Molecular study of Familial Dyslipidaemias by Next Generation Sequencing

Tese de mestrado, Bioquímica (Bioquímica Médica) Universidade de Lisboa, Faculdade de Ciências, 2022Dyslipidemia, a clinical condition defined by an abnormal concentration of lipids in the blood, can have a genetic etiology. Familial dyslipidemias constitute a group of genetically determined conditions, the majority being rare. In addition, these are often associated to serious conditions that can be prevented by the early identification of patients. Hypercholesterolemia promotes atherosclerosis, increasing patients' cardiovascular risk. Neurological manifestations and poor weight progression are frequent complications of hypocholesterolemia, and an increased risk of pancreatitis is often verified in cases of hypertriglyceridemia. This project aimed to identify the genetic cause of dyslipidemia in 96 Portuguese individuals, referred to the Cardiovascular Research Group at the National Health Institute, with a clinical diagnosis of several genetic dyslipidemias associated to different traits: hypercholesterolaemia, hypocholesterolaemia, and hypertriglyceridaemia. The lipid profile of the 96 index cases was determined for biochemical characterization. The molecular study of individuals was performed by Next Generation Sequencing with a customised target panel of 57 genes involved in lipid metabolism. Molecular diagnosis was provided after analysis of 18 genes strongly associated with several familial dyslipidemias. Rare variants detected were confirmed by PCR and Sanger Sequencing. A definite cause of monogenic dyslipidemia was established in 35 cases: 22 individuals were diagnosed with familial hypercholesterolaemia, 3 with familial hypobetalipoproteinemia, 2 with familial chylomicronemia syndrome, 7 with multifactorial chylomicronemia, and one with autosomal recessive hypertriglyceridemia. Moreover, several variants with uncertain significance were found by NGS during this project, the majority lacking functional data. As these variants can constitute the cause of disease in some cases, functional studies will be essential to assess variant’s pathogenicity. This work allowed an early and correct identification of Portuguese patients with different familial dyslipidemias, thus providing guidance for pharmacological treatment and lifestyle adaptations to reduce the chance of suffering disease complications, improving this way patients’ prognosis

ABC Transporters in Human Diseases

Mammalian ATP-binding cassette (ABC) transporters constitute a superfamily of proteins involved in many essential cellular processes. Most of these transporters are transmembrane proteins and allow the active transport of solutes, small molecules, and lipids across biological membranes. On the one hand, some of these transporters are involved in drug resistance (also referred to as MDR or multidrug resistance), a process known to be a major brake in most anticancer treatments, and the medical challenge is thus to specifically inhibit their function. On the other hand, molecular defects in some of these ABC transporters are correlated with several rare human diseases, the most well-documented of which being cystic fibrosis, which is caused by genetic variations in ABCC7/CFTR (cystic fibrosis transmembrane conductance regulator). In the latter case, the goal is to rescue the function of the deficient transporters using various means, such as targeted pharmacotherapies and cell or gene therapy. The aim of this Special Issue, “ABC Transporters in Human Diseases”, is to present, through original articles and reviews, the state-of-the-art of our current knowledge about the role of ABC transporters in human diseases and the proposed therapeutic options based on studies ranging from cell and animal models to patients

Genetické faktory v etiologii a patogenezi cholestáz s nízkou hladinou gammaglutamyltransferázy a dědičně podmíněných žloutenek

Souhrn (Česky) Identifikace genů kódujících hepatocelulární přenašeče a funkční charakterizace jednotlivých proteinů v posledním desetiletí umožnily vedle zrychlení a zpřesnění diagnostického procesu cholestázy i bližší studium regulace genové exprese a tím potenciální možnosti jejího ovlivnění. Tato práce ilustruje současný pokrok na poli geneticky podmíněných cholestáz a zaměřuje se na cholestázy s nízkou hladinou gammaglutamyltransferázy (γGT) a dědičně podmíněné žloutenky. Studie popisuje různé poruchy transportního systému hepatocytů, charakterizuje mutace a jejich fenotypické následky a rozšiřuje tyto analýzy o podrobnou charakterizaci regulačních oblastí genu ATP8B1. Oddíly týkající se cholestáz s nízkou hladinou gammaglutamyltransferázy - popisují vzácný nalezený typ inserčně-deleční mutace asociované s benigním průběhem progresivní familiární intrahepatální cholestázy 1. typu a diskutují možný mechanismus vzniku mutace a její pravděpodobný vliv na funkci proteinu FIC1. - shrnují rozsáhlou genetickou analýzu pacientů se závažnou formou progresivní intrahepatální cholestázy 2. typu, podmíněnou mutacemi v BSEP (Bile Salt Export Pump). Studie se zabývá korelací genotypu a fenotypu u 109 postižených rodin, diskutuje vliv jednotlivých mutací, zejména typ, lokalizaci a stupeň konzervace, na...(English) Recent progress in understanding the molecular mechanism of hepatobiliary disorders enabled the improvement of diagnostic accuracy and promoted the study of the regulation of gene expression and its potential modifying factors. Current achievement in the field of genetically determined cholestatic disorders is well illustrated in this thesis, focused on low gamma-glutamyltransferase (γGT) cholestasis and hereditary jaundice. The study describes several distinct defects of hepatocyte transport system, characterises underlying mutations and their phenotypic consequences and, finally, extends these studies for detailed characterisation of ATP8B1 gene regulatory regions. Chapters related to low γGT cholestasis - characterise rare type of mutation associated with benign course of PFIC type I (formerly BRIC1) and explain the putative mechanisms of mutation origin. - provide extensive study of severe forms of ABCB11 deficiency (PFIC2) including genotype-phenotype correlations in 109 affected families, evaluation of the specific ABCB11 genotypes' impact on BSEP immunostaining and risk of hepatobiliary malignancy. - identify and characterise yet unknown regulatory regions of ATP8B1, a gene mutated in Progressive Familial Intrahepatic Cholestasis type I. The studies demonstrate the complex structure...Institute of Medical Biochemistry and Laboratory Medicine First Faculty of MedicineÚstav lékařské biochemie a laboratorní diagnostiky 1. LF UK a VFN v PrazeFirst Faculty of Medicine1. lékařská fakult

The Chylomicron: Relationship to Atherosclerosis

The B-containing lipoproteins are the transporters of cholesterol, and the evidence suggests that the apo B48-containing postprandial chylomicron particles and the triglyceride-rich very low density lipoprotein (VLDL) particles play an important part in the development of the plaque both directly and indirectly by their impact on LDL composition. The ratio of dietary to synthesised cholesterol is variable but tightly regulated: hence intervention with diet at best reduces serum cholesterol by <20% andusually <10%. Statins are the mainstay of cholesterol reduction therapy, but they increase cholesterol absorption, an example of the relationship between synthesis and absorption. Inhibition of cholesterol absorption with Ezetimibe, an inhibitor of Niemann Pick C1-like 1 (NPC1-L1), the major regulator of cholesterol absorption, increases cholesterol synthesis and hence the value of adding an inhibitor of cholesterol absorption to an inhibitor of cholesterol synthesis. Apo B48, the structural protein of the chylomicron particle, is synthesised in abundance so that the release of these particles is dependent on the amount of cholesterol and triglyceride available in the intestine. This paper will discuss cholesterol absorption and synthesis, chylomicron formation, and the effect of postprandial lipoproteins on factors involved in atherosclerosis

Diagnóstico de las diátesis hemorrágicas hereditarias mediante el análisis genético por secuenciación masiva

[ES] Las diátesis hemorrágicas hereditarias (DHH) constituyen un grupo amplio y heterogéneo de trastornos hereditarios y/o congénitos poco frecuentes, relacionados con la alteración en la hemostasia primaria y en la hemostasia secundaria. Como consecuencia, el paciente experimenta episodios de sangrado con distintos grados de gravedad. No es infrecuente encontrar pacientes con estas características en la práctica clínica habitual. La gran heterogeneidad de las DHH, principalmente en los trastornos plaquetarios hereditarios, proviene de que las alteraciones en diferentes genes pueden producir un fenotipo clínico similar para cada una de ellas. Para su correcto diagnóstico es preciso, en la actualidad, combinar varias metodologías, lo que requiere un esfuerzo considerable en tiempo y en dinero. A pesar de ello, en bastantes ocasiones no se llega a un diagnóstico de certeza. Cuando los síntomas de sangrado hacen sospechar la existencia de una DHH (especialmente en niños, pero también en adultos en los que la patología no se haya manifestado en la infancia), se aplica un algoritmo que consiste en la caracterización clínica, fenotípica y funcional o del laboratorio del paciente. Todos estos estudios son necesarios para poder dirigir el análisis molecular a un gen candidato y, así, poder confirmar el diagnóstico. Esta estrategia supone un reto diagnóstico debido a que, en bastantes ocasiones, el diagnóstico de estas enfermedades es difícil, sobre todo en los casos con fenotipos hemorrágicos leves o indeterminados. También requiere una gran cantidad de pruebas, una validación interna e interlaboratorial y la repetición del estudio para que desaparezcan posibles factores intercurrentes adquiridos. Por tanto, las principales limitaciones para la detección de las DHH son: a) La falta de acuerdo en su clasificación y en su estandarización; b) la presencia de fenotipos clínico-biológicos coincidentes y su gran heterogeneidad clínica y molecular; y c) la dificultad para incorporar las herramientas diagnósticas a la rutina diaria y su complejidad técnica. Así, por ejemplo, en los trastornos plaquetarios hereditarios (TPHs), la heterogeneidad clínica, el solapamiento de fenotipos o la dificultad para definirlo obstaculizan la búsqueda de mutaciones en un gen candidato y, por tanto, el diagnóstico molecular. Por ello, sólo es factible identificar el gen afectado en el 40-50% de los TPHs. Por otro lado, en los trastornos de la hemostasia secundaria, elgen candidato a estudio suele estar bien definido, aunque pueden existir deficiencias combinadas. Sin embargo, como ocurre en la enfermedad de von Willebrand (52 exones codificantes) o, principalmente en la hemofilia A (26 exones y 186 kb de tamaño), tras descartar las mutaciones recurrentes (inversión del intrón 22; intrón 1), la secuenciación de un gran número de exones supone otra dificultad en tiempo y coste. En estos casos, también, la secuenciación convencional sería un método inapropiado para la rutina clínica diaria. La carencia de métodos diagnósticos sencillos aplicados en la práctica clínica habitual, motiva que, en ocasiones, al clínico le resulte complicado, a veces imposible, emitir un diagnóstico certero. En consecuencia, existe el riesgo de la prescripción de tratamientos inapropiados que no solucionarán su patología, por lo que el paciente no mejorará y podrá tener efectos secundarios no deseados. Además, se multiplicarán los gastos inherentes a su tratamiento y a la identificación de la anomalía causante del trastorno, al tener que emplear métodos gravosos y complejos desde el punto de vista clínico y dilatados en el tiempo. Hasta hace poco tiempo, la búsqueda de información genética a gran escala aplicada al diagnóstico clínico era, desde el punto de vista técnico, compleja, y, desde el económico, poco viable. Sin embargo, en la actualidad se dispone de un nuevo conjunto de métodos de alto rendimiento denominados secuenciación masiva o Next- Generation Sequencing (NGS), que son herramientas valiosas tanto para la investigación como para la evaluación de trastornos clínicos causados por mutaciones genéticas. Estas plataformas de alto rendimiento, en sus distintas modalidades como paneles de genes customizados (targeted genes panel), exoma completo (Whole-Exome Sequencing) o incluso, el genoma completo (Whole-Genome Sequencing) tienen la capacidad de secuenciar gran cantidad de información genética de manera paralela, simultánea y de forma precisa para identificar las mutaciones causantes de las enfermedades de base genética. Esta tecnología ha evolucionado rápidamente durante la última década hasta el punto de constituir hoy una alternativa atractiva, tanto a nivel práctico como económico, con la que abordar el diagnóstico de patologías con alta complejidad. De este modo, en el momento actual es razonable plantearse el uso de la NGS tanto para explorar cambios en todos los genes que con alta probabilidad pueden causar un fenotipo clínico y de laboratorio concreto, como para buscar la causa no conocida de patologías plaquetarias de fenotipo inespecífico que son, por otra parte, las más comunes y difícilmente abordables. Además, como bien es conocido, si los fenotipos de estas entidades pueden solaparse, es lógico pensar que el diseño de un panel que incluya a todos aquellos genes conocidos que se relacionan con cualquiera de las DHH descritas hasta el momento podría contribuir a un mejor diagnóstico de estas enfermedades. Incluso, a igualdad de tiempo y coste, estas plataformas pueden rastrear mutaciones en muchos más genes que la secuenciación clásica de Sanger. Por lo tanto, la secuenciación masiva parece una herramienta idónea para avanzar en el diagnóstico de estas enfermedades. Así, hemos planteado el análisis simultáneo de un gran número de genes y exones mediante dos tipos de paneles de genes, uno encaminado a los trastornos de la hemostasia primaria y otro, a la secundaria. Como veremos más adelante, hemos empleado la captura de secuencia que es idónea para secuenciar alteraciones genéticas hereditarias que ocurren en el 50-100% de las células, de manera precisa y asequible económicamente. De hecho, existen precedentes que demuestran cómo la tecnología NGS no sólo es capaz de identificar mutaciones ya descritas con alta eficiencia, sino que también ha logrado encontrar nuevas mutaciones responsables de los síntomas observados en el diagnóstico de las DHH, pretendiendo su incorporación al algoritmo diagnóstico de la ISTH. Como conclusión de lo expuesto, proponemos como hipótesis de trabajo que la NGS permitirá la búsqueda sistemática de mutaciones incluidas en un extenso panel de genes relacionados con alguna de las múltiples manifestaciones de las DHH, lo que contribuirá al diagnóstico correcto de estos pacientes. La finalidad de este panel es su aplicación rutinaria en el diagnóstico de todos los pacientes con sospecha de DHH. De este modo, se incrementará notablemente la posibilidad de obtener un diagnóstico correcto de estos enfermos

Evaluation of Implementation, Adaptation and Use of the Recently Proposed Urea Cycle Disorders Guidelines

BACKGROUND Implementation of guidelines and assessment of their adaptation is not an extensively investigated process in the field of rare diseases. However, whether targeted recipients are reached and willing and able to follow the recommendations has significant impact on the efficacy of guidelines. In 2012, a guideline for the management of urea cycle disorders (UCDs) has been published. We evaluate the efficacy of implementation, adaptation, and use of the UCD guidelines by applying different strategies. METHODS (i) Download statistics from online sources were recorded. (ii) Facilities relevant for the implementation of the guidelines were assessed in pediatric units in Germany and Austria. (iii) The guidelines were evaluated by targeted recipients using the AGREE instrument. (iv) A regional networking-based implementation process was evaluated. RESULTS (i) Download statistics revealed high access with an increase in downloads over time. (ii) In 18% of hospitals ammonia testing was not available 24/7, and emergency drugs were often not available. (iii) Recipient criticism expressed in the AGREE instrument focused on incomplete inclusion of patients' perspectives. (iv) The implementation process improved the availability of ammonia measurements and access to emergency medication, patient care processes, and cooperation between nonspecialists and specialists. CONCLUSION Interest in the UCD guidelines is high and sustained, but more precise targeting of the guidelines is advisable. Surprisingly, many hospitals do not possess all facilities necessary to apply the guidelines. Regional network and awareness campaigns result in the improvement of both facilities and knowledge

Biochemical and molecular characterisation of the dyslipidaemia in Portugal

Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Dyslipidaemia is one of the major modifiable independent risk factors for cardiovascular disease (CVD), with both genetic and environmental determinants. Although genetic risk factors are considered as non modifiable, their CVD-associated risk can be prevented if early identified. The correct and early identification of dyslipidaemia is important for a better patient management and could definitely contribute to CVD prevention. This thesis intended the most complete characterisation of the dyslipidaemia in the Portuguese population, both biochemically and molecularly. Reference values based on population-specific percentiles for lipid and lipoprotein biomarkers were provided for the first time in the Portuguese population, namely total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), apolipoprotein A1 (apoA1), apolipoprotein B (apoB), small, dense LDL-C (sdLDL-C), lipoprotein(a) [Lp(a)], as well apoB/apoA1 and sdLDL-C/LDL-C ratios, and non-HDL-C and remnant cholesterol. To our knowledge, the sdLDL-C percentiles were the first to be established in an European population. The percentiles were estimated through a rigorous methodology and compared with other population percentiles by a very visual and feasible method, showing relevant differences. These newly determined reference values for lipid biomarkers were then used to characterise the dyslipidaemia in our population, and can now be used in the clinic for a better patient care and management. More than cholesterol per se, our study highlighted apoB and sdLDL-C as important biomarkers to be used in dyslipidaemia evaluation. Individuals presenting extreme phenotypes were further investigated to assess possible monogenic causes, and three individuals were found to have familial hypercholesterolemia (FH), the most common genetic dyslipidaemia and one of the most common disorders that confer an increased cardiovascular risk. Finally, in an attempt to explore the causes for the FH phenotype, a polygenic risk score was validated for the first time in the Portuguese population. A total of 289 index cases were identified with monogenic FH and other causes for their dyslipidaemia, and also 100 were identified with polygenic hypercholesterolaemia, representing 53.21% of the cohort. From the monogenic causes, 91.35% have a mutation in LDLR, 4.84% in APOB, 1.04% in PCSK9 and 2.08% had mutations in phenocopies genes (LIPA, APOE, ALB), suggesting that all those monogenic and polygenic causes should be always investigated for a better patient identification. This study provided the most complete characterisation of the dyslipidaemia in the Portuguese population, and important evidences for dyslipidaemia evaluation has been produced. The results obtained have application, not only for Portugal or a south European populations, but also might have an worldwide utility for the dyslipidaemia assessment. Together, the results obtained provide useful information on an important cardiovascular risk factor and should help to tackle and identify at risk situations that need urgent measures.Fundação para a Ciência e a Tecnologia (SFRH/BD/52494/2014

Molecular genetic investigation into inherited thrombocytopenia

Inherited thrombocytopenias are a heterogeneous group of disorders characterised by abnormally low platelet counts, often with secondary qualitative defects in platelet function, which can be associated with abnormal bleeding. Next generation sequencing has only previously been employed in small-scale studies and for the confirmation of suspected variants. This study presents the first large-scale approach to fully categorise inherited thrombocytopenia. Ninety-five patients were recruited to the UK-Genotyping and Phenotyping (GAPP) study with inherited thrombocytopenia of unknown genetic aetiology. An average platelet count of 88x109/L was observed across all individuals. Platelet function testing revealed a secondary phenotypic defect in addition to the reduction in platelet count in 71% (61/86). Of the 95 patients, 69 patients, encompassing 47 index cases, were analysed by whole exome sequencing. A variant with a positive prediction of pathogenicity was identified in 40% (19/47) of patients and overall plausible candidate variants of disease were identified in 69% of index cases. Subsequently an inherited thrombocytopenia specific gene panel was developed to serve as a pre-screen for patients prior to whole exome sequencing. In total, candidate variants in genes previously known to be implicated with disease were identified in 77% (20/26) of patients analysed. In conclusion, the application of a combined phenotyping and genotyping approach to patients with inherited thrombocytopenia is an effective and efficient means of complete diagnosis. It also has the added benefit of identifying novel candidate variants in genes not previously known to cause disease that may further our understanding of the processes surrounding haemostasis through subsequent functional studies

Applications of Genetic Testing for Endocrine and Metabolic Disorders

Knowledge of inherited diseases and the ability to rapidly, efficiently and comprehensively perform genetic testing are advancing steadily. However, the ideal approach to translate this ability into clinical applications for endocrine disorders has yet to be determined. This work focuses on aspects of clinically translating knowledge of select heritable endocrine and metabolic conditions. For maturity onset diabetes of the young (MODY), a monogenic disorder with no current consensus guidelines governing testing procedures, this work addresses methods to improve detection by validating the use of next generation sequencing-based techniques to identify MODY cases and to detect copy number variations. For very severe hypertriglyceridemia, a largely polygenic trait, this work explores clinical differences associated with the underlying genotype, assesses treatment of pancreatitis, the most severe acute complication of hypertriglyceridemia, and presents a population-based study of Ontario adults to identify the most important modifiable risk factors associated with expression of hypertriglyceridemia, and to identify any gaps in appropriate care for this population. For heterozygous familial hypercholesterolemia, a condition for which universal genetic screening has been recommended, this work explores the personal impact of this diagnosis on the patient in terms of quality of life, lifestyle and self-care habits. The ultimate goal of this project is to expand the available knowledge on how best to translate the laboratory ability and findings into the clinical realm for these select endocrine and metabolic conditions