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.     

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.     

A clinical variant of familial Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive inherited disease consisting of (1) partial oculocutaneous albinism (with nystagmus, strabism, and visual acuity loss), (2) platelet storage pool deficiency (with bleeding diathesis), and (3) disorder of "ceroid" metabolism with a multisystem tissue lysosomal ceroid deposition. HPS is less uncommon in Puerto Rico, where the most important studies have been performed, but is a very rare disease in Europe. HPS basic defect remains unknown, even if an HPS-causing gene was identified in chromosome segment 10q23-q23.3, and several mutations have been reported. The aim of this article is to discuss, on the basis of a review of relevant literature, a new familial HPS clinical variant observed in 2 young sisters (aged 16 and 23 years old, respectively), characterized by the typical symptoms of this syndrome. Our patients also suffered from diffuse interstitial pulmonary disease and an unexpectedly increased platelet aggregation and were prone to bacterial infections. Interestingly, we observed urinary tract abnormality in the younger HPS sister and a porencephalic cyst in the older HPS sister; both of these developmental defects have been reported in the Cross syndrome (or oculocerebral hypopigmentation syndrome). It seems that in our patients, an overlapping of the phenotypic manifestations of different rare syndromes may be present. The presence of ceroid-like autofluorescent material in urinary sediment together with the histologic aspects and the autofluorescence of oral mucosa biopsy are consistent with a ceroid-like lipofuscin storage. HPS should be carefully tested for in suspected cases to prevent the severe visual impairment, rapidly progressive pulmonary fibrosis, and other complications associated with this disorder.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia

Familial Hypercholesterolemia (FH) results in elevated levels of blood lipids including total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG). This disease is one of the major contributors towards an early onset of coronary heart disease (CHD). The aim of the present study was to identify the genes responsible for causing FH in Pakistani population, for this purpose a large consanguineous FH family was selected for genetic analysis. Serum lipid levels, including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C), were determined in patients and healthy controls. In order to find the causative mutation in this family, direct sequencing of the low density lipoprotein receptor (LDLR) gene was performed. In addition the part of the Apolipoprotein-B (APOB) gene containing the mutations R3500Q and R3500W was also sequenced. Affected individuals of the family were found to have raised TC and LDL-C levels. Sequencing revealed an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene in all the affected individuals of the family. Common FH causing APOB mutations were not present in this family. Heterozygous individuals had TC levels ranging from ~300–500 mg/dl and the only homozygous individual with typical xanthomas had TC levels exceeding 900 mg/dl. This is the first report of a known LDLR gene mutation causing FH in the Pakistani population. Despite a large heterogeneity of LDLR mutations there are still some common mutations which are responsible for FH throughout the world.     

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.     

Molecular characterization of Polish patients with familial hypercholesterolemia: novel and recurrentLDLR mutations

Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the genes coding for the low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, a molecular analysis ofLDLR andAPOB was performed in a group of 378 unrelated ADH patients, to explore the mutation spectrum that causes hypercholesterolemia in Poland. All patients were clinically diagnosed with ADH according to a uniform protocol and internationally accepted WHO criteria. Mutational analysis included all exons, exon-intron boundaries and the promoter sequence of theLDLR, and a fragment of exon 26 ofAPOB. Additionally, the MLPA technique was applied to detect rearrangements withinLDLR. In total, 100 sequence variations were identified in 234 (62%) patients. WithinLDLR, 40 novel and 59 previously described sequence variations were detected. Of the 99LDLR sequence variations, 71 may be pathogenic mutations. The most frequentLDLR alteration was a point mutation p.G592E detected in 38 (10%) patients, followed by duplication of exons 4–8 found in 16 individuals (4.2%). Twenty-five cases (6.6%) demonstrated the p.R3527Q mutation ofAPOB. Our findings imply that major rearrangements of theLDLR gene as well as 2 point mutations (p.G592E inLDLR and p.R3527Q inAPOB) are frequent causes of ADH in Poland. However, the heterogeneity ofLDLR mutations detected in the studied group confirms the requirement for complex molecular studies of Polish ADH patients.     

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。     

禽致病性大肠杆菌脂多糖核心型分布与毒力基因的相关性分析

【目的】大肠杆菌脂多糖(LPS)核心型根据其化学结构的不同分为5种,即R1、R2、R3、R4和K12。通过对禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)安徽、江苏、上海和河南等省市分离株的脂多糖核心型分布情况的研究,分析其与大肠杆菌主要毒力基因之间的潜在联系,以期为APEC的研究和防治提供参考。【方法】对分离到的76株APEC,利用PCR方法开展对LPS核心型分型鉴定和毒力基因检测;分析LPS核心型的分布和毒力基因、致病性之间的相关性。【结果】在76株APEC分离株中,68.4% (52株)为R1核心型,15.8% (12株)为R3型,11.8% (9株)为R4型,3.9% (3株)为R2型,未检测到K12核心型。毒力基因鉴定结果中yijp、mat、fimC、ibeB和ompA的检验阳性率均达到90%以上,可作为APEC的保守基因。其中LPS核心型R1与neuC、cva/cvi、irp2均具有显著正相关性(P<0.05),R3与iroN、irp2均具有显著负相关性(P<0.05),R4核心型与aatA显著正相关(P<0.05)。【结论】APEC的LPS核心型主要为R1。LPS核心型对部分毒力基因分布具有显著影响。