apoB), the ligand for the LDL receptor. Elsewhere, we have described two unrelated patients with phenotypic homozygous FH. Both patients were offspring of consanguineous unions, and linkage to either the gene for the LDL receptor or the gene for apoB was excluded in both. Their cells in culture do not degrade LDL, despite the presence of normal surface binding of LDL to the LDL receptor. This observation suggests that the patients may be homozygous for a defective gene that encodes a component of the internalization pathway. We first excluded linkage of the defect to known genes for proteins reported to be involved in internalization of receptors in clathrin-coated pits. We then performed genomewide homozygosity mapping. Genotyping of 500 polymorphic markers in three affected and seven unaffected members of the first pedigree showed that recessive hypercholesterolemia in this family is localized to a single chromosomal region on 1p36-p35. Genotyping of two affected and five unaffected members of the second pedigree provided further evidence of linkage to this locus, thereby mapping the disease-causing gene to a 12-cM region on chromosome 1p36-p35, with a combined LOD score of 5.3 in these unrelated families. Identification of the gene in this region may lead to new insights into the mechanisms of LDL receptor-mediated uptake of LDL by cells and may help to identify further genetic risk factors for premature atherosclerosis.     

A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia

Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.     

Gene therapy with novel adeno-associated virus vectors substantially diminishes atherosclerosis in a murine model of familial hypercholesterolemia

BACKGROUND: Familial hypercholesterolemia is an inherited disease caused by mutations in the LDL receptor gene leading to severe hypercholesterolemia and atherosclerosis. The LDL receptor is predominantly expressed in the liver, making it a preferred target organ for somatic gene therapy. We recently isolated a new family of vectors based on adeno-associated viruses (AAVs) isolated from nonhuman primates, which enable efficient and stable transgene expression following in vivo gene delivery to liver. METHODS: Traditional vectors based on AAV serotype 2 and two novel AAVs from nonhuman primates, serotypes AAV7 and AAV8, were produced encoding for the human LDL receptor. Vectors were injected into the portal veins of LDL receptor deficient mice that were fed a high-fat diet to achieve severe pretreatment hypercholesterolemia. RESULTS: Animals receiving the novel AAV vectors realized nearly complete normalization of serum lipids and failed to develop the severe atherosclerosis that characterized the untreated animals; the AAV2 vector constructs demonstrated partial lipid correction and only a modest improvement in atherosclerosis. CONCLUSIONS: Using vectors based on novel nonhuman primate AAVs, which provide advantages in terms of efficiency, we were able to achieve a long-term correction of the metabolic defect in LDL receptor deficient mice.     

Use of three DNA polymorphisms of the LDL receptor gene in the diagnosis of familial hypercholesterolemia

Summary Familial hypercholesterolemia (FH) is an autosomal dominant metabolic disorder caused by several different mutations in the low density lipoprotein (LDL) receptor gene. This large number of different mutations, often undetectable in Southern blotting, makes it impossible directly to diagnose the disease. However, restriction fragment length polymorphisms (RFLPs) can be used to follow the inheritance of the defective gene in FH families. In the present study, we report the use of three RFLPs, detected by PvuII, ApaLI and AvaII restriction enzymes, to determine the haplotypes of normal and defective LDL receptor genes in 61 families with FH and in 128 normal individuals. Two of the nine haplotypes were significantly more often associated with the affected genes, whereas one was significantly less frequent. Although none of the associations was strong enough to allow diagnosis in individuals, it was possible, using the three RFLPs, to identify the haplotype of the affected gene in 57 families and to carry out unequivocal diagnosis in 67% of the cases. In four families, PvuII and AvaII detected an abnormal fragment co-segregating with the disease, thus increasing the percentage of diagnosis to 73.4% of the cases.     

A DNA probe for the LDL receptor gene is tightly linked to hypercholesterolemia in a pedigree with early coronary disease.

A large, multigenerational family with dominantly inherited hypercholesterolemia was analyzed for genetic linkage between blood levels of low-density lipoprotein (LDL) cholesterol and the locus for the LDL receptor. A genetic marker was identified by restriction fragment length polymorphism (RFLP) in a cloned segment of the LDL receptor gene. We found no exceptions to segregation of the high-LDL cholesterol phenotype with a unique allele at the LDL receptor locus in this pedigree; tight linkage was indicated by a maximum lod score of 7.52 at theta = 0. Knowledge of the LDL receptor genotype will enable investigators to study variability of phenotypic expression in response to environmental influences or to different genetic determinants.     

Elevated Atherosclerosis-Related Gene Expression,Monocyte Activation and Microparticle-Release Are Related to Increased Lipoprotein-Associated Oxidative Stress in Familial Hypercholesterolemia

Objective

Animal and in vitro studies have suggested that hypercholesterolemia and increased oxidative stress predisposes to monocyte activation and enhanced accumulation of oxidized LDL cholesterol (oxLDL-C) through a CD36-dependent mechanism. The aim of this study was to investigate the hypothesis that elevated oxLDL-C induce proinflammatory monocytes and increased release of monocyte-derived microparticles (MMPs), as well as up-regulation of CD36, chemokine receptors and proinflammatory factors through CD36-dependent pathways and that this is associated with accelerated atherosclerosis in subjects with heterozygous familial hypercholesterolemia (FH), in particular in the presence of Achilles tendon xanthomas (ATX).

Approach and Results

We studied thirty FH subjects with and without ATX and twenty-three healthy control subjects. Intima-media thickness (IMT) and Achilles tendon (AT) thickness were measured by ultrasonography. Monocyte classification and MMP analysis were performed by flow cytometry. Monocyte expression of genes involved in atherosclerosis was determined by quantitative PCR. IMT and oxLDL-C were increased in FH subjects, especially in the presence of ATX. In addition, FH subjects had elevated proportions of intermediate CD14++CD16+ monocytes and higher circulating MMP levels. Stepwise linear regression identified oxLDL-C, gender and intermediate monocytes as predictors of MMPs. Monocyte expression of pro-atherogenic and pro-inflammatory genes regulated by oxLDL-C-CD36 interaction was increased in FH, especially in ATX+ subjects. Monocyte chemokine receptor CX₃CR1 was identified as an independent contributor to IMT.

Conclusions

Our data support that lipoprotein-associated oxidative stress is involved in accelerated atherosclerosis in FH, particularly in the presence of ATX, by inducing pro-inflammatory monocytes and increased release of MMPs along with elevated monocyte expression of oxLDL-C-induced atherosclerosis-related genes.     

A nonsense mutation in the LDL receptor gene leads to familial hypercholesterolemia in the Druze sect.

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the LDL receptor gene. Here we characterize an LDL receptor mutation that is associated with a distinct haplotype and causes FH in the Druze, a small Middle Eastern Islamic sect with a high degree of inbreeding. The mutation was found in FH families from two distinct Druze villages from the Golan Heights (northern Israel). It was not found neither in another Druze FH family residing in a different geographical area nor in eight Arab and four Jewish FH heterozygote index cases whose hypercholesterolemia cosegregates with an identical LDL receptor gene haplotype. The mutation, a single-base substitution, results in a termination codon in exon 4 of the LDL receptor gene that encodes for the fourth repeat of the binding domain of the mature receptor. It can be diagnosed by allele-specific oligonucleotide hybridization of PCR-amplified DNA from FH patients.     

A novel complex mutation in the LDL receptor gene probably caused by the simultaneous occurrence of deletion and insertion in the same region

A novel complex mutation with the presence of both deletion and insertion in very close proximity in the same region was detected in exon 8 of the LDL receptor gene from two apparently unrelated Japanese families with familial hypercholesterolemia (FH). In this mutant LDL receptor gene, the nine bases from nucleotide (nt) 1115 to nt 1123 (AGGGTGGCT) were replaced by six different bases (CACTGA), and consequently the four amino acids from codon 351 to 354, Glu-Gly-Gly-Tyr, were replaced by three amino acids, Ala-Leu-Asn, in the conserved amino acid region of the growth factor repeat B of the LDL receptor. The nature of the amino acid substitution and data on the families suggest that this mutation is very likely to affect the LDL receptor function and cause FH. The generation of this complex mutation can be explained by the simultaneous occurrence of deletion and insertion through the formation of a hairpin-loop structure mediated by inverted repeat sequences. Thus this mutation supports the hypothesis that inverted repeat sequences influence the stability of a given gene and promote human gene mutations.     

Evidence for a third genetic locus causing familial hypercholesterolemia. A non-LDLR, non-APOB kindred.

Monogenically inherited hypercholesterolemia is most commonly caused by mutations at the low density lipoprotein receptor (LDLR) locus causing familial hypercholesterolemia (FH) or at the apolipoprotein B (APOB) locus causing the disorder familial defective apoB (FDB). Probands from 47 kindreds with a strict clinical diagnosis of FH were selected from the Cardiovascular Genetics Research Lipid Clinic, Utah, for molecular genetic analysis. Using a combination of single-strand conformation polymorphism (SSCP) and direct sequencing, 12 different LDLR gene mutations were found in 16 of the probands. Three of the probands were carriers of the APOB R3500Q mutation. In five of the remaining 28 pedigrees where no mutation had been detected, samples from enough relatives were available to examine co-segregation with the LDLR region using the microsatellite marker D19S221, which is within 1 Mb centromeric of the LDLR locus, and D19S394, sited within 150 kb telomeric of the LDLR locus. In four of the families there was strong evidence for co-segregation between the LDLR locus and the phenotype of hypercholesterolemia, but in one large family with 18 living affected members and clear-cut bimodal hypercholesterolemia, there were numerous exclusions of co-segregation. Using length polymorphic markers within and outside the APOB gene, linkage of phenotype in this family to the APOB region was similarly excluded. In this large family, the degree of hypercholesterolemia, prevalence of tendon xanthomata, and occurrence of early coronary disease were indistinguishable from the other families studied. In summary, the data provide unequivocal evidence that a third locus can be etiological for monogenic familial hypercholesterolemia and should be reinvigorating to research in this field.     

Three novel partial deletions of the low-density lipoprotein (LDL) receptor gene in familial hypercholesterolemia

Summary The low-density lipoprotein (LDL) receptor genes from 18 unrelated Japanese heterozygotes and 1 homozygote with classical familial hypercholesterolemia were analyzed by Southern blot hybridization using fragments of the human LDL receptor cDNA as probes. Four different deletion mutations were detected among 20 mutant LDL receptor genes (20%); they were characterized by restriction mapping. None of these mutations has previously been reported in Caucasian patients with FH: three of the mutations were novel and one was similar to the detetion mutation of FH-Tonami described previously in Japanese patients. In three of the four deletion mutations, the rearrangements were related to intron 15 of the LDL receptor gene, in which many Alu sequences exist. The data suggest that a wide range of molecular heterogeneity exists even in major rearrangements resulting in deletions in the LDL receptor gene. The data also support the hypothesis that there are preferential sites within the LDL receptor gene for major rearrangements resulting in deletions. The possibility that a higher frequency of deletion mutations occurs in classical FH than previously suspected is discussed.     

Proprotein convertase subtilisin kexin 9: the third locus implicated in autosomal dominant hypercholesterolemia

PURPOSE OF REVIEW: Autosomal dominant hypercholesterolemia is a genetic disease in which patients have elevated LDL cholesterol levels and premature atherosclerosis. Mutations in the LDL receptor and its ligand apolipoprotein B are causative for autosomal dominant hypercholesterolemia, and the study of this pathway has been crucial to understanding LDL metabolism and receptor-mediated endocytosis in general. Recently, families were identified with a clinical diagnosis of autosomal dominant hypercholesterolemia, but without linkage to the LDL receptor or apolipoprotein B genes. Identification and study of the causative genes in these families should provide additional insights into LDL metabolism. RECENT FINDINGS: Recent microarray studies and database searches identified a novel member of the proprotein convertase family called proprotein convertase subtilisin kexin 9 (PCSK9). A role for PCSK9 in cholesterol metabolism was proposed from the expression studies and confirmed by the discovery that PCSK9 missense mutations were associated with a form of autosomal dominant hypercholesterolemia, Hchola3. The cellular role for PCSK9 and the mechanism behind its mutations are under study, and a role for PCSK9 in regulating LDL receptor protein levels has been demonstrated. SUMMARY: PCSK9 is the third locus implicated in autosomal dominant hypercholesterolemia (Hchola3), and it appears to play an important role in cellular cholesterol metabolism. Understanding the function of PCSK9 will be important for broadening our knowledge of LDL metabolism and may aid in the development of novel hypocholesterolemic agents.     

Autoantibody titers against OxLDL are correlated with Achilles tendon thickness in patients with familial hypercholesterolemia

Achilles tendon xanthomas are associated with increased cardiovascular risk in patients with familial hypercholesterolemia (FH). Oxidized low density lipoprotein (OxLDL), the antibodies against OxLDL, and the LDL-associated phospholipase A(2) (Lp-PLA(2)) may play important roles in atherogenesis. We investigated the possible association between plasma levels of OxLDL, Lp-PLA(2) activity, and autoantibody titers against various types of mildly OxLDL with Achilles tendon thickness (ATT). ATT was determined by sonography in 80 unrelated heterozygous FH patients. Three different types of mildly OxLDL were prepared: OxLDL(L), OxLDL(P), and OxLDL(D), at the end of the lag, propagation, and decomposition phases of oxidation, respectively. Similar types of OxLDL were also prepared after inactivation of the LDL-associated Lp-PLA(2). These types were denoted OxLDL(-)(L), OxLDL(-)(P), and OxLDL(-)(D). FH patients exhibited significantly higher plasma OxLDL levels and serum IgG titers against OxLDL(P) and OxLDL(D) compared with 40 normolipidemic apparently healthy controls. ATT values were positively correlated with autoantibody titers against OxLDL(P) and OxLDL(D); however, in multiple regression analysis, ATT was independently associated only with the autoantibody titers against OxLDL(D). We conclude that the IgG autoantibody titers against OxLDL(D) but not OxLDL or Lp-PLA(2) may play an important role in the pathogenesis of Achilles tendon xanthomas in FH patients.     

Polymorphic DNA haplotypes at the LDL receptor locus.

Mutations in the low-density lipoprotein (LDL) receptor gene result in the autosomal dominant disorder familial hypercholesterolemia (FH). Many different LDL receptor mutations have been identified and characterized, demonstrating a high degree of allelic heterogeneity at this locus. The ability to identify mutant LDL receptor genes for prenatal diagnosis of homozygous FH or to study the role of the LDL receptor gene in polygenic hypercholesterolemia requires the use of closely linked RFLPs. In the present study we used 10 different RFLPs, including three newly described polymorphisms, to construct 123 independent haplotypes from 20 Caucasian American pedigrees. Our sample contained 31 different haplotypes varying in frequency from 0.8% to 29.3%; the five most common haplotypes account for 67.5% of the sample. The heterozygosity and PIC of each site were determined, and these values disclosed that eight of the RFLPs were substantially polymorphic. Linkage-disequilibrium analysis of the haplotype data revealed strong nonrandom associations among all 10 RFLPs, especially among those sites clustered in the 3' region of the gene. Evolutionary analysis suggests the occurrence of both mutational and recombinational events in the generation of the observed haplotypes. A strategy for haplotype analysis of the LDL receptor gene in individuals of Caucasian American descent is presented.     

pcsk9基因突变与胆固醇血症

  常染色体显性高胆固醇血症（ADH）是家族早发性动脉粥样硬化的最主要的危险因素.在与ADH有关的基因突变中,LDL-R、apoB100基因突变导致ADH的机制已经比较明确,而pcsk9基因突变与ADH相关是最近发现的.pcsk9基因编码神经凋亡调节转化酶即NARC-1, 它通过在蛋白水平降低肝细胞上LDL受体的数量,使血液中LDL不能被清除,从而与高胆固醇血症相关联.研究pcsk9与LDL之间的关系,探索pcsk9在高胆固醇血症以及动脉粥样硬化发生中的作用及机制,将有助于高胆固醇血症和动脉粥样硬化发病机制的研究,也能为防治高胆固醇血症和动脉粥样硬化提供新思路.     

家族性高胆固醇血症患者低密度脂蛋白受体基因新突变位点的鉴定

家族性高胆固醇血症(hypercholesterolemia familial,FH)是由于低密度脂蛋白受体(low density lipoprotein receptor,LDLR)基因突变导致的常染色体显性遗传性疾病,临床上表现为多发黄色瘤、高水平血浆LDL、早发性冠心病及有阳性家族史。本研究通过临床症状结合血脂测定诊断出一个FH家系,其纯合子FH患者的血浆总胆固醇水平高达19．05mmol／L,LDL达17．06mmol／L,并有黄色瘤;而杂合子FH患者的血浆总胆固醇水平为7．96mmol／L,LDL为5．55mmol/L,并有心绞痛症状和黄色瘤。我们对该FH家系患者LDLR基因的PCR扩增DNA片段进行测序,发现纯合子FH患者LDLR基因Exon4区域内发生了GAG683GCG突变,即编码LDLR第683位的谷氨酸被丙氨酸替换,而杂合子FH患者该位点呈现杂合突变。此基因型与临床诊断遗传谱完全一致。同时,利用获得Epstein-Barr(EB)病毒转化型人永生淋巴细胞株(EBV-Ls)与荧光探针DiI标记的LDL结合反应,再通过流式细胞仪检测结果显示,具有功能性LDLR的EBV-Ls细胞比例,在纯合子FH患者(7．02％)和杂合子FH患者(62．64％)均比健康对照者(84．69％)低,纯合子FH患者LDLR活性仅为健康对照者的8．29％、而杂合子FH患者LDLR活性约为健康对照者的73．96％,前者呈现非常显著的降低。这些EBV-Ls细胞LDLR的功能变化分析,有力地支持了该FH家系的临床诊断和DNA测序结果。经查阅最新的UMD-LDLR完全版证实,本研究发现鉴定的GAG683GCG突变是人LDLR基因的新突变位点。     

A missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in Sephardic Jews

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low density lipoprotein (LDL) receptor gene. Here, we characterize an LDL receptor mutation that is associated with a distinct haplotype and that causes FH in the Jewish Sephardic population originating from Safed, a town in northern Israel. The mutation was found in eight FH families originating from this community comprising 10% of heterozygote FH index cases screened in Israel. The mutation was not found in four additional FH heterozygotes whose hypercholesterolemia co-segregated with an identical LDL receptor gene haplotype. A guanine to cytosine substitution results in a missense mutation (asp¹⁴⁷ to his) in the fourth repeat of the binding domain encoded by exon 4 of the LDL receptor gene. The mutant receptor protein was synthesized in cultured cells as a 120kDa precursor form that failed to undergo normal processing to a mature cell surface form. Most of the receptor precursors were degraded in the endoplasmic reticulum. The small number of mutant receptors on the cell surface were unable to bind LDL or very low density lipoprotein. The abnormal behavior of the mutant receptor was reproduced by site-directed mutagenesis and expression of the mutant protein in CHO cells. The mutation can be diagnosed by allele-specific oligonucleotide hybridization of polymerase chain reaction amplified DNA from FH patients.     

Two mutations in LDLR gene were found in two Chinese families with familial hypercholesterolemia

Familial hypercholesterolemia (FH) (OMIM 143890) is an autosomal dominantly inherited disease mainly caused by mutations of the gene encoding the low density lipoprotein receptor (LDLR) and Apolipoprotein (Apo) B. First the common mutation R3500Q in ApoB gene was determined using PCR/RFLP method. Then the LDLR gene was screened for mutations using Touch-down PCR, SSCP and sequencing techniques. Furthermore, the secondary structure of the LDLR protein was predicted with ANTHEPROT5.0. The R3500Q mutation was absent in these two families. A heterozygous p.W483X mutation of LDLR gene was identified in family A which caused a premature stop codon, while a homozygous mutation p.A627T was found in family B. The predicted secondary structures of the mutant LDLR were altered. We identified two known mutations (p.W483X, p.A627T) of the LDLR gene in two Chinese FH families respectively.