👤 Radleigh G Santos

During the first days of development the preimplantation embryo is supplied with nutrients from the surrounding milieu. Maternal diabetes mellitus affects the uterine microenvironment, leading to a metabolic adaptation processes in the embryo. We analysed embryonic fatty acid (FA) profiles and expression of processing genes in rabbit blastocysts, separately in embryoblasts (EBs) and trophoblasts (TBs), to determine the potential consequences of maternal diabetes mellitus on intracellular FA metabolism. Insulin-dependent diabetes was induced by alloxan in female rabbits. On Day 6 post coitum, FA profiles in blastocysts (EB, TB and blastocoel fluid) and maternal blood were analysed by gas chromatography. The expression levels of molecules involved in FA elongation (fatty acid elongases, ELOVLs) and desaturation (fatty acid desaturases, FADSs) were measured in EB and TB. Maternal diabetes mellitus influenced the FA profile in maternal plasma and blastocysts. Independent from metabolic changes, rabbit blastocysts contained a higher level of saturated fatty acids (SFAs) and a lower level of polyunsaturated fatty acids (PUFAs) compared to the FA profile of the maternal plasma. Furthermore, the FA profile was altered in the EB and TB, differently. While SFAs (palmitic and stearic acid) were elevated in EB of diabetic rabbits, PUFAs, such as docosahexaenoic acid, were decreased. In contrast, in the TB, lower levels of SFAs and higher levels of oleic acid were observed. EB and TB specific alterations in gene expression were found for ELOVLs and FADSs, key enzymes for FA elongation and desaturation. In conclusion, maternal diabetes mellitus alters embryonic FA metabolism differently in EB and TB, indicating a lineage-specific metabolic adaptive response. Show less

Interleukin-27, a cytokine of the IL-12 family, is secreted by antigen-presenting cells such as macrophages and dendritic cells (DCs). Recent studies suggest an anti-inflammatory role for IL-27 by inducing IL-10 producing Tr1 cells capable of inhibiting Th1 and Th17 type responses. Our study aimed to investigate the involvement of IL-27 and Tr1 cells in the immunomodulation of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Brazil. The presence of IL-27 was evaluated in serum and biopsies of patients with PCM by ELISA, immunohistochemistry, and immunofluorescence. The presence of Tr1 in peripheral blood was analyzed by flow cytometry. In vitro assays were performed to verify the ability of P. brasiliensis yeast to induce IL-27 production by DCs and macrophages, as well as the polarization of lymphocytes to the Tr1 phenotype. Patients with the acute form and severe chronic form, the most severe and disseminated forms of PCM, presented higher serum concentrations of IL-27 and higher percentage of Tr1 cells compared to patients with mild chronic form. IL-27 was also detected in lesions of patients with PCM and associated with DCs and macrophages. P. brasiliensis Pb18 yeasts were able to induce IL-27 production by both DCs and macrophages. We found that DCs pulsed with Pb18 were able to induce Tr1 lymphocytes in vitro. Our data suggest that IL-27 and Tr1 cells could contribute to the deficient immune response to P. brasiliensis that leads to severe and disseminated forms of the disease. Show less

The centrally expressed melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R, respectively) are established targets to treat diseases of positive- and negative-energy homeostasis. We previously reported [ Doering , S. R. ; J. Med. Chem. 2017 , 60 , 4342 - 4357 ] mixture-based positional scanning approaches to identify dual MC3R agonist and MC4R antagonist tetrapeptides. Herein, 46 tetrapeptides were chosen for MC3R agonist screening selectivity profiles, synthesized, and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. Substitutions to the tetrapeptide template were selected solely based on MC3R agonist potency from the mixture-based screen. This study resulted in the discovery of compound 42 (Ac-Val-Gln-(pI)DPhe-DTic-NH Show less

The high occurrence of atherosclerotic cardiovascular disease (ASCVD) events is still a major public health issue. Although a major determinant of ASCVD event reduction is the absolute change of low-density lipoprotein-cholesterol (LDL-C), considerable residual risk remains and new therapeutic options are required, in particular, to address triglyceride-rich lipoproteins and lipoprotein(a) [Lp(a)]. In the era of Genome Wide Association Studies and Mendelian Randomization analyses aimed at increasing the understanding of the pathophysiology of ASCVD, RNA-based therapies may offer more effective treatment options. The advantage of oligonucleotide-based treatments is that drug candidates are targeted at highly specific regions of RNA that code for proteins that in turn regulate lipid and lipoprotein metabolism. For LDL-C lowering, the use of inclisiran - a silencing RNA that inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9) synthesis - has the advantage that a single s.c. injection lowers LDL-C for up to 6 months. In familial hypercholesterolemia, the use of the antisense oligonucleotide (ASO) mipomersen, targeting apolipoprotein (apoB) to reduce LDL-C, has been a valuable therapeutic approach, despite unquestionable safety concerns. The availability of specific ASOs lowering Lp(a) levels will allow rigorous testing of the Lp(a) hypothesis; by dramatically reducing plasma triglyceride levels, Volanesorsen (APOC3) and angiopoietin-like 3 (ANGPTL3)-LRx will further clarify the causality of triglyceride-rich lipoproteins in ASCVD. The rapid progress to date heralds a new dawn in therapeutic lipidology, but outcome, safety and cost-effectiveness studies are required to establish the role of these new agents in clinical practice. Show less

There is a lack of studies about polymorphisms in FADS genes in pregnant women. We aimed to verify the interaction between three FADS gene polymorphisms (rs174561; rs174575; rs3834458) and dietary α-linolenic acid (ALA) or linoleic/α-linolenic acid ratio (LA/ALA) and plasma concentrations of omega-3 (n-3) PUFAs in pregnant women. Of the 250 women evaluated, the homozygous for the rs174561 and rs3834458 minor allele had high plasma ALA concentrations at the highest ALA and LA/ALA ratio tertile (p < 0.05). Plasma concentrations of EPA and DHA were not influenced by diet. For the rs174575 SNP, pregnant women who carried the minor allele presented lower proportions of plasma EPA in the second LA/ALA ratio tertile (p < 0.05). Increased dietary intake of ALA and LA/ALA ratio promoted plasma ALA accumulation in homozygotes for the minor allele rs174561 and rs3834458. Moderate intake of LA/ALA ratio may reduce plasma concentration of EPA in pregnants carrying the rs174575 minor allele. Show less

Fusions transcripts have been proven to be strong drivers for neoplasia-associated mutations, although their incidence in T-cell lymphoblastic lymphoma needs to be determined yet. Using RNA-Seq we have selected 55 fusion transcripts identified by at least two of three detection methods in the same tumour. We confirmed the existence of 24 predicted novel fusions that had not been described in cancer or normal tissues yet, indicating the accuracy of the prediction. Of note, one of them involves the proto oncogene TAL1. Other confirmed fusions could explain the overexpression of driver genes such as COMMD3-BMI1, LMO1 or JAK3. Five fusions found exclusively in tumour samples could be considered pathogenic (NFYG-TAL1, RIC3-TCRBC2, SLC35A3-HIAT1, PICALM MLLT10 and MLLT10-PICALM). However, other fusions detected simultaneously in normal and tumour samples (JAK3-INSL3, KANSL1-ARL17A/B and TFG-ADGRG7) could be germ-line fusions genes involved in tumour-maintaining tasks. Notably, some fusions were confirmed in more tumour samples than predicted, indicating that the detection methods underestimated the real number of existing fusions. Our results highlight the potential of RNA-Seq to identify new cryptic fusions, which could be drivers or tumour-maintaining passenger genes. Such novel findings shed light on the searching for new T-LBL biomarkers in these haematological disorders. Show less

Maternal smoking is a risk factor for progeny obesity. We have previously shown, in a rat model of neonatal tobacco smoke exposure, a mild increase in food intake and a considerable increase in visceral adiposity in the adult offspring. Males also had secondary hyperthyroidism, while females had only higher T4. Since brown adipose tissue (BAT) hypofunction is related to obesity, here we tested the hypothesis that higher levels of thyroid hormones are not functional in BAT, suggesting a lower metabolic rate. We evaluated autonomic nerve activity in BAT and its function in adult rats that were exposed to tobacco smoke during lactation. At birth, litters were adjusted to 3 male and 3 female pups/litter. From postnatal day (PND) 3 to 21, Wistar lactating rats and their pups were divided into SE group, smoke-exposed in a cigarette smoking machine (4 times/day) and C group, exposed to filtered air. Offspring were sacrificed at PND180. Adult SE rats of both genders had lower interscapular BAT autonomic nervous system activity, with higher BAT mass but no change in morphology. BAT UCP1 and CPT1a protein levels were decreased in the SE groups of both genders. Male SE rats had lower β3-AR, TRα1, and TRβ1 expression while females showed lower PGC1α expression. BAT Dio2 mRNA and hypothalamic POMC and MC4R levels were similar between groups. Hypothalamic pAMPK level was higher in SE males and lower in SE females. Thus, neonatal cigarette smoke exposure induces lower BAT thermogenic capacity, which can be obesogenic at adulthood. Show less

Multiple osteochondromas is a dysplasia characterized by growth of two or more osteochondromas. It is genetically heterogeneous, caused by pathogenic variants in EXT1 or EXT2 genes in 70%-90% of patients. The EXT1 is more often mutated than EXT2 gene, with a variable prevalence between populations. There are no data about EXT1 and EXT2 pathogenic variants in patients with multiple osteochondromas in Brazilian population. The aim of this survey is to characterize these to determine the genotype profile of this population. DNA sequencing (Sanger Method) and MLPA analysis were performed to identify point mutations and deletions/duplications in the sample of 153 patients in 114 families. Germline variants were identified in 83% of families in which EXT2 variants were detected in 46% and EXT1 in 37% of cases. No variants were detected in 17% of them. We identified 50 different variants, 33 (13 frameshift, 11 nonsense, 5 missense, 2 splice site mutation, and 2 large deletions) in EXT1 and 17 (6 frameshift, 6 splice site mutation, 3 nonsense, 1 missense, and 1 large deletion) in EXT2. Of all 50 variants, 31 (62%) were novel, including 20 out of 33 (60,6%) EXT1 and 11 out of 17 (64.7%) EXT2 alleles. The vast majority of variants (88%) were "loss-of-function" and two novel hotspots in EXT2 gene were observed in our study. The prevalence of variants detected in the EXT2 gene differs from other researches from Latin America, European, and Asian population. This uncommon prevalence could be related with the newly characterized variant hotspot sites detected in EXT2 gene (p.Ala409Profs*26 and p.Ser290*). A high number of novel variants were also identified indicating that Brazilian population has a unique genetic profile. Characterizing this population and establishing its genotype is essential to understand the molecular pathogenesis of this disease in Brazil. Show less

Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered. We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes. Show less

Galectin-3 (Gal-3) is a β-galactoside binding protein that controls cell-cell and cell-extracellular matrix interactions. In lymphoid organs, gal-3 inhibits B cell differentiation by mechanisms poorly understood. The B cell development is dependent on tissue organization and stromal cell signaling, including IL-7 and Notch pathways. Here, we investigate possible mechanisms that gal-3 interferes during B lymphocyte differentiation in the bone marrow (BM) and spleen. The BM of gal-3-deficient mice (Lgals3 Show less

The role of interleukin (IL-)32 in inflammatory conditions is well-established, however, the mechanism behind its role in atherosclerosis remains unexplained. Our group reported a promoter single nucleotide polymorphism in IL-32 associated with higher high-density lipoprotein (HDL) concentrations. We hypothesize that endogenous IL-32 in liver cells, a human monocytic cell line and carotid plaque tissue, can affect atherosclerosis by regulating (HDL) cholesterol homeostasis via expression of cholesterol transporters/mediators. Human primary liver cells were stimulated with recombinant human (rh)TNFα and poly I:C to study the expression of IL-32 and mediators in cholesterol pathways. Additionally, IL-32 was overexpressed in HepG2 cells and overexpressed and silenced in THP-1 cells to study the direct effect of IL-32 on cholesterol transporters expression and function. Stimulation of human primary liver cells resulted in induction of IL-32α, IL-32β and IL-32γ mRNA expression (p < 0.01). A strong correlation between the expression of IL-32γ and ABCA1, ABCG1, LXRα and apoA1 was observed (p < 0.01), and intracellular lipid concentrations were reduced in the presence of endogenous IL-32 (p < 0.05). Finally, IL32γ and ABCA1 mRNA expression was upregulated in carotid plaque tissue and when IL-32 was silenced in THP-1 cells, mRNA expression of ABCA1 was strongly reduced. Regulation of IL-32 in human primary liver cells, HepG2 and THP-1 cells strongly influences the mRNA expression of ABCA1, ABCG1, LXRα and apoA1 and affects intracellular lipid concentrations in the presence of endogenous IL-32. These data, for the first time, show an important role for IL32 in cholesterol homeostasis. Show less

Some important environmental factors that influence the development of cardiovascular diseases (CVD) include tobacco, excess alcohol, and unhealthy diet. Methionine obtained from the diet participates in the synthesis of DNA, proteins, lipids and affects homocysteine levels, which is associated with the elevated risk for CVD development. Therefore, the aim of this study was to investigate the manner in which dietary methionine might affect cellular mechanisms underlying CVD occurrence. Swiss albino mice were fed either control (0.3% DL-methionine), methionine-supplemented (2% DL-methionine), or a methionine-deprived diet (0% DL-methionine) over a 10-week period. The parameters measured included plasma homocysteine concentrations, oxidative stress by reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, levels of inflammatory cytokines IL-1ß, TNF-α, and IL-6, as well as expression of genes associated with CVD. The levels of apolipoprotein A5 (APOA5), a regulator of plasma triglycerides, were measured. The methionine-supplemented diet increased oxidative stress by lowering the GSH/GSSG ratio in heart tissues and decreased expression of the genes Apob, Ctgf, Serpinb2, Spp1, Il1b, and Sell, but elevated expression of Thbs4, Tgfb2, Ccr1, and Vegfa. Methionine-deprived diet reduced expression of Col3a1, Cdh5, Fabp3, Bax, and Hbegf and increased expression of Sell, Ccl5, Itga2, Birc3, Msr1, Bcl2a1a, Il1r2, and Selp. Methionine-deprived diet exerted pro-inflammatory consequences as evidenced by elevated levels of cytokines IL-1ß, TNF-α, and IL-6 noted in liver. Methionine-supplemented diet increased hepatic IL-6 and cardiac TNF-α. Both methionine supplementation and deprivation lowered hepatic levels of APOA5. In conclusion, data demonstrated that a methionine-supplemented diet modulated important biological processes associated with high risk of CVD development. Show less

Bardet-Biedl syndrome is a model ciliopathy. Although the characterization of BBS proteins has evidenced their involvement in cilia, extraciliary functions for some of these proteins are also being recognized. Importantly, understanding both cilia and cilia-independent functions of the BBS proteins is key to fully dissect the cellular basis of the syndrome. Here we characterize a functional interaction between BBS4 and the secreted protein FSTL1, a protein linked to adipogenesis and inflammation among other functions. We show that BBS4 and cilia regulate FSTL1 mRNA levels, but BBS4 also modulates FSTL1 secretion. Moreover, we show that FSTL1 is a novel regulator of ciliogenesis thus underscoring a regulatory loop between FSTL1 and cilia. Finally, our data indicate that BBS4, cilia and FSTL1 are coordinated during the differentiation of 3T3-L1 cells and that FSTL1 plays a role in this process, at least in part, by modulating ciliogenesis. Therefore, our findings are relevant to fully understand the development of BBS-associated phenotypes such as obesity. Show less

Studies associate specific fatty-acids (FA) with the pathophysiology of inflammation. We aimed to analyze the impact of exercise on adipose tissue FA profile in response to a high-fat diet (HFD) and to ascertain whether these exercise-induced changes in specific FA have repercussions on obesity-related inflammation. Sprague-Dawley rats were assigned into sedentary, voluntary physical-activity (VPA) and endurance training (ET) groups fed a standard (S, 35kcal% fat) or high-fat (71kcal% fat) diets. VPA-animals had unrestricted access to wheel-running. After 9-wks, ET-animals engaged a running protocol for 8-wks, while maintained dietary treatments. The FA content in epididymal white-adipose tissue (eWAT) triglycerides was analyzed by gas-chromatography and the expression of inflammatory markers was determined using RT-qPCR, Western and slot blotting. Eight-wks of ET reversed obesity-related anatomical features. HFD increased plasma tumor necrosis factor (TNF)-α content and eWAT monocyte chemoattractant protein (MCP)-1 protein expression. HFD decreased eWAT content of saturated FA and monounsaturated FA, while increased linoleic acid and prostaglandin E2 (PGE2) levels in eWAT. VPA decreased visceral adiposity, adipocyte size and MCP-1 in HFD-fed animals. The VPA and ET interventions diminished palmitoleic acid and increased linoleic acid in HFD-fed groups. Moreover, both interventions increased PGE2 levels in standard diet-fed groups and decreased in HFD. ET increased eWAT fatty acid desaturase 1 (FADS1) and elongase 5 (ELOVL5) protein content in both diet types. ET reduced eWAT inflammatory markers (TNF-α, IL-6), macrophage recruitment (MCP-1 and F4/80) and increased IL-10/TNF-α ratio in plasma and in eWAT in both diet types. Exercise induced FA-specific changes independently of dietary FA composition, but only ET attenuated the inflammatory response in VAT of HFD-fed rats. Moreover, the exercise-induced FA changes did not correlate with the inflammatory response in VAT of rats submitted to HFD. Show less

Despite increasing research in type 2 diabetes mellitus (T2D), there are few studies showing the impact of the poor glycemic control on biological processes occurring in T2D. In order to identify potential genes related to poorly/well-controlled patients with T2D, our strategy of investigation included a primary screen by microarray (Human Genome U133) in a small group of individuals followed by an independent validation in a greater group using RT-qPCR. Ninety patients were divided as follows: poorly controlled T2D (G1), well-controlled T2D (G2), and normoglycemic individuals (G3). After using affy package in R, differentially expressed genes (DEGs) were prospected as candidate genes potentially relevant for the glycemic control in T2D patients. After validation by RT-qPCR, the obtained DEGs were as follows-G1 + G2 versus G3: Show less

Diabesity and fatty liver have been associated with low levels of high-density lipoprotein cholesterol, and thus could impair macrophage-specific reverse cholesterol transport (m-RCT). Liver X receptor (LXR) plays a critical role in m-RCT. Abcg5/g8 sterol transporters, which are involved in cholesterol trafficking into bile, as well as other LXR targets, could be compromised in the livers of obese individuals. We aimed to determine m-RCT dynamics in a mouse model of diabesity, the db/db mice. These obese mice displayed a significant retention of macrophage-derived cholesterol in the liver and reduced fecal cholesterol elimination compared with nonobese mice. This was associated with a significant downregulation of the hepatic LXR targets, including Abcg5/g8. Pharmacologic induction of LXR promoted the delivery of total tracer output into feces in db/db mice, partly due to increased liver and small intestine Abcg5/Abcg8 gene expression. Notably, a favorable upregulation of the hepatic levels of ABCG5/G8 and NR1H3 was also observed postoperatively in morbidly obese patients, suggesting a similar LXR impairment in these patients. In conclusion, our data show that downregulation of the LXR axis impairs cholesterol transfer from macrophages to feces in db/db mice, whereas the induction of the LXR axis partly restores impaired m-RCT by elevating the liver and small intestine expressions of Abcg5/g8. Show less

The sterol-responsive nuclear receptors, liver X receptors α (LXRα, We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease-exonuclease-phosphatase family domain containing 1 ( In this study, we identify Show less

Berardinelli-Seip syndrome (BSS) is a recessive autosomal genetic disorder characterized by the near loss of adipose tissue with disturbance in lipid metabolism. Biochemical and hormonal parameters and Pro12Ala, Pvull, Avall, Sstl and ADIPOQ polymorphisms in 22 patients with BSS were analyzed and examined for a possible association with lipid profiles. Parental consanguinity, insulin resistance and diabetes mellitus were observed in 63.6, 81.8 and 59.1% of patients, respectively. All individuals presented high triglyceride levels, and 68.1% of patients showed high cholesterol levels. The Pro/Pro genotype of the Pro12Ala polymorphism of the PPARγ2 gene was found in 86.3% of patients; the Ala/Ala variant was not observed in any patient. The PvuII polymorphism of the LPL gene showed a frequency of 50% for the P1P2 variant. The AvaII polymorphism of the LDLR gene showed a similar frequency of 40.9% for both CT and TT variants. The S1S1 genotype of the Sstl polymorphism of the APOC3 gene had a frequency of 86.3%. The CC allele of the ADIPOQ polymorphism of the adiponectin gene was found in 54.6% of patients. No association was found between lipid parameters and the relevant Pvull, Avall and Sstl polymorphisms. However, we did observe an association of the Pro12Ala and ADIPOQ polymorphisms with higher lipid levels, suggesting a close relationship between these factors. Show less

The aim of this study was to evaluate the proteic changes in high-density lipoproteins (HDL) induced by methionine-induced hyperhomocysteinemia in mice and its relationship with two of their main antiatherogenic properties. The oral administration of methionine resulted in an elevation (~8 times) in the plasma concentration of homocysteine. Hyperhomocysteinemia was inversely correlated with the plasma concentration of HDL cholesterol and its main protein component of HDL, apolipoprotein (apo) A-I, respectively. The cholesterol efflux in vivo from macrophages to HDL was decreased in hyperhomocysteinemic mice compared with the control mice. However, the reverse cholesterol transport from macrophages to feces remained unchanged. On the other hand, the ability of HDL from hyperhomocysteinemic mice to prevent the oxidative modification of low-density lipoproteins (LDL) was found decreased and associated with a concomitant reduction in the plasma activity of paraoxonase-1 (PON1) and the plasma concentration of apoA-I, and with a relative reduction in the apoA-IV content (~1.5 times) in the hyperhomocysteinemic HDL, respectively. The decrease in the ability of HDL from hyperhomocysteinemic mice to prevent LDL from oxidation was associated with a decrease in the apoA-I, PON1 and apoA-IV. Show less

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. 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. 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). 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. Show less

HSD17B12 is a member of the hydroxysteroid dehydrogenase superfamily, a multifunctional group of enzymes involved in the metabolism of steroids, retinoids, bile and fatty acids. Whether the main role of HSD17B12 in mammals is in steroid or fatty acid metabolism is a subject of intense debate. In mollusks it has been shown that an HSD17B12 orthologue can convert estrone into estradiol in vitro, although its primary in vivo function remains unknown. To gain insight into its role in gastropods, we provide here the first cloning of Hsd17b12 in Nucella lapillus and its detailed tissue distribution through quantitative PCR. Furthermore, given that the endocrine disruptor tributyltin (TBT) has been reported to unbalance steroid and lipid levels in gastropods, we tested its impact in on NlHsd17b12 transcript expression. Our results show that NlHsd17b12 is ubiquitously expressed in all tissues analyzed, with higher levels in organs with high metabolic rates, such as kidney and digestive gland, a pattern consistent with an involvement in lipid metabolism. Exposure to TBT chloride at 100 ng Sn/L caused a decrease in NlHsd17b12 mRNA levels in digestive gland, after one and two months, while no effect was observed in gonads. Overall, these results suggest that in mollusks, as in mammals, this enzyme is likely to be involved in lipid metabolism, and emphasize the need to perform more detailed studies on its in vivo function, in order to understand its physiological role and the biological impact of its disruption by pollutants such as TBT. Show less

Severe hypertriglyceridemia (HTG) has been linked to defects in LPL, APOC2, APOA5, LMF1 and GBIHBP1 genes. However, a number of severe HTG cases are probably caused by as yet unidentified mutations. Very high triglyceride plasma levels (>112 mmol/L at diagnosis) were found in two sisters of a Chilean consanguineous family, which is strongly suggestive of a recessive highly penetrant mutation. The aim of this study was to determine the genetic locus responsible for the severe HTG in this family. We carried out a genome-wide linkage study with nearly 300,000 biallelic markers (Illumina Human CytoSNP-12 panel). Using the homozygosity mapping strategy, we searched for chromosome regions with excess of homozygous genotypes in the affected cases compared to non-affected relatives. A large homozygous segment was found in the long arm of chromosome 11, with more than 2,500 consecutive homozygous SNP shared by the proband with her affected sister, and containing the APOA5/A4/C3/A1 cluster. Direct sequencing of the APOA5 gene revealed a known homozygous nonsense Q97X mutation (p.Gln97Ter) found in both affected sisters but not in non-affected relatives nor in a sample of unrelated controls. The Q97X mutation of the APOA5 gene in homozygous status is responsible for the severe hypertriglyceridemia in this family. We have shown that homozygosity mapping correctly pinpointed the genomic region containing the gene responsible for severe hypertriglyceridemia in this consanguineous Chilean family. Show less

Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant. Show less

Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5'UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations. Show less

Langer-Giedion syndrome (LGS) (OMIM 150230) is defined as a contiguous gene syndrome caused by loss of functional copies of the TRPS1 and EXT1 genes usually secondary to 8q microdeletion. Tibial hemimelia (TH) is the least common lower limb deficiency characterized by hypoplasia of the tibia with relatively intact fibula. We describe the third report of LGS with bilateral TH and an 8q23.1-q24.12 interstitial deletion. It is not possible to exclude that this association is fortuitous, but our report reinforces the suggestion of a putative gene involved in limb development in this chromosomal region interval. Show less

Left ventricular noncompaction (LVNC) is a rare and potentially progressive cardiomyopathy, characterized by the persistence of multiple trabeculations and deep intratrabecular recesses in the ventricular myocardium. Although two-dimensional and color Doppler echocardiography are the most useful diagnostic modalities, cardiac magnetic resonance imaging has proved to have high sensitivity and specificity in the diagnosis of this anomaly. To characterize the clinical and imaging features of LVNC in a pediatric population and to assess their evolution. We performed a retrospective chart review of five pediatric patients with LVNC, followed at Coimbra Pediatric Hospital between January 1999 and December 2007. Median age at presentation was five months (ranging from one day to 13 years), and they were mainly male (1.5:1). Two of the children had a family history of sudden death. In one case the clinical presentation was cardiac arrest due to ventricular fibrillation and in three others, congestive cardiac failure. None of the five cases had associated congenital cardiac anomalies. Involvement of the ventricular apical region was found in all cases. Four children additionally had ventricular dysfunction which improved with diuretic and vasodilator therapy. Mean follow-up was 34 months, ranging from six months to seven years. In one case a change in the morphological phenotype was noted, from a dilated to a hypertrophic form. In this case and in the child's father a mutation in the MYBPC3 gene was identified, which is associated with hypertrophic cardiomyopathy. No thromboembolic phenomena or deaths occurred during the study period. In the pediatric population, congestive cardiac failure is the most common clinical presentation of LVNC, which can coexist with other cardiomyopathies, particularly dilated and hypertrophic forms. The sample presented in this analysis is statistically non-significant due to its limited size and the authors highlight the need for larger prospective studies in the pediatric population in order to clarify this disease and its diagnostic criteria. Show less

Hypertrophic cardiomyopathy (HCM), a complex myocardial disorder with an autosomal dominant genetic pattern and prevalence of 1:500, is the most frequent cause of sudden death in apparently healthy young people. The benefits of gene-based diagnosis of HCME for both basic research and clinical medicine are limited by the considerable costs of current genetic testing due to the large number of genes and mutations involved in this pathology. However, coupling two high-throughput techniques--mass spectrometry genotyping (MSG) and high resolution melting (HRM)--is an encouraging new strategy for HCM diagnosis. Our aim was to evaluate the diagnostic efficacy of both techniques in this pathology by studying 13 individuals with a clinical phenotype of HCM. Peripheral blood samples were collected from: (i) seven subjects with a clinical diagnosis of HCM, all bearing known mutations previously identified by dideoxy sequencing and thus being used as blinded samples (sample type 1); (ii) one individual with a clinical diagnosis of HCM negative for mutations after dideoxy sequencing of the five most common HCM genes, MYH7, MYBPC3, TNNI3, TNNT2 and MYL2 (sample type 2); and (iii) five individuals individual with a clinical diagnosis of HCM who had not previously been genetically studied (sample type 3). The 13 samples were analyzed by MSG for 534 known mutations in 32 genes associated with HCM phenotypes and for all coding regions and exon-intron boundaries of the same HCM genes by HRM. The 32 studied genes include the most frequent HCM-associated sarcomere genes, as well as 27 genes with lower reported HCM phenotype association. This coupled genotyping strategy enabled us to identify a c.128delC (p.A43Vfs165) frame-shift mutation in the CSRP3 gene, a gene not usually studied in current HCM genetics. The heterozygous CSRP3 mutation was found in two patients (sample types 2 and 3) aged 50 and 52 years, respectively, both with diffuse left ventricular hypertrophy. Furthermore, this coupled strategy enabled us to find a novel mutation, c.817C >T (p.Arg273Cys), in MYBPC3 in an individual from sample type 3, subsequently confirmed by dideoxy sequencing. This novel mutation in MYBPC3, not present in 200 chromosomes from 200 healthy individuals, affects a codon known to harbor an HCM-causing mutation--p.Arg253His. In conclusion, in the cohort used in this work coupling two technologies, MSG and HRM, with high sensitivity and low false positive results, enabled rapid, innovative and low-cost genotyping of HCM patients, which may in the short-term be suitable for accurate genetic diagnosis of HCM. Show less

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). PGC-1α enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1α levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1α-mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1α and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1α enhances lipogenesis in skeletal muscle through liver X receptor α-dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1α and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1α coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo. Show less

Gómez-López-Hernández (GLH) syndrome or cerebello-trigeminal dysplasia is a neurocutaneous syndrome whose etiology is unknown at the present time. We report two additional Brazilian patients, including the oldest one known to date (age 29). Here, we review the expanded phenotype in four patients with new clinical, psychiatric, radiological, and molecular investigations. One patient may have hypomania within the bipolar spectrum disorder with onset in childhood and adolescence. Primary growth hormone (GH) deficiency was ruled out in all patients, although one of them might have developed secondary GH deficiency due to partial hypopituitarism following severe hydrocephalus. Brain magnetic resonance angiography disclosed no azygous anterior cerebral artery (ACA) but only normal variants. Molecular analysis of the lysosomal acid phosphatase gene (ACP2) was performed, but no pathogenic mutations were identified. We present an overview of the phenotypic features of all patients described to date. There are currently 12 unrelated patients reported in the literature, 5 of whom are Brazilian. We discuss new molecular insights and speculate about the pathogenesis of GLH syndrome. Show less