一个家族性高胆固醇血症家系的基因突变筛查与评估

目的：对一个临床诊断的家族性高胆固醇血症（FH）家系进行全基因组外显子测序,以寻找该家系的致病基因。方法：采集该家系成员外周静脉血样本,检测血清总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）和高密度脂蛋白胆固醇（HDL-C）这四项指标,并提取白细胞DNA进行全基因组外显子测序,筛选出4个FH相关基因LDLR、APOB、PCSK9、LDLRAP1的单核苷酸多态性（SNP）位点情况并进行生物信息学分析,采用Polyphen-2和SIFT软件对SNP位点进行致病性分析。结果：该家系APOB基因的SNP位点rs676210、rs679899、c.10094A > T和c.9937C > G均可能与血脂增高有关,但这些位点均不与疾病表型共分离。经Polyphen-2和SIFT软件预测未发现LDLR,PCSK9和LDLRAP1基因的致病性变异。结论：该FH家系存在可能与血脂增高相关的APOB基因SNP位点,其功能尚需进一步的实验验证。     

Novel mutations of APOB cause ApoB truncations undetectable in plasma and familial hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL) is a genetic disorder characterized by low levels of apoB-100 and LDL cholesterol. Truncation-producing mutations of apoB (chromosome 2) are among several potential causes of FHBL in patients. Ten new families with FHBL linked to chromosome 2 were identified. In Family 8, a 4432delT in exon 26 produces a frame-shift and a premature stop codon predicted to produce a truncated apoB-30.9. Even though this truncation is just 10 amino acid shorter than the well-documented apoB-31, which is readily detectable in plasma, apoB-30.9 is undetectable. Most truncations shorter than apoB-30 are not detectable in plasma. In Family 34, an acceptor splicing mutation at position -1 of exon 14 changes the acceptor splice site AG to AA. Two families (Family 50 and 52) had mutations (apoB-9 and apoB-29) reported previously. In Family 98, a novel point mutation in exon 26 (11163T>G) causes a premature stop codon, and produces a truncated apoB-80.5 readily detectable in plasma. Sequencing of the ApoB gene in families 1, 5, 18, 58, and 59 did not reveal mutations.     

Association of the apolipoprotein B gene polymorphisms with essential hypertension in Northern Chinese Han population

Objective To study the association of the apolipoprotein B gene polymorphisms with essential hypertension in Northern Chinese Han population. Methods XbaI and EcoRI polymorphisms of the apolipoprotein B (APOB) gene were genotyped by polymerase chain reaction (PCR) and restriction fragment-length polymorphism (RFLP) method in 503 unrelated hypertensive patients and 490 healthy controls recruited from international collaborative study of cardiovascular disease in Asia (InterAsia). Results The difference in the genotypic distributions could be neglected across the groups. The prevalence of X allele in healthy controls (4.8%) was less frequent in Chinese, and there was no significant difference in the frequency of the X allele between cases (5.7%) and controls (P=0.38). The observed E- allele frequencies were closely similar among groups (5.9% in cases vs 5.0% in controls, P=0.39). Logitstic regression analyses revealed that the lack of association still persisted after adjustment of other environmental factors. Haplotype analysis showed that X-E was most frequent and no haplotype could significantly contribute to essential hypertension. Conclusion The APOB gene XbaI and EcoRI polymorphisms are not associated with essential hypertension in the Northern Chinese Han population. Future studies on single nucleotide polymorphisms in larger samples are needed to further investigate the possible contribution of the APOB gene to essential hypertension.     

Genetic variation in APOB, PCSK9, and ANGPTL3 in carriers of pathogenic autosomal dominant hypercholesterolemic mutations with unexpected low LDL-Cl Levels

Autosomal Dominant Hypercholesterolemia (ADH) is caused by LDLR and APOB mutations. However, genetically diagnosed ADH patients do not always exhibit the expected hypercholesterolemic phenotype. Of 4,669 genetically diagnosed ADH patients, identified through the national identification screening program for ADH, 75 patients (1.6%) had LDL-cholesterol (LDL-C) levels below the 50th percentile for age and gender prior to lipid-lowering therapy. The genes encoding APOB, PCSK9, and ANGPTL3 were sequenced in these subjects to address whether monogenic dominant loss-of-function mutations underlie this paradoxical phenotype. APOB mutations, resulting in truncated APOB, were found in five (6.7%) probands, reducing LDL-C by 56%. Rare variants in PCSK9, and ANGPTL3 completely correcting the hypercholesterolemic phenotype were not found. The common variants p.N902N, c.3842+82T>A, p.D2312D, and p.E4181K in APOB, and c.1863+94A>G in PCSK9 were significantly more prevalent in our cohort compared to the general European population. Interestingly, 40% of our probands carried at least one minor allele for all four common APOB variants compared to 1.5% in the general European population. While we found a low prevalence of rare variants in our cohort, our data suggest that regions in proximity of the analyzed loci, and linked to specific common haplotypes, might harbor additional variants that correct an ADH phenotype.     

Semiquantitative multiplex PCR: a useful tool for large rearrangement screening and characterization

Methods presently employed for detection of large rearrangements have several drawbacks, such as the amount of sample and time required, technical difficulty, or the probability of false-negative carriers. Using the low-density-lipoprotein receptor (LDLR) gene, whose mutations are responsible for familial hypercholesterolemia (FH), we have developed a procedure to detect large rearrangements in this gene based on semiquantitative PCR, with important improvements as compared to previous methods. Our method covers the complete LDLR gene and introduces an internal control in the reaction. The procedure discriminates the four different large rearrangements (two deletions and two insertions) that we have used as positive mutation controls (Valencia-1 to -5). All altered exons from each rearrangement are identified. Furthermore, when families from probands carrying these large rearrangements (34 members) were analyzed, our results agreed with those obtained previously with Southern blot. We have also analyzed a sample of 110 unrelated FH probands and the method has correctly identified the two different large rearrangements present and insertions or deletions as small as 1 bp. In conclusion, the method we present allows the identification of large rearrangements affecting exons of the gene, including small insertions or deletions or complete gene deletion. In addition, it constitutes a first characterization step of rearrangements, and is easy to carry out fast, and can be applied to the analysis of any gene.     

Interaction between SREBP-1a and APOB polymorphisms influences total and low-density lipoprotein cholesterol levels in patients with coronary artery disease

In this study, we examined the insertion/deletion (Ins/Del) and XbaI polymorphisms of the apolipoprotein B (APOB) gene and the -36delG polymorphism in the sterol regulatory element binding protein-1a (SREBP-1a) gene in 298 patients with non-diabetic angiographically assessed coronary artery disease (CAD), and 188 healthy controls, from a Brazilian population of European descent. Del/X+ haplotype carriers had higher levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in patients (TC, p = 0.05; LDL-C, p = 0.049) and controls (TC, p = 0.004; LDL-C, p = 0.013). No association was detected between the SREBP-1a-36delG polymorphism and lipid levels, but a significant interaction effect between APOB and SREBP-1a polymorphisms was observed in the patient sample on TC (p = 0.005) and on LDL-C (p = 0.019) levels. Carriers of the APOB Del/X+ haplotype and SREBP-1a G-G- genotype showed the highest levels of these lipid parameters. This effect of interaction was not observed in the control sample. Despite the associations with lipids, these polymorphisms were not associated with CAD risk or severity in this sample.     

载脂蛋白与Ⅱ型糖尿病及其并发症的关系

目的观察载脂蛋白与Ⅱ型糖尿病(T2DM)及其并发症的关系。方法载脂蛋白APOA1、APOB采用免疫透射比浊法测定;TC、TG、HDL-C采用酶法测定。试剂为上海科华公司。仪器为美国贝克曼CX7全自动生化分析仪。结果T2DM合并冠心病组、肾病组的APOB分别高于正常人、视网膜微血管病组、无血管并发症组。T2DM各组的APOA1/B比值均高于正常人,且合并冠心病组、肾病组,分别高于视网膜微血管病组与无血管并发症组。结论APOA1、APOB与T2DM动脉硬化关系密切,APOA1/B比值是反映T2DM脂代谢的敏感指标。     

冠心病血浆HDL-C,APOAI,APOB和LP(a)相关性探讨

笔者测量了冠心病患者和对照组血液中的脂类及载脂蛋白的含量 ,发现当高密度脂蛋白胆固醇 ( HDL-C) <0 .9mm ol/L、载脂蛋白 AI( A POAI) <1.0 g/L、载脂蛋白 B( APOB) >0 .9g/L 及脂蛋白〔LP( a)〕>2 0 0 m g/L,患者的发病率明显高于对照组 ( P<0 .0 1)。对于 CHD的这四个危险因素 ,在临床上可用于对一般人群 CH D的调查及预防治疗后的观察 ,都有非常重要的意义。     

Haplotype analysis of signal peptide (insertion/deletion) and XbaI polymorphisms of the APOB gene in gallbladder cancer.

PURPOSE: The incidence of gallbladder cancer (GBC) is usually paralleled by the prevalence of gallstone disease, and genes of cholesterol metabolism have been implicated in gallstone disease. The XbaI and insertion/deletion (ins/del) polymorphism of Apolipoprotein B (APOB) appears to influence cholesterol homoeostasis and possibly risk for gallstone disease. We examined the effect of these polymorphisms individually as well as their haplotypes on GBC and gallstone patients in North Indian population. METHODS: The study comprises 123 consecutive cases of proven GBC, 172 cases of gallstone and 232 healthy subjects of similar age and sex. The genomic DNA was extracted from peripheral blood leucocytes and genotyping was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. RESULTS: In a case-control study, APOB XbaI and ins/del polymorphisms were not significantly associated with risk of GBC. Using the expectation maximization algorithm, four haplotypes were obtained, and haplotype X(+),D was found to be significantly higher in GBC patients without stone in comparison with healthy subjects [odds ratio (OR) 2.9, 95% confidence interval 1.2-6.6 P=0.012]. CONCLUSIONS: The X(+),D haplotype of APOB is associated with increased risk for development of GBC and the risk is not modified in the presence of gallstones.