Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.

A mutation in the promoter of the lipoprotein lipase (LPL) gene in a patient with familial combined hyperlipidemia and low LPL activity.

We have identified a naturally occurring mutation in the promoter of the lipoprotein lipase (LPL) gene. One of 20 patients with familial combined hyperlipidemia (FCHL) and reduced levels of postheparin plasma LPL activity was found to be a heterozygote carrier of this mutation. The mutation, a T-->C substitution at nt -39, occurred in the binding site of the transcription factor Oct-1. As a result, the transcriptional activity of the mutant promoter was < 15% of wild type, as determined by transfection studies in the human macrophage-like cell line THP-1. This decrease in promoter activity was observed in undifferentiated as well as in phorbol ester-differentiated THP-1 cells. Furthermore, the inductive effect of elevating the levels of intracellular cAMP was equally reduced. This mutation was not present among 20 FCHL patients with normal plasma LPL levels nor has it been reported among individuals with familial LPL deficiency. Thus, heterozygosity for LPL promoter mutations may be one of several factors that contribute to the etiology of FCHL.

Mutações inativadoras no gene MKRN3 são causa de puberdade precoce central familial

A maioria dos casos de puberdade precoce central (PPC) em meninas permanece idiopática. A hipótese de uma causa genética vem se fortalecendo após a descoberta de alguns genes associados a este fenótipo, sobretudo aqueles implicados com o sistema kisspeptina (KISS1 e KISS1R). Entretanto, apenas casos isolados de PPC foram relacionados à mutação na kisspeptina ou em seu receptor. Até recentemente, a maioria dos estudos genéticos em PPC buscava genes candidatos selecionados com base em modelos animais, análise genética de pacientes com hipogonadismo hipogonadotrófico, ou ainda, nos estudos de associação ampla do genoma. Neste trabalho, foi utilizado o sequenciamento exômico global, uma metodologia mais moderna de sequenciamento, para identificar variantes associadas ao fenótipo de PPC. Trinta e seis indivíduos com a forma de PPC familial (19 famílias) e 213 casos aparentemente esporádicos foram inicialmente selecionados. A forma familial foi definida pela presença de mais de um membro afetado na família. DNA genômico foi extraído dos leucócitos do sangue periférico de todos os pacientes. O estudo de sequenciamento exômico global realizado pela técnica ILLUMINA, em 40 membros de 15 famílias com PPC, identificou mutações inativadoras em um único gene, MKRN3, em cinco dessas famílias. Pesquisa de mutação no MKRN3 realizada por sequenciamento direto em duas famílias adicionais (quatro pacientes) identificou duas novas variantes nesse gene. O MKRN3 é um gene de um único éxon, localizado no cromossomo 15 em uma região crítica para a síndrome de Prader Willi. O gene MKRN3 sofre imprinting materno, sendo expresso apenas pelo alelo paterno. A descoberta de mutações em pacientes com PPC familial despertou o interesse para a pesquisa de mutações nesse gene em 213 pacientes com PPC aparentemente esporádica por meio de reação em cadeia de polimerase seguida de purificação enzimática e sequenciamento automático direto (Sanger). Três novas mutações e duas já anteriormente identificadas, incluindo quatro frameshifts e uma variante missense, foram encontradas, em heterozigose, em seis meninas não relacionadas. Todas as novas variantes identificadas estavam ausentes nos bancos de dados (1000 Genomes e Exome Variant Server). O estudo de segregação familial em três dessas meninas com PPC aparentemente esporádica e mutação no MKRN3 confirmou o padrão de herança autossômica dominante com penetrância completa e transmissão exclusiva pelo alelo paterno, demonstrando que esses casos eram, na verdade, também familiares. A maioria das mutações encontradas no MKRN3 era do tipo frameshift ou nonsense, levando a stop códons prematuros e proteínas truncadas e, portanto, confirmando a associação com o fenótipo. As duas mutações missenses (p.Arg365Ser e p.Phe417Ile) identificadas estavam localizadas em regiões de dedo ou anel de zinco, importantes para a função da proteína. Além disso, os estudos in silico dessas duas variantes demonstraram patogenicidade. Todos os pacientes com mutação no MKRN3 apresentavam características clínicas e hormonais típicas de ativação prematura do eixo reprodutivo. A mediana de idade de início da puberdade foi de 6 anos nas meninas (variando de 3 a 6,5) e 8 anos nos meninos (variando de 5,9 a 8,5). Tendo em vista o fenômeno de imprinting, análise de metilação foi também realizada em um subgrupo de 52 pacientes com PPC pela técnica de MS-MLPA, mas não foram encontradas alterações no padrão de metilação. Em conclusão, este trabalho identificou um novo gene associado ao fenótipo de PPC. Atualmente, mutações inativadoras no MKRN3 representam a causa genética mais comum de PPC familial (33%). O MKRN3 é o primeiro gene imprintado associado a distúrbios puberais em humanos. O mecanismo preciso de ação desse gene na regulação da secreção de GnRH necessita de estudos adicionais

Will Familial Hypercholesterolaemia Cohorts Hide Many More Lisosomal Acid Lipase Deficiency Patients?

Aims: Lisosomal Acid Lipase Deficiency (LALD), historical known as Cholesterol Ester Storage Disease (CESD), is an autosomal lisosomal storage recessive disorder and an unrecognized cause of dyslipidaemia. Mutations in LIPA gene are the underlying cause of LALD, being a mutation in the splice site of exon 8 the most common cause of the disease. Patients with LALD present dyslipidaemia and altered liver function. The aim of this work was to analyze LIPA gene in patients with unexplained dyslipidaemia.

Construction of a New Familial Hypercholesterolemia Variant Data Base. A Systematic Review for a 2015 Update

Aims: Familial hypercholesterolemia (FH) is an autosomal dominant disorder with increased cardiovascular risk, caused by mutations in LDLR, APOB and PCSK 9 genes. Although it is described that over 1700 variants have been found, none of the existing databases are completely updated. The aim of this work is to construct a FH database in order to provide a unique source of verified information about variants associated with FH for a more accurate genetic diagnosis.

Network Analyis Approach to Find New Candidate Genes and Pathways Involved in the Pathophysiology of Familial Hypercholesterolemia

Introduction: Familial hypercholesterolaemia (FH) is a common genetic cause of premature coronary heart disease (CHD) due to lifelong elevated plasma low-density lipoprotein (LDL) levels. Worldwide only 40 % of patients (FH+) with a clinical diagnosis of FH carry a mutation in any of the three genes (namely: LDLR, APOB, PCSK 9) that are currently known to be associated to the disease. We guess that the remaining 60 % of the patients (FH-) probably includes a high percentage of individuals with a polygenic form of dyslipidemia or an environmental form of hypercholesterolemia and a small percentage of individuals with mutations in some novel genes, never associated before with dyslipidemias. Here we present the preliminary results of an integrative approach intended to identify new candidate genes and to dissect pathways that can be dysregulated in the disease.

Genetic Analysis of Familial Hypercholesterolemia in Iberoamerican Countries

The Iberoamerican Familial Hypercholesterolemia network (IBAFH_N) was created in 2013 to promote awareness for Familial Hypercholesterolemia (FH) in these countries – Argentina, Brazil, Chile, Mexico, Portugal, Spain, Uruguay and more recently Colombia – that share a past and history. The aim of this work was to perform a molecular analysis of FH mutations in Iberoamerica.

Using percentiles to diagnose familial hypercholesterolemia in Portugal

Aims: Familial hypercholesterolemia (FH) is a genetic disorder of lipid metabolism, clinically characterised by high levels of low-density lipoprotein cholesterol (LDL-C) that leads to cholesterol accumulation in tendons and arteries, premature atherosclerosis and increased risk of premature coronary heart disease. In 1999, the Portuguese FH Study was established at the National Institute of Health to identify the genetic cause of hypercholesterolemia in individuals with a clinical diagnosis of FH and to perform an epidemiologic study to determine the prevalence and distribution of FH in Portugal. In the last 16 years, a genetic defect was identified in 749 patients, representing 3. 7 % of the cases estimated to exist in Portugal. Index patients were included in this study using the Simon Broome (SB) criteria. However, there are different FH clinical criteria to diagnose index cases. Since there are no clinical criteria to identify relatives with FH, the aim of this work was to investigate if a diagnostic tool based on population specific 95 th percentile improves the clinical identification of Portuguese FH patients comparing with SB criteria.

Electrocardiographic features of disease progression in arrhythmogenic right ventricular cardiomyopathy/dysplasia.

BACKGROUND Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is considered a progressive cardiomyopathy. However, data on the clinical features of disease progression are limited. The aim of this study was to assess 12-lead surface electrocardiographic (ECG) changes during long-term follow-up, and to compare these findings with echocardiographic data in our large cohort of patients with ARVC/D. METHODS Baseline and follow-up ECGs of 111 patients from three tertiary care centers in Switzerland were systematically analyzed with digital calipers by two blinded observers, and correlated with findings from transthoracic echocardiography. RESULTS The median follow-up was 4 years (IQR 1.9-9.2 years). ECG progression was significant for epsilon waves (baseline 14% vs. follow-up 31%, p = 0.01) and QRS duration (111 ms vs. 114 ms, p = 0.04). Six patients with repolarization abnormalities according to the 2010 Task Force Criteria at baseline did not display these criteria at follow-up, whereas in all patients with epsilon waves at baseline these depolarization abnormalities also remained at follow-up. T wave inversions in inferior leads were common (36% of patients at baseline), and were significantly associated with major repolarization abnormalities (p = 0.02), extensive echocardiographic right ventricular involvement (p = 0.04), T wave inversions in lateral precordial leads (p = 0.05), and definite ARVC/D (p = 0.05). CONCLUSIONS Our data supports the concept that ARVC/D is generally progressive, which can be detected by 12-lead surface ECG. Repolarization abnormalities may disappear during the course of the disease. Furthermore, the presence of T wave inversions in inferior leads is common in ARVC/D.

Prospective longitudinal voxel-based morphometry study of major depressive disorder in young individuals at high familial risk

BACKGROUND Previous neuroimaging studies indicate abnormalities in cortico-limbic circuitry in mood disorder. Here we employ prospective longitudinal voxel-based morphometry to examine the trajectory of these abnormalities during early stages of illness development. METHOD Unaffected individuals (16-25 years) at high and low familial risk of mood disorder underwent structural brain imaging on two occasions 2 years apart. Further clinical assessment was conducted 2 years after the second scan (time 3). Clinical outcome data at time 3 was used to categorize individuals: (i) healthy controls ('low risk', n = 48); (ii) high-risk individuals who remained well (HR well, n = 53); and (iii) high-risk individuals who developed a major depressive disorder (HR MDD, n = 30). Groups were compared using longitudinal voxel-based morphometry. We also examined whether progress to illness was associated with changes in other potential risk markers (personality traits, symptoms scores and baseline measures of childhood trauma), and whether any changes in brain structure could be indexed using these measures. RESULTS Significant decreases in right amygdala grey matter were found in HR MDD v. controls (p = 0.001) and v. HR well (p = 0.005). This structural change was not related to measures of childhood trauma, symptom severity or measures of sub-diagnostic anxiety, neuroticism or extraversion, although cross-sectionally these measures significantly differentiated the groups at baseline. CONCLUSIONS These longitudinal findings implicate structural amygdala changes in the neurobiology of mood disorder. They also provide a potential biomarker for risk stratification capturing additional information beyond clinically ascertained measures.

Prospective longitudinal study of subcortical brain volumes in individuals at high familial risk of mood disorders with or without subsequent onset of depression

Subcortical volumetric brain abnormalities have been observed in mood disorders. However, it is unknown whether these reflect adverse effects predisposing to mood disorders or emerge at illness onset. Magnetic resonance imaging was conducted at baseline and after two years in 111 initially unaffected young adults at increased risk of mood disorders because of a close family history of bipolar disorder and 93 healthy controls (HC). During the follow-up, 20 high-risk subjects developed major depressive disorder (HR-MDD), with the others remaining well (HR-well). Volumes of the lateral ventricles, caudate, putamen, pallidum, thalamus, hippocampus and amygdala were extracted for each hemisphere. Using linear mixed-effects models, differences and longitudinal changes in subcortical volumes were investigated between groups (HC, HR-MDD, HR-well). There were no significant differences for any subcortical volume between groups controlling for multiple testing. Additionally, no significant differences emerged between groups over time. Our results indicate that volumetric subcortical brain abnormalities of these regions using the current method appear not to form familial trait markers for vulnerability to mood disorders in close relatives of bipolar disorder patients over the two-year time period studied. Moreover, they do not appear to reduce in response to illness onset at least for the time period studied.

Electrophysiologic assessment of conduction abnormalities and atrial arrhythmias associated with amyloid cardiomyopathy.

BACKGROUND Arrhythmias in cardiac amyloidosis (CA) result in significant comorbidity and mortality but have not been well characterized. OBJECTIVE The purpose of this study was to define intracardiac conduction, atrial arrhythmia substrate, and ablation outcomes in a group of advanced CA patients referred for electrophysiologic study. METHODS Electrophysiologic study with or without catheter ablation was performed in 18 CA patients. Findings and catheter ablation outcomes were compared to age- and gender-matched non-CA patients undergoing catheter ablation of persistent atrial fibrillation (AF). RESULTS Supraventricular tachycardias were seen in all 18 CA patients (1 AV nodal reentrant tachycardia, 17 persistent atrial tachycardia [AT]/AF). The HV interval was prolonged (>55 ms) in all CA patients, including 6 with normal QRS duration (≤100 ms). Thirteen supraventricular tachycardia ablations were performed in 11 patients. Of these, 7 underwent left atrial (LA) mapping and ablation for persistent AT/AF. Compared to non-CA age-matched comparator AF patients, CA patients had more extensive areas of low-voltage areas LA (63% ± 22% vs 34% ± 22%, P = .009) and a greater number of inducible ATs (3.3 ± 1.9 ATs vs 0.2 ± 0.4 ATs, P <.001). The recurrence rate for AT/AF 1 year after ablation was greater in CA patients (83% vs 25%), and the hazard ratio for postablation AT/AF recurrence in CA patients was 5.4 (95% confidence interval 1.9-35.5, P = .007). CONCLUSION In this group of patients with advanced CA and atrial arrhythmias, there was extensive conduction system disease and LA endocardial voltage abnormality. Catheter ablation persistent AT/AF in advanced CA was associated with a high recurrence rate and appears to have a limited role in control of these arrhythmias.

The Collective Protagonist: Multiple Points of View and the Search for Truth in Familial Narratives

Thesis (Master's)--University of Washington, 2016-06

Universal Screening for Familial Colorectal Cancer Syndromes at Seattle Cancer Care Alliance: Qualitative Study of Providers involved in Implementation and Patients Experience with First Wave of Testing

Thesis (Master's)--University of Washington, 2016-06