👤 Yigal M Pinto

Identifying genes or genomic regions influencing carcass-quality traits such as fatness (FTN) is essential to optimize the genetic selection processes in beef cattle. The aim of this study was to identify genomic regions associated with FTN in Nellore cattle as well as to elucidate the metabolic pathways related to the phenotypic expression. Ultrasound-based measurements of FTN were collected in 11 750 animals, with 39 903 animals in the pedigree file. Additionally, 1440 animals were genotyped using the GGP-indicus 35K SNP panel, which contained 33 623 SNPs after quality control. Twenty genes related to FTN were found on 11 chromosomes, explaining 12.96% of the total additive genetic variance. Gene ontology revealed seven genes: NR1L2, PKD2, GSK3β, EXT1, RAD51B, SORCS1 and DPH6, associated with important processes related to FTN. In addition, novel candidate genes (MAATS1, LYPD1, CDK5RAP2, RAD51B, c13H2Oorf96 and TRAPPC11) were detected and could provide further knowledge to uncover genetic regions associated to carcass fatness in beef cattle. Show less

Genome-wide association study (GWAS) is a powerful tool to identify candidate genes and genomic regions underlying key biological mechanisms associated with economically important traits. In this context, the aim of this study was to identify genomic regions and metabolic pathways associated with backfat thickness (BFT) and rump fat thickness (RFT) in Nellore cattle, raised in pasture-based systems. Ultrasound-based measurements of BFT and RFT (adjusted to 18 months of age) were collected in 11,750 animals, with 39,903 animals in the pedigree file. Additionally, 1,440 animals were genotyped using the GGP-indicus 35K SNP chip, containing 33,623 SNPs after the quality control. The single-step GWAS analyses were performed using the BLUPF90 family programs. Candidate genes were identified through the Ensembl database incorporated in the BioMart tool, while PANTHER and REVIGO were used to identify the key metabolic pathways and gene networks. A total of 18 genomic regions located on 10 different chromosomes and harbouring 23 candidate genes were identified for BFT. For RFT, 22 genomic regions were found on 14 chromosomes, with a total of 29 candidate genes identified. The results of the pathway analyses showed important genes for BFT, including TBL1XR1, AHCYL2, SLC4A7, AADAT, VPS53, IDH2 and ETS1, which are involved in lipid metabolism, synthesis of cellular amino acids, transport of solutes, transport between Golgi Complex membranes, cell differentiation and cellular development. The main genes identified for RFT were GSK3β, LRP1B, EXT1, GRB2, SORCS1 and SLMAP, which are involved in metabolic pathways such as glycogen synthesis, lipid transport and homeostasis, polysaccharide and carbohydrate metabolism. Polymorphisms located in these candidate genes can be incorporated in commercial genotyping platforms to improve the accuracy of imputation and genomic evaluations for carcass fatness. In addition to uncovering biological mechanisms associated with carcass quality, the key gene pathways identified can also be incorporated in biology-driven genomic prediction methods. Show less

Diabetic kidney disease (DKD) is associated with lipid derangements that worsen kidney function and enhance cardiovascular (CVD) risk. The management of dyslipidemia, hypertension and other traditional risk factors does not completely prevent CVD complications, bringing up the participation of nontraditional risk factors such as advanced glycation end products (AGEs), carbamoylation and changes in the HDL proteome and functionality. The HDL composition, proteome, chemical modification and functionality were analyzed in nondialysis subjects with DKD categorized according to the estimated glomerular filtration rate (eGFR) and urinary albumin excretion rate (AER). Individuals with DKD were divided into eGFR> 60 mL/min/1.73 m Targeted proteomic analyses quantified 28 proteins associated with HDL in all groups, although only 2 were more highly expressed in the eGFR< 60 + A3 group than in the controls: apolipoprotein D (apoD) and apoA-IV. HDL from the eGFR< 60 + A3 group presented higher levels of total AGEs (20%), pentosidine (6.3%) and carbamoylation (4.2 x) and a reduced ability to remove The increase in apoD and apoA-IV could contribute to counteracting the HDL chemical modification by AGEs and carbamoylation, which contributes to HDL loss of function in well-established DKD. Show less

We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 ( Show less

Amyloidosis is a group of disorders characterised by the accumulation of extracellular deposits of insoluble protein aggregates. Clinical management depends on the accurate identification of the amyloid precursor and underlying cause. We describe a rare case of apolipoprotein A-IV cardiac amyloidosis, the diagnosis of which required mass spectrometry-based proteomic analysis. To be able to perform the differential diagnosis of cardiac amyloidosis subtypes using imaging methods, analytical results and tissue analysis.To recognise the added value of mass spectrometry (MS)-based proteomic analysis. Show less

Metabolic syndrome (MS) is a condition that, when associated with ischemic heart disease and cardiovascular events, can be influenced by genetic variants and determine more severe coronary atherosclerosis. To examine the contribution of genetic polymorphisms to the extension and severity of coronary disease in subjects with MS and recent acute coronary syndrome (ACS). Patients (n = 116, 68% males) aged 56 (9) years, with criteria for MS, were prospectively enrolled to the study during the hospitalization period after an ACS. Clinical and laboratory parameters, high-sensitivity C-reactive protein, thiobarbituric acid reactive substances, adiponectin, endothelial function, and the Gensini score were assessed. Polymorphisms of paraoxonase-1 (PON-1), methylenotetrahydrofolate reductase (MTHFR), endothelial nitric oxide synthase (ENOS), angiotensin-converting enzyme (ACE), angiotensin II type 1 receptor (AT1R), apolipoprotein C3 (APOC3), lipoprotein lipase (LPL) were analysed by polymerase chain reaction (PCR) technique, followed by the identification of restriction fragment length polymorphisms (RFLP, and a genetic score was calculated. Parametric and non-parametric tests were used, as appropriate. Significance was set at p < 0.05. Polymorphisms of PON-1, MTHFR and ENOS were not in the Hardy-Weinberg equilibrium. The DD genotype of LPL was associated with higher severity and greater extension of coronary lesions. Genetic score tended to be higher in patients with Gensini score < P50 (13.7 ± 1.5 vs. 13.0 ± 1.6, p = 0.066), with an inverse correlation between genetic and Gensini scores (R = -0.194, p = 0.078). The LPL polymorphism contributed to the severity of coronary disease in patients with MS and recent ACS. Combined polymorphisms were associated with the extension of coronary disease, and the lower the genetic score the more severe the disease. Show less

Aerobic exercise training (AET) improves the reverse cholesterol transport (RCT) in cholesteryl ester transfer protein-transgenic (CETP-tg) mice. We aimed at investigating the role of AET in the expression of genes and proteins involved in lipid flux in the aorta and macrophages of CETP-tg mice. Three-month-old male mice were randomly divided into trained (T; treadmill 15 m/min; 30 min/day) and sedentary (S) groups. After 6 weeks, peritoneal macrophages and the aortic arch were obtained immediately (0 h) or 48 h after the last exercise session. mRNA was determined by RT-qPCR, protein levels by immunoblot and Show less

Koolen de Vries syndrome (KDVS; MIM 610443) is a genomic disorder caused by a recurrent microdeletion derived from nonallelic homologous recombination mediated by flanking segmental duplications. Clinical manifestations of this syndrome are characterized by intellectual disability, hypotonia, a friendly behavior, distinctive facial features, and epilepsy. Herein, we report a case of 2 girls who revealed global developmental delay, mild facial dysmorphisms, friendly behavior, and epileptic seizure with a de novo 17q21.31 microdeletion detected by chromosomal microarray analysis (CMA). Conventional cytogenetics analysis by GTG-banding showed a female karyotype 46,XX for both girls. CMA revealed a microdeletion spanning approximately 500 kb in 17q21.31 in both girls, encompassing the following genes: Show less

The influence of genotype (lean v. fatty) and dietary protein level (normal v. reduced) on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid-sensitive factors is reported for the first time, using the pig as an experimental model. The experiment was conducted on forty entire male pigs (twenty lean pigs of Large White×Landrace×Pietrain cross-breed and twenty fatty pigs of Alentejana purebreed) from 60 to 93 kg of live weight. Each pig genotype was divided into two subgroups, which were fed the following diets: a normal protein diet (NPD) equilibrated for lysine (17·5 % crude protein and 0·7 % lysine) and a reduced protein diet (RPD) not equilibrated for lysine (13·1 % crude protein and 0·4 % lysine). The majority of plasma metabolites were affected by genotype, with lean pigs having higher contents of lipids, whereas fatty pigs presented higher insulin, leptin and urea levels. RPD increased plasma TAG, free fatty acids and VLDL-cholesterol compared with NPD. Hepatic total lipids were higher in fatty pigs than in the lean genotype. RPD affected hepatic fatty acid composition but had a slight influence on gene expression levels in the liver. Sterol regulatory element-binding factor 1 was down-regulated by RPD, and fatty acid desaturase 1 (FADS1) and fatty acid binding protein 4 (FABP4) were affected by the interaction between genotype and diet. In pigs fed RPD, FADS1 was up-regulated in the lean genotype, whereas FABP4 increased in the fatty genotype. Although there is a genotype-specific effect of dietary protein restriction on hepatic lipid metabolism, lipogenesis is not promoted in the liver of lean or fatty pigs. Show less

In ruminants, unsaturated dietary fatty acids are biohydrogenated in the rumen and are further metabolised in various tissues, including liver, which has an important role in lipid and lipoprotein metabolism. Therefore, manipulation of muscle fatty acid composition should take into account liver metabolism. In the present study, the influence of breed and diet on liver lipid composition and gene expression was investigated in order to clarify the role of this organ in the lipid metabolism of ruminants. Forty purebred young bulls from two phylogenetically distant autochthonous cattle breeds, Alentejana and Barrosã, were assigned to two different diets (low vs. high silage) and slaughtered at 18 months of age. Liver fatty acid composition, mRNA levels of enzymes and transcription factors involved in lipid metabolism, as well as the plasma lipid profile, were assessed. In spite of similar plasma non-esterified fatty acids levels, liver triacylglycerols content was higher in Barrosã than in Alentejana bulls. Moreover, the fatty acid composition of liver was clearly distinct from the remaining tissues involved in fatty acid metabolism of ruminants, as shown by Principal Components Analysis. The hepatic tissue is particularly rich in α-linolenic acid and their products of desaturation and elongation. Results indicate that DGAT1, ELOVL2, FADS1 and FADS2 genes influence the fatty acid composition of the liver the most. Moreover, genes such as DGAT1 and ELOVL2 appear to be more sensitive to genetic background than to dietary manipulation, whereas genes encoding for desaturases, such as FADS1, appear to be modulated by dietary silage level. Our results indicate that liver plays an important role in the biosynthesis of n-3 LC-PUFA. It is also suggested that dietary silage level influences the hepatic fatty acid metabolism in a breed-dependent manner, through changes in the expression of genes encoding for enzymes associated with the desaturation and elongation pathway. The importance of devising custom-made feeding strategies taking into account the genetic background is, therefore, stressed by the results from this experiment. 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

The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses. Show less

TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155-161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca(2+) sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca(2+) sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca(2+) was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg(2+)-, and Ca(2+)/Mg(2+)-bound states indicated that all of the cTnC mutants (except I148V in the Ca(2+)/Mg(2+) condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca(2+) as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM. Show less

We investigated the presence of a clinical diagnosis of hypertrophic cardiomyopathy (HCM) and of risk factors for sudden cardiac death (SCD) at the first cardiological evaluation after predictive genetic testing in asymptomatic carriers of an MYBPC3 gene mutation. Two hundred and thirty-five mutation carriers were cardiologically evaluated on the presence of HCM and risk factors. A clinical diagnosis of HCM was made in 53 carriers (22.6%). Disease penetrance at 65 years was incomplete for all types of MYBPC3 gene mutations. Women were affected less often than men (15 and 32% respectively, P = 0.003) and disease penetrance was lower in females than in males (13 and 30% at 50 years, respectively, P = 0.024). One risk factor was present in 87 carriers and 9 had two or more risk factors. Twenty-five carriers (11%) with one or more risk factors and manifest HCM could be at risk for SCD. At first cardiological evaluation almost one-quarter of asymptomatic carriers was diagnosed with HCM. Risk factors for SCD were frequently present and 11% of carriers could be at risk for SCD. Predictive genetic testing in HCM families and frequent cardiological evaluation on the presence of HCM and risk factors for SCD are justified until advanced age. Show less

Toll-like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of atherosclerosis. The activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine macrophages were treated with CpG-ODN, TLR9 agonist, and oxidized particles of LDL (Paz-PC) and FCF was analyzed by means of Oil Red O staining. The administration of CpG-ODN plus Paz-PC onto macrophages increased the amount of lipid droplets, correlated to increased levels of tumor necrosis factor (TNF)-alpha, IFNbeta, and IP-10. The underlying mechanism by which TLR9 ligation influenced Paz-PC in the FCF was NF-kappaB- and IRF7-dependent, as observed by higher levels of phosphorylated IkappaBalpha, increased nuclear translocation of the p65 subunit, lower levels of the total IKKalpha protein and higher release of interferon-dependent cytokines, such as IP-10. Liver X receptors (LXRs) regulate lipid cellular transport and negatively modulate TLR-dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG-ODN plus Paz-PC stimulation. In this condition, we observed decreased levels of the nuclear translocation of the p65 subunit, related to the higher presence of LXRalpha into the nucleus. TNF-alpha, IP-10, and IFNbeta levels were reduced by the administration of GW3965 following CpG-ODN and Paz-PC treatment. In conclusion, the activation of TLR9 facilitates the formation of foam cells in an NF-kappaB- and IRF7-dependent manner, countered by the activation of LXRs. This study further support LXRs as potential anti-atherosclerotic target. Show less

A series of 53 Portuguese patients (derived from 43 families) born in the period 1963-1999 have been diagnosed with neuronal ceroid lipofuscinosis (NCL) based on clinicopathological findings. Plotting the cumulative number of new cases per year against the year of birth resulted in a slightly S-shaped curve, with a nearly straight central segment over a period of 14 years (1977-1990) indicating a continuous registration of new cases born during the corresponding time period. In this period the prevalence of overall NCL in the Portuguese population was calculated to be 1.55 per 100.000 live births.Twenty-six patients from 20 unrelated families were further evaluated by combining clinicopathological with biochemical and genetic data. No intra-familial heterogeneity was observed. Four sub-types of childhood NCL were identified: infantile NCL (INCL) with granular osmiophilic inclusions (GROD) and PPT1 deficiency (1/26), classical LINCL with curvilinear (CV) inclusions and tripeptidyl peptidase (TPP1) deficiency (3/26), variant late infantile NCL (LINCL) with fingerprint/curvilinear (FP/CV) inclusions and normal TPP1 enzyme activity (11/26) and juvenile NCL (JNCL) with a mix of FP/CV (11/26). Eight of 11 JNCL patients were homozygous for the 1.02-kb deletion in the CLN3 gene, and 3 were heterozygous with an unidentified mutation in the second allele. The 1.02-kb deletion in the CLN3 gene accounted for 86.3 % (19/22) of CLN3-causing alleles and 36.5 % (19/52) of childhood NCL defects. The causal mutations for CLN1 and CLN2 were V181M (2/2) and R208X (4/6), respectively. CLN1, CLN2 and CLN3 affected 3.8 %, 11.5 % and 42.3 % of NCL Portuguese patients, respectively. In 42.3 % of patients affected by the vLINCL form, CLN3, CLN5 and CLN8 gene defects were excluded by direct sequencing of cDNA. Genetic variants such as CLN6 might therefore cause a significant portion of childhood NCL in the Portuguese population. The relative frequency of classical childhood forms of NCL in the Portuguese population is reported and contributes to the knowledge of genetic epidemiology of these world-widely distributed disorders. Show less