Genomic analysis to screen potential genes and mutations in children with non-syndromic early onset severe obesity: a multicentre study in Turkey.

BACKGROUND: Obesity is a complex genetic-based pediatric disorder which triggers life-threatening conditions. Therefore, the understanding the molecular mechanisms of obesity has been a significant approach in medicine. Computational methods allow rapid and comprehensive pathway analysis, which is important for generation of diagnosis and treatment of obesity. METHODS AND RESULTS: Aims of our study are to comprehensively investigate genetic characteristics of obesity in children with non-syndromic, early-onset (< 7 years), and severe obesity (BMI-SDS > 3) through computational approaches. First, the mutational analyses of 41 of obesity-related genes in 126 children with non-syndromic early-onset severe obesity and 76 healthy non-obese controls were performed using the next generation sequencing (NGS) technique, and the NGS data analyzed by using bioinformatics methods. Then, the relationship between pathogenic variants and anthropometric/biochemical parameters was further evaluated. Obtained results demonstrated that the 15 genes (ADIPOQ, ADRB2, ADRB3, IRS1, LEPR, NPY, POMC, PPARG, PPARGC1A, PPARGC1B, PTPN1, SLC22A1, SLC2A4, SREBF1 and UCP1) which directly related to obesity found linked together via biological pathways and/or functions. Among these genes, IRS1, PPARGC1A, and SLC2A4 stand out as the most central ones. Furthermore, 12 of non-synonymous pathogenic variants, including six novels, were detected on ADIPOQ (G90S and D242G), ADRB2 (V87M), PPARGC1A (E680G, A477T, and R656H), UCP1 (Q44R), and IRS1 (R302Q, R301H, R301C, H250P, and H250N) genes. CONCLUSION: We propose that 12 of non-synonymous pathogenic variations detected on ADIPOQ, ADRB2, PPARGC1A, UCP1, and IRS1 genes might have a cumulative effect on the development and progression of obesity.

Robustness of Massively Parallel Sequencing Platforms.

The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are significant improvements in accuracy and reproducibility of HTS based analyses, the usability of these types of data for diagnostic and prognostic applications necessitates a near perfect data generation. To assess the usability of a widely used HTS platform for accurate and reproducible clinical applications in terms of robustness, we generated whole genome shotgun (WGS) sequence data from the genomes of two human individuals in two different genome sequencing centers. After analyzing the data to characterize SNPs and indels using the same tools (BWA, SAMtools, and GATK), we observed significant number of discrepancies in the call sets. As expected, the most of the disagreements between the call sets were found within genomic regions containing common repeats and segmental duplications, albeit only a small fraction of the discordant variants were within the exons and other functionally relevant regions such as promoters. We conclude that although HTS platforms are sufficiently powerful for providing data for first-pass clinical tests, the variant predictions still need to be confirmed using orthogonal methods before using in clinical applications.

Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts.

Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF-F × IVV-F, IVF-V × IVV-V, IVF-F × IVF-V, and IVV-F × IVV-V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up-regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up-regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo-produced blastocysts. After vitrification, however, in vitro-produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro-produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up-regulation of genes that are involved in stress responses.

Genetic control of lipids in the mouse cross DU6i x DBA/2.

An F(2) pedigree based on the mouse lines DU6i and DBA/2 with extremely different growth and obesity characteristics was generated to search for QTLs affecting serum concentrations of triglycerides (TG), total cholesterol (CHOL), HDL cholesterol (HDL-C), and LDL cholesterol (LDL-C). Compared with many other studies, we searched for spontaneous genetic variants contributing to high lipid levels under a standard breeding diet. Significant QTLs for CHOL were identified on chromosomes 4 and 6, and a female-specific locus on chromosome 3. QTLs for HDL-C were detected on chromosome 11 for both sexes, and on chromosome 1 for females. These QTLs are located in syntenic human regions that have QTLs that have not been previously confirmed in animal studies. LDL-C QTLs have been mapped for both sexes to chromosome 8 and in males on chromosome 13. Epistatic interactions that significantly accounted for the phenotypic variance of HDL-C, CHOL, and LDL-C serum concentrations were also detected with one interaction between chromosomes 8 and 15, accounting for 22% of the observed variance in LDL-C levels. The identified loci coincide in part with regions controlling growth and obesity. Thus, multiple genes or pleiotropic effects may be assumed. The identified QTLs for cholesterol and its transport proteins as subcomponents of risk for coronary heart disease will further improve our understanding of the genetic net controlling plasma lipid concentrations.

Differentially expressed genes in adipose tissues of high body weight-selected (obese) and unselected (lean) mouse lines.

Recently, quantitative trait loci (QTLs) for body weight and obesity have been mapped in an intercross population between the high body weight-selected mouse line DU6i and the inbred line DBA/2. Most QTLs were highly significant, but had small effects only. Under the hypothesis that small-effect QTLs might result from changes in gene activity, our strategy to identify candidate genes for the observed effects was directed towards the identification of differentially expressed genes. Therefore, here we compare the transcription profile of about 11 000 genes in epididymal fat tissues of males of two high body weight-selected (DU6 and DU6i) and two unselected mouse lines (DUKs and DBA/2). For the hybridisation of GeneChips, we used pooled samples of 20 individual mice. By pair-wise comparisons between selected and unselected mouse lines, a set of 77 genes was identified representing genes whose level of expression differed between obese and lean mouse strains. According to the functional classification of genes, 69 differentially expressed genes were involved in regulatory and metabolic pathways, cell division, cell stability, or immune response, and thus might have an effect on body weight and fat accumulation. 14 out of these genes, occur in QTL regions for body weight or abdominal fat weight. Further analyses are necessary to discriminate between genes directly causing QTL effects and indirectly regulated differentially expressed genes.

Genetic, sex, and diet effects on body weight and obesity in the Berlin Fat Mouse Inbred lines.

Mouse lines long-term selected for high fatness offer the possibility to identify individual genes involved in the development of obesity. The Berlin Fat Mouse (BFM) line has been selected for low protein content and afterward for high fatness. Three Berlin Fat Mouse Inbred (BFMI) lines, which are derivates of the selection line BFM and an unselected control line (C57BL/6; B6) were systematically phenotyped between 3 and 20 wk. The body weights and body compositions were measured on a weekly basis. We demonstrated that the BFMI lines dispose of more body weight, body fat mass, and body lean mass than the control line B6 because of a better feed efficiency in these lines. In contrast to other growth-selected mouse lines, the BFMI lines exhibited a general increase in body fat mass but only a marginal increase in body lean mass. The three BFMI lines also showed line- and sex-specific patterns and varied in their response to high-fat diet. The phenotypic differences between the BFMI lines can be traced back to different sets of fixed alleles contributing to fat accumulation and diet-induced obesity. Our results demonstrate that the genetically related BFMI lines are novel models to study the genetic as well as the nutritional aspects of obesity.

Chromosome-wise dissection of the genome of the extremely big mouse line DU6i.

The extreme high-body-weight-selected mouse line DU6i is a polygenic model for growth research, harboring many small-effect QTL. We dissected the genome of this line into 19 autosomes and the Y chromosome by the construction of a new panel of chromosome substitution strains (CSS). The DU6i chromosomes were transferred to a DBA/2 mice genetic background by marker-assisted recurrent backcrossing. Mitochondria and the X chromosome were of DBA/2 origin in the backcross. During the construction of these novel strains, >4000 animals were generated, phenotyped, and genotyped. Using these data, we studied the genetic control of variation in body weight and weight gain at 21, 42, and 63 days. The unique data set facilitated the analysis of chromosomal interaction with sex and parent-of-origin effects. All analyzed chromosomes affected body weight and weight gain either directly or in interaction with sex or parent of origin. The effects were age specific, with some chromosomes showing opposite effects at different stages of development.