中国人群家族性高胆固醇血症LDLR基因突变研究进展

家族性高胆固醇血症(Familial hypercholesterolemia,FH)主要是由于低密度脂蛋白受体(Low-density lipoprotein receptor,LDLR)基因突变导致的单基因显性遗传性疾病。FH患者LDLR基因突变导致细胞膜表面LDLR减少或缺如,机体代谢胆固醇能力降低,血浆胆固醇增高并沉积在不同的组织和器官,常伴有全身黄色瘤和早发冠心病,因此FH也是最常见的严重代谢性疾病。世界范围内对LDLR基因突变的报道总共有1741种,经整理我国目前报道的140例FH指示病例,包括108种LDLR基因突变类型。文章对已报道的中国FH患者LDLR基因突变特点进行系统分析和综述,旨在为FH诊断治疗提供参考依据。     

Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients

The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to date there have been only a few reports of the spectrum of mutations in FH patients from Pakistan. In order to identify the causative LDLR variants the gene was sequenced in a Pakistani FH family, while high resolution melting analysis followed by sequencing was performed in a panel of 27 unrelated sporadic hypercholesterolemia patients. In the family a novel missense variant (c.1916T > G, p.(V639G)) in exon 13 of LDLR was identified in the proband. The segregation of the identified nucleotide change in the family and carrier status screening in a group of 100 healthy subjects was done using restriction fragment length polymorphism analysis. All affected members of the FH family carried the variant and none of the non-affected members nor any of the healthy subjects. In one of the sporadic cases, two sequence changes were detected in exon 9, one of these was a recurrent missense variant (c.1211C > T; p.T404I), while the other was a novel substitution mutation (c.1214 A > C; N405T). In order to define the allelic status of this double heterozygous individual, PCR amplified fragments were cloned and sequenced, which identified that both changes occurred on the same allele. In silico tools (PolyPhen and SIFT) were used to predict the effect of the variants on the protein structure, which predicted both of these variants to have deleterious effect. These findings support the view that there will be a novel spectrum of mutations causing FH in patients with hypercholesterolaemia from Pakistan.     

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.     

Global defects in the expression and function of the low density lipoprotein receptor (LDLR) associated with two familial hypercholesterolemia mutations resulting in misfolding of the LDLR epidermal growth factor-AB pair

The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterol-rich lipoproteins from the circulation. Mutations to the receptor result in familial hypercholesterolemia, and over 60 of these occur in the calcium-binding epidermal growth factor-like domain pair. Two selected mutations in this region (G322S and R329P) were introduced into the domain pair and analyzed by in vitro refolding. Both exhibited differing levels of protein misfolding with R329P being the most pronounced. Solution NMR studies of the mutant domain pairs after purification established that a fraction of protein maintains a native-like fold and that this fraction contains two intact calcium-binding sites. An in vivo analysis of intact receptors containing these binding sites showed significantly reduced cell-surface expression compared with the native LDL receptor levels, again with R329P showing the most severe decrease. The sum of these results suggests that either local changes in structure or domain misfolding may be associated with the mutations. There is also the possibility that the misfolding of the calcium-binding epidermal growth factor-like pair region is propagated to other regions of the intact receptor, resulting in more global defects. Surprisingly, for both mutants, those full-length receptors that fold and reach the cell surface retain the ability to bind LDL and release the ligand upon exposure to low pH. This analysis provides significant insight into the protein defect resulting from each of the two mutations and allows their classification to be 2B (partially transport-defective). The results also highlight a range of misfolding defects that may be associated with familial hypercholesterolemia and may enable the prediction of the consequences of homologous disease-causing mutations to other proteins.     

Two novel frameshift mutations associated with the presence of direct repeats of the LDL receptor gene in familial hypercholesterolemia

Two novel frameshift mutations were detected in the mutant LDL receptor genes responsible for familial hypercholesterolemia. One was a 5-bp insertion at codon 395 in exon 9, and the other was a one nucleotide deletion at codon 531 in exon 11. Both mutations alter the reading frame and consequently produce a premature stop codon in the region of the mature LDL receptor homologous to the epidermal growth factor (EGF) precursor. With regard to the mechanism responsible for the generation of these frameshift mutations, strand slipped mispairing mediated by short direct repeats is considered to be the most likely. The findings seem to support the hypothesis that a short direct repeat in DNA sequence can have a profound influence on the stability of a given gene and promote human gene mutations.     

A novel pathogenic variant of the LDLR gene in the Asian population and its clinical correlation with familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a disease implicated with defects in either, Low density lipoprotein receptor gene (LDLR), Apolipoprotein B-100 gene (APOB), the Proprotein convertase subtilisin/kexin type 9 gene (PCSK9) or other related genes of the lipid metabolism pathway. The general characterization of heterozygous FH is by elevated low-density lipoprotein (LDL) cholesterol and early-onset cardiovascular diseases, while the more severe type, the homozygous FH results in extreme elevated levels of LDL cholesterol and usually death of an affected individual by early twenties. We present here a novel non-synonymous, missense mutation in exon 14 of the LDLR gene in two siblings of the Malay ethnicity discovered during an in-house genetic test. We postulate that their elevated cholesterol is due to this novel mutation and they are positive for homozygous FH. This is the first report of a C711Y mutation in patients with elevated cholesterol in Asia.     

Screening for known mutations in the LDL receptor gene causing familial hypercholesterolemia

Familial hypercholesterolemia (FH) is caused by defective low density lipoprotein (LDL) receptors and is characterized by hypercholesterolemia and premature coronary heart disease. Two strategies can be used to identify the mutation in the LDL receptor gene underlying FH. One strategy is to search for novel mutations by DNA sequencing with or without prior mutation screening. The other strategy is to screen for known mutations. In this study we employed the latter strategy to screen 75 unrelated, Norwegian FH subjects for 38 known mutations. Three of the 38 mutations were detected in our group of FH subjects. Two subjects had FH-Padova, one had FH-Cincinnati-2 and one had FH-Gujerat. When additional unrelated FH heterozygotes were screened for the three mutations, the gene frequencies were 1.3%, 1.0% and 3.0%, respectively. In addition to identifying known mutations we also detected a novel stop codon in codon 541 (S541X). We conclude that screening for known mutations in the LDL receptor gene should be used as a complementary strategy to screening for novel mutations in order to understand the molecular genetics of FH.     

New mutations in low-density lipoprotein receptor gene in familial hypercholesterolemia patients from Petrozavodsk

Using an automated fluorescent single-strand conformation polymorphism (SSCP) analysis of the entire coding region, promoter zone, and exon-intron junctions of the low-density lipoprotein (LDL) receptor gene, we examined 80 DNA samples of patients with familial hypercholesterolemia (FH) from Petrozavodsk. We revealed mutations that might cause FH in five probands, including FH-North Karelia (c.925-931del7) mutation and four previously unknown mutations. These novel mutations included a transversion c.618T>G (p.S206R), one nucleotide insertion c.195_196insT (p.FsV66:D129X), a complex gene rearrangement c.192del10/ins8 (p.FsS65:D129X), and a single nucleotide deletion c.2191delG (p.FsV731:V736X). Three out of four novel mutations produce an open reading frame shift and the premature termination of translation. An analysis of the cDNA sequence of the LDL receptor showed that this might result in the formation of a transmembrane-domain-deficient receptor that is unable to bind and internalize the ligand. Our results suggest the absence of a strong founder effect associated with FH in the Petrozavodsk population.     

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.     

Multiplex ligation-dependent probe amplification of LDLR enhances molecular diagnosis of familial hypercholesterolemia

Autosomal dominant (AD) familial hypercholesterolemia [FH; Mendelian Inheritance in Man (MIM) 143890] typically results from mutations in the LDL receptor gene (LDLR), which are now commonly diagnosed using exon-by-exon screening methods, such as exon-by-exon sequence analysis (EBESA) of genomic DNA (gDNA). However, many patients with FH have no LDLR mutation identified by this method. Part of the diagnostic gap is attributable to the genetic heterogeneity of AD FH, but another possible explanation is inadequate sensitivity of EBESA to detect certain mutation types, such as large deletions or insertions in LDLR. Multiplex ligation-dependent probe amplification (MLPA) is a new method that detects larger gDNA alterations that are overlooked by EBESA. We hypothesized that some FH patients with no LDLR mutation detectable by EBESA would have an abnormal LDLR MLPA pattern. In 70 unrelated FH patients, 44 had LDLR mutations detected by EBESA, including missense, RNA splicing, nonsense, or small deletion mutations, and 5 had the APOB R3500Q mutation. Among the remaining 21 AD FH patients with no apparent LDLR mutation, we found abnormal LDLR MLPA patterns in 12 and then demonstrated the deleted sequence in 5 of these. These findings indicate that MLPA may be a useful new adjunctive tool for the molecular diagnosis of FH.     

Two new mutations in the sterol 27-hydroxylase gene in two families lead to cerebrotendinous xanthomatosis

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C→T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C→T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively. Received: 24 July 1996 / Revised: 9 August 1996     

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.     

Two partial deletion mutations involving the same Alu sequence within intron 8 of the LDL receptor gene in Korean patients with familial hypercholesterolemia

Twenty-eight unrelated persons heterozygous for familial hypercholesterolemia (FH) were screened to assess the frequency and nature of major structural rearrangements at the low-density lipoprotein (LDL) receptor gene in Korean FH patients. Genomic DNA was analyzed by Southern blot hybridization with probes encompassing exons 1–18 of the LDL receptor gene. Two different deletion mutations (FH29 and FH110) were detected in three FH patients (10.7%). Each of the mutations was characterized by the use of exon-specific probes and detailed restriction mapping mediated by long-PCR (polymerase chain reaction). Mutation FH29 was a 3.83-kb deletion extending from intron 6 to intron 8 and FH110 was a 5.71-kb deletion extending from intron 8 to intron 12. In FH29, the translational reading frame was preserved and the deducible result was a cysteine-rich A and B repeat truncated protein that might be unable to bind LDL but would continue to bind β-VLDL. FH110 is presumed to be a null allele, since the deletion shifts the reading frame and results in a truncated protein that terminates in exon 13. Sequence analysis revealed that both deletions have occurred between two Alu-repetitive sequences that are in the same orientation. This suggested that in these patients the deletions were caused by an unequal crossing over event following mispairing of two Alu sequences on different chromatids during meiosis. Moreover, in both deletions, the recombinations were related to an Alu sequence in intron 8 and the deletion breakpoints are found within a specific sequence, 27 bp in length. This supports the hypothesis that this region might have some intrinsic instability, and act as one of the important factors in large recombinational rearrangements. Received: 3 April 1996 / Revised: 19 August 1996     

Two missense mutations in exon 9 of caprine PRLR gene were associated with litter size

Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions of the prolactin receptor (PRLR) gene by DNA sequencing and PCR‐RFLP. Two SNPs (c.1457G>A and c.1645G>A) were identified that caused amino acid variations p.Ser485Asn and p.Val548Met respectively. Statistical results indicated that the c.1457G>A and c.1645G>A SNPs were significantly associated with litter size in Boer and Guanzhong goat breeds. Further analysis revealed that combined genotype C4 (GGGG) and haplotype G‐G were better than the others for litter size in both goat breeds. These results might contribute to goat genetic resources and breeding.     

Multiple mutations underlying familial hypercholesterolemia in the South African population

Summary Ten restriction fragment length polymorphisms of the LDL receptor gene were used for haplotype analysis in 12 unrelated patients with homozygous familial hypercholesterolemia. These patients were drawn from the Black, Coloured, and White population groups and collectively represent 24 mutant alleles underlying the FH phenotype. Five distinct haplotypes were detected. Hybridization analysis using DNA codigested with EcoRI and PstI revealed that haplotype IV was associated with two distinct mutations. When coupled to the recent demonstration by other workers of two receptor defects in South African Afrikaners homozygous for FH and haplotype I, these data are suggestive of at least seven distinct LDL receptor mutations in the FH patients examined and thus in the general South African population.     

家族性渗出性玻璃体视网膜病变患者LRP5基因突变研究

家族性渗出性玻璃体视网膜病变(familial exudative vitreoretinopathy, FEVR)是一种遗传性眼科疾病,主要特征为视网膜周边血管发育异常和病变。由于病变程度不同,临床表型可从无明显症状到眼睛完全失明。本文通过研究3个中国FEVR家系和1个散发型患者,探索其致病突变与疾病表型之间的关系。收集家系中患者及亲属的外周血制备基因组DNA,设计引物以扩增FEVR致病基因FZD4、LRP5、NDP和TSPAN12的外显子区域并通过Sanger法进行测序。结果发现,在LRP5基因上存在3个未在dbSNP数据库及千人基因组计划数据库中报道过的杂合突变p.M181R、p.R399S和p.G503R,以及2个已发现但未在FEVR中报道过的杂合突变p.R494Q和p.G876S。生物信息学分析表明这5个突变位点均高度保守,为有害突变。荧光素酶报告基因实验证明了这5个突变均不能激活Norrin/β-catenin信号通路,进一步证明了这5个突变的致病性。该研究扩充了我国FEVR疾病的遗传数据库,可为该疾病的分子诊断提供依据。     

Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families

Autosomal dominant non‐syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next‐generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three‐generation Chinese families with late‐onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co‐segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI‐OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild‐type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.     

South African founder mutations in the low-density lipoprotein receptor gene causing familial hypercholesterolemia in the Dutch population

In South African Afrikaners, three point mutations in the gene coding for the low-density lipoprotein (LDL)-receptor are responsible for more than 95% of the cases of familial hypercholesterolemia (FH). To investigate whether one or more of these mutations originated in The Netherlands, a large group of Dutch heterozygous FH patients was screened for the presence of these three mutations. Of these, a missense mutation in exon 9 of the LDL-receptor gene, resulting in a substitution of Met for Val⁴⁰⁸, responsible for 15% of FH in Afrikaners, was found in 19 (1.5%) of 1268 FH patients of Dutch descent. Nine of the patients carrying the exon 9 mutation on one allele shared the LDL-receptor DNA haplotype with an FH patient from South Africa, who was homozygous for the same mutation. This would suggest that the mutation in these patients and in the South African patient have a common ancestral background. The remaining ten FH patients all shared a common haplotype, partly identical to the Afrikaner haplotype, which chould have arisen from a single recombinational event. This mutation has not been described in persons other than of Dutch ancestry and supports the hypothesis that this mutation in exon 9 originated in The Netherlands and, in all likelihood, was introduced into South Africa by early Dutch settlers in the seventeenth century.