Ablating both Fabp1 and Scp2/Scpx (TKO) induces hepatic phospholipid and cholesterol accumulation in high fat-fed mice

Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.     

Cloning and Tissue Expression Characterization of the Chicken APOB Gene

Apolipoprotein B (APOB) serves an essential role in the assembly and secretion of triglyceride-rich lipoproteins and lipids transport. This study was designed to clone the full-length cDNA of the chicken APOB gene, to characterize the expression profile, and investigate the differential expression between layer and broiler of the chicken APOB gene. The full-length cDNA sequence (14,150-bp) that contained a 13,896-bp ORF encoding 4,631 amino acids was obtained by RT-PCR, RACE, and bioinformatics analysis. qReal-Time PCR analysis showed that the chicken APOB gene was highly expressed in kidney, liver, and intestine. The results of differential expression showed that the APOB gene was more highly expressed in intestine and kidney in Bai'er layer than in broiler, but there was no significant difference in liver between the two breeds. The results of this study provided basic molecular information for studying the role of APOB in the energy transportation in avian species.     

A novel method for whole blood PCR without pretreatment

PCR is usually performed on purified DNA. However, the extraction of DNA from whole blood is time consuming and involves the risk of contamination at every step. Hence, it is desirable to amplify DNA directly from whole blood. Earlier, investigators tried to achieve this target by either pretreatment of whole blood samples with different agents or by altering the conventional thermal cyclic conditions. This would make the technique cumbersome and time consuming. Here, we describe a simple protocol to amplify DNA directly from whole blood without the need of pretreatment. PCR buffer system was optimized in the laboratory and Apolipoprotein B gene was used as a model for this experiment. 480 bp was the target site for amplification. Fresh whole blood samples were used both from healthy and diseased individuals (coronary artery disease patients). Successful amplification was achieved with 1 μl volume of whole blood and it was comparable to that of genomic DNA. No pretreatment of whole blood samples was required with the optimized buffer system. 3mM concentration of MgCl(2) was observed to be optimal and hence used in the reaction mixture. Amplification was relatively better with this buffer system as compared to that of commercially available PCR buffer. With the present technique, amplicon detection did not require the centrifugation/dilution of the PCR products which further saves time. Successful amplification was achieved in both the healthy and diseased blood samples, indicating the robustness of the technique as changed blood composition and presence of increased inhibitory molecules in the diseased state did not seem to affect the efficacy of the present technique. In conclusion, as compared to the existing protocols for whole blood PCR, the present technique is relatively novel, simple, requires minimal steps and eliminates the need for additional standardizations.     

The polymorphism − 1131T > C in apolipoprotein A5 gene is associated with dyslipidemia in Brazilian subjects

Background

Polymorphisms in apolipoprotein A5 gene (APOA5) have been associated with higher triglyceride levels in many populations. The aim of the study was to determine the allelic and genotypic distribution of the APOA5 − 1131T > C polymorphism and to identify the association of the genetic variant and the risk for dyslipidemia.

Methods

We genotyped 109 dyslipidemic subjects and 107 controls. The total cholesterol, triglycerides and HDL-c were determined enzymatically. Comparison of means among groups was calculated by ANOVA. Significant differences among groups were evaluated by Student–Newman–Keuls test.

Results

The minor allele C was more frequent in dyslipidemic subjects than controls (p = 0.019) and confers an increased individual risk for dyslipidemia (OR = 1.726, CI 95% = 1.095–2.721). The genotype analysis by gender showed that this allele was more frequent in dyslipidemic males (p = 0.037; OR = 2.050, CI 95% = 1.042–4.023). When participants were analyzed according to genotypes TT and TC/CC, C-carriers presented higher cholesterol and triglycerides levels than TT homozygous (p = 0.046 and 0.049, respectively).

Conclusions

The allele C confers higher total cholesterol and triglycerides levels in dyslipidemic adults. The APOA5 − 1131T > C polymorphism is associated with dyslipidemia in male subjects.     

Familial hypobetalipoproteinemia: Analysis of three Spanish cases with two new mutations in the APOB gene

Extremely low LDL-cholesterol concentrations are very unusual and generally related with comorbidities accompanying malnutrition. Less frequently low LDL-cholesterol levels result from mutations in the APOB, PCSK9, ANGPTL3, SAR1B and MTTP genes (primary hypobetalipoproteinemia). We investigated three patients with plasma LDL-cholesterol levels below the fifth percentile of the Spanish population. We recorded data on demographic and anthropometric characteristics, life style habits, physical examination, liver ultrasound and lipid and lipoprotein levels, in the probands and their first-degree relatives. Secondary causes of hypocholesterolemia were ruled out by clinical study, complementary tests and follow-up. The APOB, MTTP and SAR1B genes were sequenced. Patients were found to be heterozygotes for point mutations located in the exon 26 of the APOB gene. One patient, with fatty liver, carried a previously described mutation (c.7600C > T) (Arg2507X), causing the formation of truncated Apo B-55.25. The other two mutations producing truncations are new. One asymptomatic patient carried the Arg3672X (Apo B-80.93) and the other with fatty liver and steatorrhea carried the Ser2184fsVal2193X (Apo B-48.32). Our study reinforces the concept that in the heterozygous carriers of truncated Apo Bs, the clinical manifestations of FHBL are dependent on the size of the truncations.     

APOB-516 T allele homozygous subjects are unresponsive to dietary changes in a three-month primary intervention study targeted to reduce fat intake

Dietary guidelines aim to control fat intake and reduce cardiovascular risk but an important interindividual variability occurs among subjects. The objective was to investigate whether the response of lipid and glucose homeostasis parameters after a three-month diet aimed at reducing cardiovascular risk could be modulated by the −516C/T polymorphism in the apolipoprotein B gene (APOB). Middle-aged men (n = 69) and women (n = 100) with moderate cardiovascular disease risk were advised to reduce total energy and fat intakes and replace saturated dietary fat by monounsaturated and polyunsaturated fat. Subjects were genotyped for APOB-516C/T polymorphism. At the entry and at the end of the three-month period, fasting and postprandial plasma lipid analyses were performed. At entry, subjects homozygous for the APOB-516 T allele exhibited significantly lower fasting plasma concentrations of apolipoprotein B 48, triglycerides and triglyceride-rich lipoproteins-triglycerides compared to C carrier subjects. After the diet period, while C carrier subjects presented a clear improvement of most biological parameters, paradoxically T/T subjects did not modify them. In addition, the apoB 48 postprandial response after a standardized mixed test meal was not improved in T/T subjects after the three-month diet, contrary to C allele carriers. Even though their phenotype at entry does not show any significant increase of risk factors when compared to other groups, subjects homozygous for the APOB-516 T allele are unresponsive to a healthy diet that improves cardiovascular risk status in the whole population.     

SNP-based large-scale identification of allele-specific gene expression in human B cells

Polymorphism and variations in gene expression provide the genetic basis for human variation. Allelic variation of gene expression, in particular, may play a crucial role in phenotypic variation and disease susceptibility. To identify genes with allelic expression in human cells, we genotyped genomic DNA and cDNA isolated from 31 immortalized B cell lines from three Centre d'Etude du Polymorphisme Humain (CEPH) families using high-density single-nucleotide polymorphism (SNP) chips containing 13,900 exonic SNPs. We identified seven SNPs in five genes with monoallelic expression, 146 SNPs in 125 genes with allelic imbalance in expression with preferentially higher expression of one allele in a heterozygous individual. The monoallelically expressed genes (ERAP2, MDGA1, LOC644422, SDCCAG3P1 and CLTCL1) were regulated by cis-acting, non-imprinted differential allelic control. In addition, all monoallelic gene expression patterns and allelic imbalances in gene expression in B cells were transmitted from parents to offspring in the pedigree, indicating genetic transmission of allelic gene expression. Furthermore, frequent allele substitution, probably due to RNA editing, was also observed in 21 genes in 23 SNPs as well as in 48 SNPs located in regions containing no known genes. In this study, we demonstrated that allelic gene expression is frequently observed in human B cells, and SNP chips are very useful tools for detecting allelic gene expression. Overall, our data provide a valuable framework for better understanding allelic gene expression in human B cells.     

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.     

Bi-PASA genotyping of a new polymorphism in the APOB gene shows no evidence for an association with fatness in pigs

Sequence analysis of PCR products of a 343-bp fragment from exon 29 of the porcine APOB gene of four Erhualian and four Landrace pigs revealed a missense G/C substitution at position of 6117 in this gene. Two allele-specific primers were designed to genotype this polymorphism using the Bi-PASA technique. Genotyping of 146 animals from Erhualian, Hampshire, Large White, Landrace and Duroc breeds revealed large breed differences in allele frequency. No association with fatness was observed within each of the four European breeds, where animals had been selected to be those with the highest and those with the lowest backfat depth at 100 kg liveweight from a large carcass dissection project.