👤 Fernanda Degobbi Lopes

Brain derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) play crucial roles in neuronal development, synaptic transmission, and neuroplasticity. Deficits in BDNF/TrkB signalling and trafficking have been identified in several neurodegenerative diseases, including Alzheimer's disease (AD). Individuals with Down syndrome (DS) are at an increased risk of developing AD compared to the general population. Basal forebrain neurons (BFNs) are among the first to degenerate in AD and DS, but the mechanisms underlying their vulnerability remain unclear. Using BFNs derived from the Dp1Tyb mouse model of DS, we investigated neurotrophic signalling and trafficking deficits in AD-DS. We found enlarged early endosomes and elevated levels of active Rab5, a GTPase critical for early endosome formation, in Dp1Tyb BFNs. These abnormalities were associated with impaired transport of internalised TrkB from axon terminals to the soma. Using microfluidic devices, we demonstrated that axonal BDNF stimulation enhanced signalling endosome dynamics in wild-type but not Dp1Tyb BFNs, which is likely due to impaired axonal ERK1/2 signalling. Our findings establish a link between Rab5 hyperactivation, endosomal dysfunction, and impaired ERK1/2 signalling, highlighting the interplay between trafficking and neurotrophic signalling, and underscore the importance of targeting endolysosomal and signalling pathways to mitigate neuronal dysfunction in AD-DS. Show less

Elevated lipoprotein(a) (Lp(a)) and Lp(a)-raising genetic variants (e.g. rs3798220) are independent cardiovascular risk factors lacking preventive strategies. Given the prothrombotic properties attributed to high Lp(a), aspirin was hypothesized to confer benefit in primary prevention. We performed a systematic review and meta-analysis to evaluate the impact of aspirin on cardiovascular and bleeding outcomes in this population. MEDLINE, Web of Science and CENTRAL were searched (November 2025) for randomized and observational studies assessing aspirin use in primary prevention among individuals with Lp(a) ≥ 50 mg/dL or Lp(a)-associated genetic variants. The primary outcome was major adverse cardiovascular events (MACE). Secondary outcomes included myocardial infarction (MI), coronary artery disease (CAD), cardiovascular mortality, and bleeding. Random-effects meta-analyses pooled the Hazard ratios (HR) with 95% confidence intervals (CI). Certainty of evidence was assessed using GRADE. Seven studies including 6498 participants met inclusion criteria. Aspirin was not associated with a reduction in MACE (HR 0.99, 95% CI 0.79-1.24; I Aspirin was not associated with a reduction of MACE among individuals with elevated Lp(a). A potential benefit for MI requires confirmation in adequately designed and powered prospective studies. Pooled data from rs3798220-C carriers suggest a potential significant benefit that warrants further investigation REGISTRATION: PROSPERO identifier no. CRD42024520731. Show less

Understanding the immunopathogenesis of COVID-19 has yielded valuable insights into predicting adverse outcomes-particularly mortality. However, significant gaps persist in our comprehension of the complex interplay among the proposed pathophysiological mechanisms. Here, we aim to investigate the immunological factors associated with mortality in critically ill, unvaccinated COVID-19 patients admitted to the intensive care unit (ICU). We conducted a single-center, prospective study involving 56 unvaccinated COVID-19 patients admitted to the ICU. Plasma cytokine levels at admission were quantified using enzyme-linked immunosorbent assay (ELISA). Continuous variables were presented as median (IQR), and categorical variables as frequencies and percentages. Non-parametric tests assessed group differences. Logistic regression and receiver operating characteristic (ROC) curve analyses identified predictors of mortality, with bootstrapping (1000 re-samplings; 95 % BCa CI) applied for model validation. Deceased patients exhibited significantly higher levels of interleukin (IL)-1β, IL-2, IL-6, transforming growth factor (TGF)-β, and interferon (IFN)-γ compared to survivors. Conversely, IL-10 and IL-27 were associated with favorable outcomes. Logistic regression modeling identified elevated IL-2 and IFN-γ levels as significant predictors of mortality. Notably, individual ROC curve analyses demonstrated that IL-1β and TGF-β had excellent discriminatory ability for mortality, while IFN-γ, IL-2, and IL-27 showed very good to excellent discriminatory capacity. Our results indicate that distinct cytokine profiles differentiate survivors from non-survivors in critically ill, unvaccinated COVID-19 patients. These findings highlight the importance of cytokine dysregulation in severe COVID-19 cases and suggest potential targets for prognostic approaches. Further research is warranted to validate these results and translate them into effective clinical management strategies. Show less

Complex diseases arise from the interplay of genetic and environmental factors. We present a case where complex diseases seem to coexist. A 12-month-old girl was referred for short stature and hypotonia. Initial evaluation revealed central hypothyroidism, growth hormone deficiency and a small pituitary gland with ectopic neurohypophysis. Replacement therapy improved growth, but developmental delay and strabismus ensued. At age 10, she experienced a first seizure treated with levetiracetam. At age 12, she presented diabetic ketoacidosis and functional insulin therapy was started; positive autoantibodies confirmed autoimmune etiology. Initial genetic testing performed by microarray analysis retrieved normal results, but exome sequencing revealed a heterozygous pathogenic variant in KANSL1 gene, allowing for the diagnosis of Koolen-de Vries syndrome. In this patient, Koolen-de Vries syndrome presented initially as hypopituitarism and only later epilepsy. Afterwards, type 1 diabetes mellitus ensued, highlighting the complexity of intertwined conditions. Show less

Interventions have focused on evaluating effective strategies for increasing physical activity (PA) and reducing sedentary behavior (SB) in children and adolescents, which is still a challenge mainly in low- and middle-income countries. Thus, this study aimed to assess the effect of the Movimente Study on device-measured PA and SB in two-time segments of the school day amongst Brazilian adolescents. Six elementary schools were randomized into the intervention (IG) or control group (CG). Participants were in 7th -9th grades. A school year (2017) multicomponent intervention was delivered consisting of three components: (1) teacher training, (2) education curriculum, and (3) school environment. PA and SB were assessed using GT3x + ActiGraph hip-worn accelerometers. The trial's primary outcome was overall device-measured PA and SB. Exploratory secondary analyses examined PA and SB within in-School (08:00-11:59) and out-of-school (12:00-22:00) time segments. A two-level linear mixed model assessed the effect of the intervention on light-intensity PA (LPA), moderate- to vigorous-intensity PA (MVPA), SB, and MVPA/SB ratio within and between groups. There was a significant effect on the IG compared to the CG for MVPA (Coefficient [Coef.] = 16.2; 95% Confidence Interval [95%CI] = 6.9;25.5; p-value = 0.001), SB (Coef. = -22.7; 95%CI = -44.7;-0.7; p-value = 0.043), and MVPA/SB ratio (Coef. = 3.2; 95%CI = 1.2;5.3; p-value = 0.002) performed in the out-of-school segment, but not in the In-school segment. However, there were no significant differences within- nor between-group differences in LPA in both day segments. The Movimente Study was associated with greater increases in MVPA, improvements in the MVPA/SB ratio, and reductions in SB during the out-of-school period compared with control peers. Clinical Trials - NCT02944318. Registration Date: 10/24/2016. Show less

Most deleterious variants are recessive and segregate at relatively low frequency. Therefore, high sample sizes are required to identify these variants. In this study we report a large-scale sequence based genome-wide association study (GWAS) in pigs, with a total of 120,000 Large White and 80,000 Synthetic breed animals imputed to sequence using a reference population of approximately 1,100 whole genome sequenced pigs. We imputed over 20 million variants with high accuracies (R2>0.9) even for low frequency variants (1-5% minor allele frequency). This sequence-based analysis revealed a total of 14 additive and 9 non-additive significant quantitative trait loci (QTLs) for growth rate and backfat thickness. With the non-additive (recessive) model, we identified a deleterious missense SNP in the CDHR2 gene reducing growth rate and backfat in homozygous Large White animals. For the Synthetic breed, we revealed a QTL on chromosome 15 with a frameshift variant in the OBSL1 gene. This QTL has a major impact on both growth rate and backfat, resembling human 3M-syndrome 2 which is related to the same gene. With the additive model, we confirmed known QTLs on chromosomes 1 and 5 for both breeds, including variants in the MC4R and CCND2 genes. On chromosome 1, we disentangled a complex QTL region with multiple variants affecting both traits, harboring 4 independent QTLs in the span of 5 Mb. Together we present a large scale sequence-based association study that provides a key resource to scan for novel variants at high resolution for breeding and to further reduce the frequency of deleterious alleles at an early stage in the breeding program. Show less

Encephalocraniocutaneous lipomatosis (ECCL), also known as Haberland syndrome, is a sporadic tumor predisposition neurocutaneous disorder, included in the oculoectodermal syndrome group of mosaic RASopathies. ECCL primarily affects the skin, central nervous system and eyes. Key diagnostic features include nevus psiloliparus, a hallmark subcutaneous lipomatous hamartoma associated with alopecia, along with subcutaneous lipomas, focal skin aplasia, and patchy alopecia. Neurologically, intracranial lipomas, particularly in the cerebellopontine angle, are prevalent, along with cortical dysplasia, ventriculomegaly, and vascular malformations. Ocular findings commonly involve choristomas, lipodermoids, and dermoids, which may impair vision. Diagnosis can be made clinically, but further confirmatory genetic testing can in some cases identify a pathogenic variant in the FGFR1 or KRAS genes. Molecular testing aids diagnosis but is not always conclusive. Management is multidisciplinary with focus on symptomatic management, typically involving dermatological, neurological, and ophthalmologic evaluations with consideration of brain and spine neuroimaging and surgical management of tumors. The prognosis varies, with most individuals leading generally normal lives, though there is a risk of developmental delay, seizures, and low-grade gliomas. The severity of CNS involvement does not consistently correlate with cutaneous or ocular abnormalities. Show less

Activation of fibroblast growth factor receptor 1 (FGFR1) signaling improves the metabolic health of animals and humans, while inactivation leads to diabetes in mice. Direct human genetic evidence for the role of FGFR1 signaling in human metabolic health has not been fully established. We hypothesized that individuals with naturally occurring Participants with rare These findings suggest that impaired FGFR1 signaling may contribute to an early insulin resistance phase of diabetes pathogenesis and support the candidacy of the FGFR1 signaling pathway as a therapeutic target for improving the human metabolic health. Show less

Visceral leishmaniasis (VL) is an important tropical and neglected disease and represents a serious global health problem. The initial interaction between the phagocytes and the parasite is crucial to determine the pathogen's capacity to initiate infection and it shapes the subsequent immune response that will develop. While type-1 T-cells induce IL-6, IL-1β, TNF-α, and IL-12 production by monocytes/macrophages to fight the infection, type-2 T-cells are associated with a regulatory phenotype (IL-10 and TGF-β) and successful infection establishment. Recently, our group demonstrated the role of an important Th1/Th17 T-cell population, the mucosal-associated invariant T (MAIT) cells, in VL. MAIT cells can respond to Here, we describe the impact of the MR1-blockage on Overall, our data showed that VL patients presents higher percentage of activated neutrophils than asymptomatic and non-infected controls. In addition, MR1 blockade led to lower TNF-α and TGF-β production by non-activated neutrophils from asymptomatic individuals. Moreover, TNF-α and IL-10 production by monocytes was higher in VL patients. In the analysis of soluble mediators produced These data corroborate the hypothesis that MR1-restricted responses are associated to a protective role during Show less

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiomyopathy and a leading cause of sudden death. Genetic testing and familial cascade screening play a pivotal role in the clinical management of HCM patients. However, conventional genetic tests primarily focus on the detection of exonic and canonical splice site variation. Oversighting intronic non-canonical splicing variants potentially contributes to a proportion of HCM patients remaining genetically undiagnosed. Here, using a non-integrative reprogramming strategy, we generated induced pluripotent stem cell (iPSC) lines from four individuals carrying one of two variants within intronic regions of MYBPC3: c.1224-52G > A and c.1898-23A > G. Upon differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), mis-spliced mRNAs were identified in cells harbouring these variants. Both abnormal mRNAs contained a premature termination codon (PTC), fitting the criteria for activation of nonsense mediated decay (NMD). However, the c.1898-23A > G transcripts escaped this mRNA quality control mechanism, while the c.1224-52G > A transcripts were degraded. The newly generated iPSC lines represent valuable tools for studying the functional consequences of intronic variation and for translational research aimed at reversing splicing abnormalities to prevent disease progression. Show less

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Show less

Germ cell tumors (GCTs) constitute diverse neoplasms arising in the gonads or extragonadal locations. Testicular GCTs (TGCTs) are the predominant solid tumors in adolescents and young men. Despite cisplatin serving as the primary therapeutic intervention for TGCTs, 10‑20% of patients with advanced disease demonstrate resistance to cisplatin‑based chemotherapy, and epithelial‑mesenchymal transition (EMT) is a potential contributor to this resistance. EMT is regulated by various factors, including the snail family transcriptional repressor 2 ( Show less

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies. Show less

Urea cycle disorders (UCD) are a group of genetic diseases caused by deficiencies in the enzymes and transporters involved in the urea cycle. The impairment of the cycle results in ammonia accumulation, leading to neurological dysfunctions and poor outcomes to affected patients. The aim of this study is to investigate and describe UCD patients' principal clinical and biochemical presentations to support professionals on urgent diagnosis and quick management, aiming better outcomes for patients. We explored medical records of 30 patients diagnosed in a referral center from Brazil to delineate UCD clinical and biochemical profile. Patients demonstrated a range of signs and symptoms, such as altered levels of consciousness, acute encephalopathy, seizures, progressive loss of appetite, vomiting, coma, and respiratory distress, in most cases combined with high levels of ammonia, which is an immediate biomarker, leading to a UCD suspicion. The most prevalent UCD detected were ornithine transcarbamylase deficiency, followed by citrullinemia type 1, hyperargininemia, carbamoyl phosphate synthase 1 deficiency, and argininosuccinic aciduria. Clinical symptoms were highly severe, being the majority developmental and neurological disabilities, with 20% of death rate. Laboratory analysis revealed high levels of ammonia (mean ± SD: 860 ± 470 μmol/L; reference value: ≤80 μmol/L), hypoglycemia, metabolic acidosis, and high excretion of orotic acid in the urine (except in carbamoyl phosphate synthetase 1 [CPS1] deficiency). We emphasize the need of urgent identification of UCD clinical and biochemical conditions, and immediate measurement of ammonia, to enable the correct diagnosis and increase the chances of patients' survival, minimizing neurological and psychomotor damage caused by hepatic encephalopathy. Show less

Dementia due to Alzheimer's disease (AD) is a complex neurodegenerative disorder, which much of heritability remains unexplained. At the clinical level, one of the most common physiological alterations is the slowing of oscillatory brain activity, measurable by electroencephalography (EEG). Relative power (RP) at the conventional frequency bands (i.e., delta, theta, alpha, beta-1, and beta-2) can be considered as AD endophenotypes. The aim of this work is to analyze the association between sixteen genes previously related with AD: APOE, PICALM, CLU, BCHE, CETP, CR1, SLC6A3, GRIN2 β, SORL1, TOMM40, GSK3 β, UNC5C, OPRD1, NAV2, HOMER2, and IL1RAP, and the slowing of the brain activity, assessed by means of RP at the aforementioned frequency bands. An Iberian cohort of 45 elderly controls, 45 individuals with mild cognitive impairment, and 109 AD patients in the three stages of the disease was considered. Genomic information and brain activity of each subject were analyzed. The slowing of brain activity was observed in carriers of risk alleles in IL1RAP (rs10212109, rs9823517, rs4687150), UNC5C (rs17024131), and NAV2 (rs1425227, rs862785) genes, regardless of the disease status and situation towards the strongest risk factors: age, sex, and APOE ɛ4 presence. Endophenotypes reduce the complexity of the general phenotype and genetic variants with a major effect on those specific traits may be then identified. The found associations in this work are novel and may contribute to the comprehension of AD pathogenesis, each with a different biological role, and influencing multiple factors involved in brain physiology. Show less

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development. Show less

Predictive genetic screening of relatives of patients with hypertrophic cardiomyopathy (HCM) caused by sarcomere protein (SP) gene mutations is current standard of care, but there are few data on long-term outcomes in mutation carriers without HCM. The aim of this study was to determine the incidence of new HCM diagnosis in SP mutation carriers. This was a retrospective analysis of adult and pediatric SP mutation carriers identified during family screening who did not fulfill diagnostic criteria for HCM at first evaluation. The authors evaluated 285 individuals from 156 families (median age 14.2 years [interquartile range: 6.8 to 31.6 years], 141 [49.5%] male individuals); 145 (50.9%) underwent cardiac magnetic resonance (CMR). Frequency of causal genes was as follows: MYBPC3 n = 123 (43.2%), MYH7 n = 69 (24.2%), TNNI3 n = 39 (13.7%), TNNT2 n = 34 (11.9%), TPM1 n = 9 (3.2%), MYL2 n = 6 (2.1%), ACTC1 n = 1 (0.4%), multiple mutations n = 4 (1.4%). Median follow-up was 8.0 years (interquartile range: 4.0 to 13.3 years) and 86 (30.2%) patients developed HCM; 16 of 50 (32.0%) fulfilled diagnostic criteria on CMR but not echocardiography. Estimated HCM penetrance at 15 years of follow-up was 46% (95% confidence interval [CI]: 38% to 54%). In a multivariable model adjusted for age and stratified for CMR, independent predictors of HCM development were male sex (hazard ratio [HR]: 2.91; 95% CI: 1.82 to 4.65) and abnormal electrocardiogram (ECG) (HR: 4.02; 95% CI: 2.51 to 6.44); TNNI3 variants had the lowest risk (HR: 0.19; 95% CI: 0.07 to 0.55, compared to MYBPC3). Following a first negative screening, approximately 50% of SP mutation carriers develop HCM over 15 years of follow-up. Male sex and an abnormal ECG are associated with a higher risk of developing HCM. Regular CMR should be considered in long-term screening. Show less

The authors report the clinical and genetic investigation of a family with hypertrophic cardiomyopathy (HCM). The individuals described are three affected first-degree relatives (father, daughter and son), one affected niece and unaffected nephew and niece. Those affected all share a very similar phenotype consisting of asymmetric HCM, with hypertrophy particularly affecting the septum and the anterior wall, and similar electrocardiographic features, including a short PR interval. Case 1 (proband) presented with obstructive HCM and had undergone myectomy and mitral valve replacement. Case 2 (oldest offspring of Case 1) had non-obstructive HCM with exertional angina and NYHA II heart failure (HF) symptoms; she developed non-sustained ventricular tachycardia during follow-up and received a single-chamber ICD for primary prevention of sudden cardiac death. Case 3 (son of case 1) presented with asymptomatic non-obstructive HCM and developed NYHA II HF symptoms during follow-up. Case 4 had non-obstructive HCM, mainly with NYHA II HF symptoms. Testing of the proband for sarcomeric mutations and phenocopies was initially negative. After eight years of clinical follow-up, the suspicion of an undiscovered pathogenic gene mutation shared among the members of this family led us to enroll the proband in a whole-genome sequencing research project, which revealed a heterozygous pathogenic intronic MYBPC3 variant (c.1227-13G>A [rs397515893]), cosegregating with the phenotype. Show less

Feed efficiency (FE) is one of the most important traits in pig production. However, it is difficult and costly to measure it, limiting the collection of large amount of data for an accurate selection for better FE. Therefore, the identification of single-nucleotide polymorphisms (SNPs) associated with FE-related traits to be used in the genetic evaluation is of great interest of pig breeding programs for increasing the prediction accuracy and the genetic progress of these traits. The objective of this study was to identify SNPs significantly associated with FE-related traits: average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR). We also aimed to identify potential candidate genes for these traits. Phenotypic information recorded on a population of 2386 three-way crossbreed pigs that were genotyped for 51 468 SNPs was used. We identified three loci of quantitative trait (QTL) regions associated with ADG and three QTL regions associated with ADFI; however, no significant association was found for FCR. A false discovery rate (FDR) ≤ 0.005 was used as the threshold for declaring an association as significant. The QTL regions associated with ADG on Sus scrofa chromosome (SSC) 1 were located between 177.01 and 185.47 Mb, which overlaps with the QTL regions for ADFI on SSC1 (173.26 and 185.47 Mb). The other QTL region for ADG was located on SSC12 (2.87 and 3.22 Mb). The most significant SNPs in these QTL regions explained up to 3.26% of the phenotypic variance of these traits. The non-identification of genomic regions associated with FCR can be explained by the complexity of this trait, which is a ratio between ADG and ADFI. Finally, the genes CDH19, CDH7, RNF152, MC4R, PMAIP1, FEM1B and GAA were the candidate genes found in the 1 Mb window around the QTL regions identified in this study. Among them, the MC4R gene (SSC1) has a well-known function related to ADG and ADFI. In this study, we identified three QTL regions for ADG (SSC1 and SSC12) and three for ADFI (SSC1). These regions were previously described in purebred pig populations; however, to our knowledge, this is the first study to confirm the relevance of these QTL regions in a crossbred pig population. The potential use of the SNPs and genes identified in this study in prediction models that combine genomic selection and marker-assisted selection should be evaluated for increasing the prediction accuracy of these traits in this population. Show less

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by ventricular dilatation and impaired systolic function. Familial forms account for 30-50% of cases. Autosomal dominant inheritance is the predominant pattern of transmission. Causal genetic variants have been identified in several genes and molecular diagnosis has implications for genetic counseling and risk stratification. We aimed to estimate the frequency of genetic variants and the molecular basis of DCM in Portugal. We performed a multicenter study of unrelated patients, recruited between 2013 and 2014. Variants in 15 genes were screened using PCR with direct sequencing (next-generation sequencing with at least 30-fold coverage combined with Sanger sequencing). A total of 107 patients were included, 64 (60%) men, mean age at diagnosis 38±13 years, with 48 (45%) familial cases. In total, 31 rare variants in eight genes (mainly in MYBPC3, TNNT2 and LMNA) were identified, in 28 patients (26%). Only four variants had been previously described in association with DCM, 11 with hypertrophic cardiomyopathy, and nine variants were novel. Four variants were likely pathogenic and the remainder were of uncertain significance. We found no major differences in the main clinical and imaging characteristics between patients with or without rare variants and patients with likely pathogenic variants. Our results reflect the complexity and diversity of DCM genetics. For better interpretation of the pathogenicity of the variants found and their causative roles in DCM, molecular cascade screening of families is imperative. Further insight into genotype-phenotype correlations and risk stratification is desirable. Show less

We present an ancillary study of the Portuguese Registry of Hypertrophic Cardiomyopathy (PRo-HCM). This is one of the largest HCM genetic studies based on a registry. Collected genetic variants were re-analysed for pathogenicity. Demographic, clinical, imaging and outcome data were analysed for associations with genotype, focusing on comparisons between patients with (G+) vs without (G-) a pathogenic/likely pathogenic (P/LP) variant in one the 9 main causal sarcomeric genes. From the 1042 patients in the registry, 528 (51%) had genetic testing. 152 (28%) were G+ and 98 pts. (19%) had variants of unknown significance. From the patients with the 9 mentioned genes sequenced (424 pts), 14.6% had P/LP variants in MYBPC3, 8.7% MYH7, 4.5% TNNT2, 1.7% TNNI3. Patients were 51 ± 16 years-old, 59% males. Genotype was associated with the following: birthplace (p = 0.005); age (p < 0.001); family history of HCM (p < 0.0005); hypertension (p < 0.0005); chest pain (p = 0.015); pattern of hypertrophy (p = 0.006); left ventricular hypertrophy on the ECG (p < 0.0005); family history of sudden cardiac death (SCD) (p = 0.002). G+ patients more frequently had more than one risk factor for SCD (p = 0.002) and a higher ESC-SCD risk score (p = 0.003). In survival analysis, G+ was associated with SCD (p = 0.017) and MYH7+ with LV systolic dysfunction (p = 0.038). Half of the registry patients had genetic testing. Sarcomere-positive patients had distinct demographics, ECG, imaging characteristics and family history and are at increased risk of SCD. The presence of a MYH7 mutation was associated with evolution towards LV systolic dysfunction. Show less

Gestational exposure to air pollution is associated with negative outcomes in newborns and children. In a previous study, we demonstrated a synergistic negative effect of pre- and postnatal exposure to PM Show less

High throughput sequencing technologies have revolutionized the identification of mutations responsible for genetic diseases such as hypertrophic cardiomyopathy (HCM). However, approximately 50% of individuals with a clinical diagnosis of HCM have no causal mutation identified. This may be due to the presence of pathogenic mutations located deep within the introns, which are not detected by conventional sequencing analysis restricted to exons and exon-intron boundaries. The aim of this study was to develop a whole-gene sequencing strategy to prioritize deep intronic variants that may play a role in HCM pathogenesis. The full genomic DNA sequence of 26 genes previously associated with HCM was analysed in 16 unrelated patients. We identified likely pathogenic deep intronic variants in VCL, PRKAG2 and TTN genes. These variants, which are predicted to act through disruption of either splicing or transcription factor binding sites, are 3-fold more frequent in our cohort of probands than in normal European populations. Moreover, we found a patient that is compound heterozygous for a splice site mutation in MYBPC3 and the deep intronic VCL variant. Analysis of family members revealed that carriers of the MYBPC3 mutation alone do not manifest the disease, while family members that are compound heterozygous are clinically affected. This study provides a framework for scrutinizing variation along the complete intronic sequence of HCM-associated genes and prioritizing candidates for mechanistic and functional analysis. Our data suggest that deep intronic variation contributes to HCM phenotype. 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

The role of copy-number variants (CNV) as a cause of hypertrophic cardiomyopathy (HCM) is poorly studied. The aim of this study was to use high-throughput sequence (HTS) data combined with a read-depth strategy, to screen for CNV in cardiomyopathy-associated genes in a large consecutive cohort of HCM patients. Five-hundred-and-five unrelated HCM patients were genotyped using a HTS approach for 41 cardiovascular genes. We used a previously validated read-depth strategy (ExomeDepth) to call CNVs from the short-read sequence data. Detected CNVs in 19 cardiomyopathy-associated genes were then validated by comparative genomic hybridization array. Twelve CNVs were identified. Four CNVs in 4 patients (0.8% of the cohort) were validated: one large deletion in MYBPC3, one large deletion in PDLIM3, one duplication of the entire TNNT2 gene and one large duplication in LMNA. Our data suggest that the proportion of HCM cases with pathogenic CNVs is small (<1%). For the small subset of patients with clearly interpretable CNVs, our findings have direct clinical implications. Short read sequence data can be used for CNV calling, but the high false positive rate requires a validation step. The two-step strategy described here is effective at identifying novel genetic causes of HCM and similar techniques should be applied whenever possible. Show less

The genetic basis of familial hypertrophic cardiomyopathy (HCM) is well described, but the relation between genotype and clinical phenotype is still poorly characterised. To summarise and critically review the current literature on genotype-phenotype associations in patients with HCM and to perform a meta-analysis on selected clinical features. PubMed/Medline was searched up to January 2013. Retrieved articles were checked for additional publications. Observational, cross-sectional and prospectively designed English language human studies that analysed the relationship between the presence of mutations in sarcomeric protein genes and clinical parameters. The pooled analysis was confined to studies reporting on cohorts of unrelated and consecutive patients in which at least two sarcomere genes were sequenced. A random effect meta-regression model was used to determine the overall prevalence of predefined clinical features: age at presentation, gender, family history of HCM, family history of sudden cardiac death (SCD), and maximum left ventricular wall thickness (MLVWT). The I(2) statistic was used to estimate the proportion of total variability in the prevalence data attributable to the heterogeneity between studies. Eighteen publications (corresponding to a total of 2459 patients) were selected for the pooled analysis. The presence of any sarcomere gene mutation was associated with a younger age at presentation (38.4 vs 46.0 years, p<0.0005), a family history of HCM (50.6% vs 23.1%, p<0.0005), a family history of SCD (27.0% vs 14.9%, p<0.0005) and greater MLVWT (21.0 vs 19.3 mm, p=0.03). There were no differences when the two most frequently affected genes, MYBPC3 and MYH7, were compared. A total of 53 family studies were also included in the review. These were characterised by pronounced variability and the majority of studies reporting on outcomes analysed small cross-sectional cohorts and were unsuitable for pooled analyses. The presence of a mutation in any sarcomere gene is associated with a number of clinical features. The heterogeneous nature of the disease and the inconsistency of study design precludes the establishment of more precise genotype-phenotype relationships. Large scale studies examining the relation between genotype, disease severity, and prognosis are required. Show less

Clinical interpretation of the large number of rare variants identified by high throughput sequencing (HTS) technologies is challenging. The aim of this study was to explore the clinical implications of a HTS strategy for patients with hypertrophic cardiomyopathy (HCM) using a targeted HTS methodology and workflow developed for patients with a range of inherited cardiovascular diseases. By comparing the sequencing results with published findings and with sequence data from a large-scale exome sequencing screen of UK individuals, we sought to quantify the strength of the evidence supporting causality for detected candidate variants. 223 unrelated patients with HCM (46±15 years at diagnosis, 74% males) were studied. In order to analyse coding, intronic and regulatory regions of 41 cardiovascular genes, we used solution-based sequence capture followed by massive parallel resequencing on Illumina GAIIx. Average read-depth in the 2.1 Mb target region was 120. Rare (frequency<0.5%) non-synonymous, loss-of-function and splice-site variants were defined as candidates. Excluding titin, we identified 152 distinct candidate variants in sarcomeric or associated genes (89 novel) in 143 patients (64%). Four sarcomeric genes (MYH7, MYBPC3, TNNI3, TNNT2) showed an excess of rare single non-synonymous single-nucleotide polymorphisms (nsSNPs) in cases compared to controls. The estimated probability that a nsSNP in these genes is pathogenic varied between 57% and near certainty depending on the location. We detected an additional 94 candidate variants (73 novel) in desmosomal, and ion-channel genes in 96 patients (43%). This study provides the first large-scale quantitative analysis of the prevalence of sarcomere protein gene variants in patients with HCM using HTS technology. Inclusion of other genes implicated in inherited cardiac disease identifies a large number of non-synonymous rare variants of unknown clinical significance. Show less