DNA methylation microarrays identify epigenetically regulated lipid related genes in obese patients with hypercholesterolemia.

BACKGROUND: Epigenetics can contribute to lipid disorders in obesity. The DNA methylation pattern can be the cause or consequence of high blood lipids. The aim of the study was to investigate the DNA methylation profile in peripheral leukocytes associated with elevated LDL-cholesterol level in overweight and obese individuals. METHODS: To identify the differentially methylated genes, genome-wide DNA methylation microarray analysis was performed in leukocytes of obese individuals with high LDL-cholesterol (LDL-CH, ≥ 3.4 mmol/L) versus control obese individuals with LDL-CH, < 3.4 mmol/L. Biochemical tests such as serum glucose, total cholesterol, HDL cholesterol, triglycerides, insulin, leptin, adiponectin, FGF19, FGF21, GIP and total plasma fatty acids content have been determined. Oral glucose and lipid tolerance tests were also performed. Human DNA Methylation Microarray (from Agilent Technologies) containing 27,627 probes for CpG islands was used for screening of DNA methylation status in 10 selected samples. Unpaired t-test and Mann-Whitney U-test were used for biochemical and anthropometric parameters statistics. For microarrays analysis, fold of change was calculated comparing hypercholesterolemic vs control group. The q-value threshold was calculated using moderated Student's t-test followed by Benjamini-Hochberg multiple test correction FDR. RESULTS: In this preliminary study we identified 190 lipid related CpG loci differentially methylated in hypercholesterolemic versus control individuals. Analysis of DNA methylation profiles revealed several loci engaged in plasma lipoprotein formation and metabolism, cholesterol efflux and reverse transport, triglycerides degradation and fatty acids transport and ß-oxidation. Hypermethylation of CpG loci located in promoters of genes regulating cholesterol metabolism: PCSK9, LRP1, ABCG1, ANGPTL4, SREBF1 and NR1H2 in hypercholesterolemic patients has been found. Novel epigenetically regulated CpG sites include ABCG4, ANGPTL4, AP2A2, AP2M1, AP2S1, CLTC, FGF19, FGF1R, HDLBP, LIPA, LMF1, LRP5, LSR, NR1H2 and ZDHHC8 genes. CONCLUSIONS: Our results indicate that obese individuals with hypercholesterolemia present specific DNA methylation profile in genes related to lipids transport and metabolism. Detailed knowledge of epigenetic regulation of genes, important for lipid disorders in obesity, underlies the possibility to influence target genes by changing diet and lifestyle, as DNA methylation is reversible and depends on environmental factors. These findings give rise for further studies on factors that targets methylation of revealed genes.

A novel TAZ gene mutation and mosaicism in a Polish family with Barth syndrome.

Barth syndrome (BTHS) is an X-linked recessive disease primarily affecting males. Clinically, the disease is characterized by hypertrophic or dilated cardiomyopathy, skeletal myopathy, chronic/cyclic neutropenia, 3-methylglutaconic aciduria, growth retardation and respiratory chain dysfunction. It is caused by mutations in the TAZ gene coding for the tafazzin protein which is responsible for cardiolipin remodeling. In this work, we present a novel pathogenic TAZ mutation c.83T>A, p.Val28Glu, found in mosaic form in almost all female members of a Polish family. Sanger sequencing of DNA from peripheral blood and from epithelial cells showed female mosaicism in three generations. This appears to be a new mechanism of inheritance and further research is required in order to understand the mechanism of this mosaicism. We conclude that BTHS genetic testing should include two or more tissues for women that appear to be noncarriers when blood DNA is initially tested. The results of our study should not only be applicable to BTHS families, but also to families with other X-linked diseases.

Analysis of tafazzin and deoxyribonuclease 1 like 1 transcripts and X chromosome sequencing in the evaluation of the effect of mosaicism in the TAZ gene on phenotypes in a family affected by Barth syndrome.

Barth syndrome is a rare disease affecting mitochondria structure and function in males. In our previous study, we have shown a new mutation (c.83T>A, p.Val28Glu) in the TAZ gene in two affected patients with congenital cardiomyopathy. Furthermore, women in this family had no mutations in their blood cells, whereas they only had mutations in the oral epithelial cells. The objective of the project was to evaluate the effect of intertissue mosaicisms on the Barth syndrome phenotypes, searching for another disease-related loci on chromosome X and finally to assess the consequences of the mutation. We conducted the advanced genetic study including cytogenetic research (constitutional karyotyping in blood and fibroblasts), NGS sequencing (with custom chromosome X sequencing together with the evaluation of loss of heterozygosity (LOH) and aberrations (CNV) in the whole genome) in four different tissues and sequencing of tafazzin and deoxyribonuclease 1 like 1 transcripts. The presence of deletions within the 5'untranslated region of the TAZ gene and/or the noncoding regions of the DNASE1L1 gene were detected in several tissues. Whereas, there is no intertissue mosaicism regarding point mutation in TAZ gene in all investigated tissues in female carriers. Only the male patient presented biochemical markers and neurological symptoms of Barth syndrome. All the female carriers are healthy and have normal tafazzin and deoxyribonuclease 1 like 1 transcripts in 2 analyzed tissues. The conclusion of this study is that we cannot rule out or confirm mosaicism in the noncoding regions of TAZ or DNASE1L1 genes, but this is not clinically relevant in female carriers because they are healthy. Finally, it has been proven that mutation (c.83T>A, p.Val28Glu) is responsible for disease in males in this family.

Epigenetic Regulation of Processes Related to High Level of Fibroblast Growth Factor 21 in Obese Subjects.

We hypothesised that epigenetics may play an important role in mediating fibroblast growth factor 21 (FGF21) resistance in obesity. We aimed to evaluate DNA methylation changes and miRNA pattern in obese subjects associated with high serum FGF21 levels. The study included 136 participants with BMI 27-45 kg/m2. Fasting FGF21, glucose, insulin, GIP, lipids, adipokines, miokines and cytokines were measured and compared in high serum FGF21 (n = 68) group to low FGF21 (n = 68) group. Human DNA Methylation Microarrays were analysed in leukocytes from each group (n = 16). Expression of miRNAs was evaluated using quantitative PCR-TLDA. The study identified differentially methylated genes in pathways related to glucose transport, insulin secretion and signalling, lipid transport and cellular metabolism, response to nutrient levels, thermogenesis, browning of adipose tissue and bone mineralisation. Additionally, it detected transcription factor genes regulating FGF21 and fibroblast growth factor receptor and vascular endothelial growth factor receptor pathways regulation. Increased expression of hsa-miR-875-5p and decreased expression of hsa-miR-133a-3p, hsa-miR-185-5p and hsa-miR-200c-3p were found in the group with high serum FGF21. These changes were associated with high FGF21, VEGF and low adiponectin serum levels. Our results point to a significant role of the epigenetic regulation of genes involved in metabolic pathways related to FGF21 action.

Quality of life assessment in patients with HNF1A-MODY and GCK-MODY.

AIM: The impact of maturity onset diabetes of the young (MODY) on quality of life (QoL) has never been examined. We assessed disease impact on QoL among patients with HNF1A-MODY and GCK mutation carrier status. METHODS: The study included 80 patients with HNF1A-MODY and 89 GCK gene mutation carriers. We also examined 128 type 1 diabetes (T1DM) patients for comparison. Diabetes-specific QoL was assessed using the Audit of Diabetes Dependent Quality of Life questionnaire. RESULTS: HNF1A-MODY and GCK-MODY groups had similar mean age (41.7 vs. 38.0 years, respectively) and BMI (24.1 vs. 24.3 kg/m2), whereas T1DM patients were on average younger (34.2 years) with similar BMI (25.0 kg/m2). Less than a third of GCK mutation carriers were on pharmacotherapy (n = 20, 31%), while the majority of HNF1A mutation carriers used oral drugs or insulin (n = 66, 82.5%). While current QoL was similar across the three groups (p = 0.66), two other major indices-the impact of diabetes on QoL and the average weighted impact (AWI)-differed among them (p < 0.001 for both comparisons). The impact of diabetes on patient QoL and AWI observed in both MODY groups was smaller than in T1DM. Etiological diagnosis of diabetes and a diagnosis of retinopathy were the only independent factors influencing the impact of diabetes on QoL and AWI in regression analysis. In HNF1A-MODY, all three major indices of QoL were more heavily influenced for patients on insulin in comparison to other treatment sub-groups. CONCLUSION: MODY has a smaller negative impact on QoL compared to T1DM. Mode of treatment further stratifies QoL decline for HNF1A-MODY subjects.

Case report of dysregulation of primary bile acid synthesis in a family with X-linked adrenoleukodystrophy.

RATIONALE: X-linked adrenoleukodystrophy (X-ALD) is a rare disorder caused by mutations in the ABCD1 gene, coding for peroxisomal membrane transporter adrenoleukodystrophy (ALD) protein. The disease is characterized by accumulation of very long chain fatty acids (VLCFAs) in tissues. Adult adrenomyeloneuropathy (AMN) and the cerebral inflammatory form of ALD are the main phenotypes presenting various symptoms. PATIENT CONCERNS: We report a case of 37-year-old patient with diagnosis of X-ALD, confirmed based on elevated VLCFA concentrations and genetic testing of ABCD1 gene. The complete clinical picture in the patient indicates AMN phenotype with cerebral involvement. DIAGNOSES: The reduced synthesis of unconjugated cholic and chenodeoxycholic acids, and the reduction to 28% to 29% of peroxisomal beta-oxidation of behenic acid and normal peroxisomal metabolism of pristanic and palmitic acid were observed in the X-ALD patient. Sanger sequencing of major genes involved in primary bile acid (BA) synthesis failed to identify pathogenic mutations of the investigated set of genes. INTERVENTIONS: Plasma concentrations of BAs, VLCFAs, and beta-oxidation of C22:0, C16:0, and pristanic acid were studied in primary skin fibroblasts of the patient. In addition, we performed sequencing of the ABCD1, ABCD3, CYP7A1, CYP7B1, CYP27A1, HSD3B7, AKR1D1, and SLC27A5 genes in the X-ALD family. OUTCOMES: In the Polish family affected with AMN a dysregulation of the primary BA synthesis pathway was found. LESSONS: We have demonstrated the coincidence of the adult form of X-ALD with abnormalities in BA synthesis. We suggest that decreased synthesis of BAs may be an additional dysfunction as a consequence of the ABCD1 c.659T>C, p.(Leu220Pro) mutation and may be further evidence that disturbed cholesterol metabolism is important in the pathology of ALD.

Genetic testing for monogenic diabetes using targeted next-generation sequencing in patients with maturity-onset diabetes of the young.

INTRODUCTION: Molecular diagnosis of monogenic diabetes mellitus is important for individualized patient care. Next-generation sequencing (NGS) enables a simultaneous analysis of multiple genes in a single test. OBJECTIVES: We aimed to assess the feasibility of using NGS for detecting mutations in a set of known monogenic diabetes gene mutations in a cohort of Polish patients with maturity-onset diabetes of the young (MODY) with earlier negative Sanger sequencing results for HNF1A-MODY or GCK-MODY. PATIENTS AND METHODS: We selected a panel of 28 chromosomal genes in which mutations have been reported to cause monogenic diabetes. The MiSeq platform was used for NGS. An exon-capture assay was designed to include coding regions and splice sites. A total of 54 patients with existing negative Sanger sequencing screening results for HNF1A or GCK gene mutations were selected for the study. RESULTS: NGS results were generated for all 54 patients and 9 positive controls with previously identified HNF1A or GCK gene mutation. All selected positive controls were confirmed by NGS. Among 28 genes, mutations were detected in 16. The type of the analyzed genetic changes was described in the NGS study as high (n = 3) or moderate (n = 76). Among the detected mutations, there were 4 known GCK gene mutations that had been previously missed in Sanger sequencing. So far, Sanger sequencing allowed us to confirm 21 gene mutations detected by NGS, and segregation with diabetes in 14 pedigrees. CONCLUSIONS: Our pilot study using NGS for monogenic diabetes screening in the MODY cohort confirmed that it improves the detection of diabetes-related sequence differences. The screening with NGS should also include diabetic patients for whom Sanger-based screening for particular subtypes of MODY provided negative results.