Hoxa5 undergoes dynamic DNA methylation and transcriptional repression in the adipose tissue of mice exposed to high-fat diet.

BACKGROUND/OBJECTIVES: The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes. SUBJECTS/METHODS: We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox (Hox) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed. RESULTS: Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance (P<0.001) and insulin sensitivity (P<0.05). MeDIP-seq data analysis revealed a uniform distribution of differentially methylated regions (DMR) through the entire adipocyte genome, with a higher number of hypermethylated regions in HFD mice (P<0.005). This different methylation profile was accompanied by increased expression of the Dnmt3a DNA methyltransferase (Dnmt; P<0.05) and the methyl-CpG-binding domain protein Mbd3 (P<0.05) genes in HFD mice. Gene ontology analysis revealed that, in the HFD-treated mice, the Hox family of development genes was highly enriched in differentially methylated genes (P=0.008). To validate this finding, Hoxa5, which is implicated in fat tissue differentiation and remodeling, has been selected and analyzed by bisulphite sequencing, confirming hypermethylation in the adipose tissue from the HFD mice. Hoxa5 hypermethylation was associated with downregulation of Hoxa5 mRNA and protein expression. Feeding animals previously exposed to the HFD with a standard chow diet for two further months improved the metabolic phenotype of the animals, accompanied by return of Hoxa5 methylation and expression levels (P<0.05) to values similar to those of the control mice maintained under standard chow. CONCLUSIONS: HFD induces adipose tissue abnormalities accompanied by epigenetic changes at the Hoxa5 adipose tissue remodeling gene.

Epigenetics: spotlight on type 2 diabetes and obesity.

Type 2 diabetes (T2D) and obesity are the major public health problems. Substantial efforts have been made to define loci and variants contributing to the individual risk of these disorders. However, the overall risk explained by genetic variation is very modest. Epigenetics is one of the fastest growing research areas in biomedicine as changes in the epigenome are involved in many biological processes, impact on the risk for several complex diseases including diabetes and may explain susceptibility. In this review, we focus on the role of DNA methylation in contributing to the risk of T2D and obesity.

Difference between apnea-hypopnea index (AHI) and oxygen desaturation index (ODI): proportional increase associated with degree of obesity.

PURPOSE: Obesity is one of the main predisposing factors for obstructive sleep apnea (OSA) hypopnea syndrome. It has been described that body mass index (BMI) influences the accuracy of oxygen desaturation index (ODI) for the diagnosis of OSA by polysomnography (PSG). We analyzed the relationship between traditional indicators: apnea-hypopnea index (AHI) and ODI in a population at high risk for OSA, by respiratory polygraphy (RP) and PSG. METHODS: This is a retrospective study of 1898 patients with suspicion of OSA, from which 1053 underwent RP and 582 underwent PSG with OSA. We compared results considering gender, age, and degree of obesity. RESULTS: This study included 1333 records of patients with OSA-more than 80 % of whom were overweight or obese. We observed that AHI and ODI increased progressively with obesity grade and said increase was associated with BMI only in men. The evaluation of the agreement between AHI and ODI found a difference between normal weight and obese patients, regardless of gender. CONCLUSIONS: Study findings contribute to understand the role of oximetry in the diagnosis of OSA in obese patients. Our results were observed using full PSG and a simplified home method. The correlation between these indicators could improve our clinical interpretation of OSA severity among obese patients when abbreviated tests are used.

Identification of Endoglin as an epigenetically regulated tumour-suppressor gene in lung cancer.

BACKGROUND: The transforming growth factor-beta (TGF- ß) pathway has been implicated in proliferation, migration and invasion of various cancers. Endoglin is a TGF-ß accessory receptor that modulates signalling. We identified Endoglin as an epigenetically silenced tumour-suppressor gene in lung cancer by means of a genome-wide screening approach, then sought to characterise its effect on lung cancer progression. METHODS: Methylation microarray and RNA sequencing were carried out on lung cancer cell lines. Epigenetic silencing of Endoglin was confirmed by methylation and expression analyses. An expression vector and a 20-gene expression panel were used to evaluate Endoglin function. Pyrosequencing was carried out on two independent cohorts comprising 112 and 202 NSCLC cases, respectively, and the impact of Endoglin methylation on overall survival (OS) was evaluated. RESULTS: Methylation in the promoter region resulted in silencing of Endoglin, which could be reactivated by demethylation. Increased invasion coupled with altered EMT marker expression was observed in cell lines with an epithelial-like, but not those with a mesenchymal-like, profile when Endoglin was absent. Methylation was associated with decreased OS in stage I but not in stages II-III disease. CONCLUSIONS: We show that Endoglin is a common target of epigenetic silencing in lung cancer. We reveal a link between Endoglin silencing and EMT progression that might be associated with decreased survival in stage I disease.

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Increased hexosamine biosynthetic pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway.

AIMS/HYPOTHESIS: Beta cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (decline of glucose-stimulated insulin secretion, downregulation of specific gene expression). Apoptosis and dysfunction are caused, at least in part, by lipoglucotoxicity. The mechanisms implicated are oxidative stress, increase in the hexosamine biosynthetic pathway (HBP) flux and endoplasmic reticulum (ER) stress. Oxidative stress plays a role in glucotoxicity-induced beta cell dedifferentiation, while glucotoxicity-induced ER stress has been mostly linked to beta cell apoptosis. We sought to clarify whether ER stress caused by increased HBP flux participates in a dedifferentiating response of beta cells, in the absence of relevant apoptosis. METHODS: We used INS-1E cells and murine islets. We analysed the unfolded protein response and the expression profile of beta cells by real-time RT-PCR and western blot. The signal transmission pathway elicited by ER stress was investigated by real-time RT-PCR and immunofluorescence. RESULTS: Glucosamine and high glucose induced ER stress, but did not decrease cell viability in INS-1E cells. ER stress caused dedifferentiation of beta cells, as shown by downregulation of beta cell markers and of the transcription factor, pancreatic and duodenal homeobox 1. Glucose-stimulated insulin secretion was inhibited. These effects were prevented by the chemical chaperone, 4-phenyl butyric acid. The extracellular signal-regulated kinase (ERK) signal transmission pathway was implicated, since its inhibition prevented the effects induced by glucosamine and high glucose. CONCLUSIONS/INTERPRETATION: Glucotoxic ER stress dedifferentiates beta cells, in the absence of apoptosis, through a transcriptional response. These effects are mediated by the activation of ERK1/2.

Methylation of the calcium channel regulatory subunit α2δ-3 (CACNA2D3) predicts site-specific relapse in oestrogen receptor-positive primary breast carcinomas.

BACKGROUND: Calcium is an important intracellular messenger that mediates many biological processes that are relevant to the malignant process. Calcium ion channels are key in controlling the intracellular calcium, and little is known about their role in human cancer. METHODS: We used qPCR and pyrosequencing to investigate expression and epigenetic regulation of the calcium channel regulatory subunit α(2)δ-3 (CACNA2D3) in breast cancer cell lines, primary cancers and metastatic lesions. RESULTS: Expression of CACNA2D3 mRNA is regulated in breast cancer cell lines by methylation in the CpG island located in the 5' regulatory region of the gene. Expression is upregulated by azacytidine (AZA) in cells with CpG island methylation but unaffected in cells lacking methylation. In primary breast carcinomas, methylation is more common in cancers, which subsequently relapse with loco-regional and, particularly, visceral metastatic disease in both oestrogen receptor-α (ER)-positive and -negative cases. Furthermore, CACNA2D3 CpG island is frequently methylated in breast cancer that has metastasised to the central nervous system. CONCLUSION: Methylation-dependent transcriptional silencing of CACNA2D3 may contribute to the metastatic phenotype of breast cancer. Analysis of methylation in the CACNA2D3 CpG island may have potential as a biomarker for risk of development of metastatic disease.

Site-specific CpG methylation in the CCAAT/enhancer binding protein delta (CEBPδ) CpG island in breast cancer is associated with metastatic relapse.

BACKGROUND: The CCAAT/enhancer binding protein delta (CEBPδ) is a member of a highly conserved family of basic region leucine zipper transcription factors. It has properties consistent with a tumour suppressor; however, other data suggest that CEBPδ may be involved in the metastatic process. METHODS: We analysed the expression of CEBPδ and the methylation status of the CpG island in human breast cancer cell lines, in 107 archival cases of primary breast cancer and in two series of metastatic breast cancers using qPCR and pyrosequencing. RESULTS: Expression of CEBPδ is downregulated in primary breast cancer by site-specific methylation in the CEBPδ CpG island. Expression is also downregulated in 50% of cases during progression from primary carcinoma to metastatic lesions. The CEBPδ CpG island is methylated in 81% metastatic breast cancer lesions, while methylation in the CEBPδ CpG island in primary cancers is associated with increased risk of relapse and metastasis. CONCLUSION: CCAAT/enhancer binding protein delta CpG island methylation is associated with metastasis in breast cancer. Detection of methylated CEBPδ genomic DNA may have utility as an epigenetic biomarker of primary breast carcinomas at increased risk of relapse and metastasis.

High frequency of complex TP53 mutations in CNS metastases from breast cancer.

BACKGROUND: Brain metastasis from breast cancer is usually associated with a poor prognosis and early death. Alteration of p53 may contribute to malignant progression by abrogation of apoptosis induced by oncogene activation and by acquisition of gain-of-function properties, which promote tumour aggression. Mutation in TP53 occurs at high frequency in carcinomas of the lung and gastro-intestinal tract, but is much less frequent, at 25%, in primary breast cancer. The frequency of TP53 alteration in the central nervous system (CNS) metastatic breast cancer is not known. METHODS: In all, 23 cases of histologically confirmed CNS metastatic breast cancer were identified and the coding sequence of TP53 determined. TP53 was also sequenced in two control series of primary breast carcinomas from independent clinical centres. RESULTS: We demonstrate a strikingly high frequency of TP53 mutation in the CNS metastatic lesions with an over-representation of complex mutations (non-sense/deletions/insertions). Complex mutations occur in metastatic lesions in both triple-negative breast cancer and hormone receptor/HER2-positive cases. Analysis of paired primary carcinomas and brain metastatic lesions revealed evidence for both clonal selection and generation of new mutations (missense and complex) in progression from a primary breast carcinoma to brain metastasis. CONCLUSION: Mutation in TP53 is the most common genetic alteration reported during metastasis to the brain in breast cancer.