Avaliação e validação da utilidade clínica do sequenciamento de nova geração (NGS) para confirmação do diagnóstico de doenças lisossômicas selecionadas

Introdução: As doenças lisossômicas (DLs) são patologias genéticas que, apesar de serem classificadas como raras, acometem uma significativa porcentagem da população. Muitos fatores tornam seu diagnóstico desafiador, entre eles a variabilidade no fenótipo, com poucos sinais e sintomas clínicos específicos. O desenvolvimento de ferramentas inovadoras de investigação, destacando-se os novos métodos de sequenciamento massivo, reduziria a “odisseia diagnóstica” enfrentada pelo paciente e sua família, proporcionaria um diagnóstico mais precoce com melhor resultado no tratamento nas situações em que há terapia disponível, além de um adequado aconselhamento genético. A tecnologia de sequenciamento de nova geração (next-generation sequencing, NGS) tem claras vantagens sobre as técnicas de sequenciamento convencional, oferecendo um alto rendimento diagnóstico ao permitir definir um espectro mutacional abrangente. O NGS permite o sequenciamento de vários genes simultaneamente com custo global relativamente baixo, tornando painéis de genes uma alternativa atrativa para o screening genético. Esta abordagem é capaz de detectar, de maneira altamente específica, variantes missense, nonsense, de sítio de splicing, e pequenas indels, e algumas grandes deleções em homozigose ou hemizigose. No entanto, para que essa tecnologia seja aplicável à prática clínica, é necessária uma etapa de validação prévia para a determinação dos parâmetros críticos de análise desde o processamento da amostra até a análise e interpretação de dados. Após a validação dos métodos, será possível a avaliação da utilidade clínica de painéis NGS para diversas aplicações, incluindo seu uso como método confirmatório nos casos de triagem neonatal alterada para doenças lisossômicas. Objetivos: 1) Desenhar e validar uma estratégia baseada no NGS para a análise de 24 genes, incluídos em 3 painéis distintos de acordo com parâmetros pré definidos, e associados com 22 DLs; 2) Avaliar a qualidade e eficiência do ADN extraído de sangue impregnado em papel filtro (SIPF) para uso no NGS; 3) Avaliar a utilidade clínica do NGS para: a) diagnóstico molecular, b) diagnóstico diferencial e, c) confirmação diagnóstica de casos alterados na triagem neonatal para pelo menos 4 dessas condições (doença de Gaucher, Fabry, Pompe e Niemann-Pick tipo A/B). Metodologia: Estudo descritivo, amostragem por conveniência, incluindo pacientes com diagnóstico clínico e bioquímico das DLs estudadas. Foi utilizada a plataforma NGS Ion Torrent Personal Genome Machine (Thermo Fisher Scientific) para sequenciamento de três painéis de genes, desenhados através da ferramenta Ion AmpliseqTM Designer (Thermo Fisher Scientific). Para a validação foram incluídos pacientes com diagnóstico molecular prévio por sequenciamento de Sanger. Para a extração de ADN de SIPF, foi avaliado o método não comercial de fenol-clorofórmio. A utilidade clínica do NGS foi estabelecida através do: a) diagnóstico molecular de um grupo de pacientes com suspeita de lipofuscinose ceróide neuronal tipo 2 (CLN2) para o estabelecimento do genótipo (TPP1) associado à doença, assim como estabeleceu-se o diagnóstico molecular de outros pacientes com suspeita clínica e/ou bioquímica de algumas das DLs selecionadas, b) diagnóstico diferencial de um paciente utilizando um dos painéis NGS desenhados e, c) avaliação das amostras de recém-nascidos (obtidas em estudo paralelo) que tiveram resultados inicialmente alterados na triagem neonatal bioquímica para algumas das DLs selecionadas. Resultados: 1) Três painéis foram desenhados, cada um consistindo em dois pools de primers que amplificam as regiões codificantes e 20pb da junção exon-intron. Os painéis A, B e C têm amplitude de cobertura de 97.74, 99.6 e 98.38%, respectivamente. Nesta validação foi possível estabelecer a sensibilidade, especificidade e limitações de cada painel (Artigo 1); 2) foi estabelecida a metodologia para a extração de ADN para seu uso em vários processos moleculares downstream: PCR convencional, Real-Time PCR, PCR-RFLP, MLPA, Sequenciamento de Sanger e NGS na plataforma Ion Torrent PGMTM (Artigo 2); 3) A utilidade clínica dos painéis desenhados foi estabelecida através de: a) Genotipagem de 48 pacientes com suspeita clínica e bioquímica de CLN2 (painel C) (Artigo 4), e de 3 outros casos: 1 caso de Niemann-Pick tipo B (SMPD1) (painel B) e 2 casos de doença de Danon (LAMP2) (painel A) (Artigo 1), b) o diagnóstico diferencial foi realizado através do caso de um paciente com suspeita inicial de Niemann-Pick C que, após a análise molecular, foi diagnosticado com Niemann-Pick tipo B utilizando o painel B, (Artigo 1) c) a aplicabilidade dos painéis de genes para a confirmação diagnóstica em casos de triagem neonatal foi realizada em 2 casos com resultados alterados na triagem neonatal: um caso de Pompe (painel A) e outro de Gaucher (painel B) (Artigo 3 e Anexo I). Conclusão: A abordagem por NGS através do uso de painéis, foi capaz de identificar diferentes alterações genéticas nos genes estudados, incluindo variantes do tipo missense, nonsense, de sítio de splicing, e pequenas indels. Estes painéis oferecem uma estratégia de triagem dos 24 genes associadas às DLs selecionadas. Este trabalho foi inovador ao utilizar o NGS para a análise molecular dos genes associados às DLs no Brasil; foi desenvolvido no Laboratório de Genética e Biologia Molecular do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre, conhecido como um centro de referência em diagnóstico e pesquisa de DLs, e que atualmente tem os painéis NGS como principal ferramenta de diagnóstico molecular.Introduction: Lysosomal diseases (LDs) are genetic pathologies that, although classified as rare, affect a significant percentage of the population. Many factors make its diagnosis challenging, including variability in phenotype, with few specific clinical signs and symptoms. The development of innovative research tools, highlighting new methods of massive sequencing, would reduce the "diagnostic odyssey" faced by the patient and his family, would provide an earlier diagnosis with better treatment outcome in situations where therapy is available, in addition to adequate genetic counseling. Next-generation sequencing (NGS) technology has clear advantages over conventional sequencing techniques, offering a high diagnostic yield by enabling a comprehensive mutational spectrum to be defined. The NGS allows the sequencing of several genes simultaneously with relatively low overall cost, making gene panels an attractive alternative for genetic screening. This approach is able to detect, in a highly specific manner, missense, nonsense, splicing, and small indels variants, and some large deletions in homozygosis or hemizygosis. However, for this technology to be applicable to clinical practice, a prior validation step is required to determine the critical parameters of analysis from sample processing to data analysis and interpretation. After validation of the methods, it will be possible to evaluate the clinical usefulness of NGS panels for several applications, including their use as a confirmatory method in cases of altered neonatal screening for lysosomal diseases. Objectives: 1) To design and validate an NGS-based strategy for the analysis of 24 genes, included in 3 different panels according to pre-defined parameters, and associated with 22 LDs; 2) To evaluate the quality and efficiency of DNA extracted from dried blood spots (DBS) for its use in NGS; 3) To evaluate the clinical use of NGS for: a) molecular diagnosis, b) differential diagnosis and c) diagnostic confirmation of altered cases in neonatal screening for at least 4 of these conditions (Gaucher, Fabry, Pompe and Niemann-Pick type A / B). Methodology: Descriptive study, convenience sampling, including patients with clinical and biochemical diagnosis of selected LDs. The NGS Ion Torrent Personal Genome Machine (Thermo Fisher Scientific) platform was used for the sequencing of three gene panels, designed using the Ion Ampliseq ™ Designer tool (Thermo Fisher Scientific). For the validation, patients with previous molecular diagnosis by Sanger sequencing were included. For the extraction of DBS DNA, a non-commercial method of phenol-chloroform was evaluated. The clinical utility of NGS was established through: a) molecular diagnosis of a group of patients with suspected type 2 neuronal cercoid lipofuscinosis (CLN2) to establish the disease-associated genotype (TPP1), as well as the molecular diagnosis of other patients with clinical and / or biochemical suspicion of some of the selected LDs, b) differential diagnosis of a patient using one of the designed NGS panels, and c) evaluation of the newborn samples (obtained in a parallel study) that initially had altered results in the neonatal screening for some of the selected LDs. Results: 1) Three panels were designed, each consisting of two pools of primers that amplify the coding regions and 20pb of the exon-intron junction. Panels A, B and C have coverage range of 97.74, 99.6 and 98.38%, respectively. In this validation it was possible to establish the sensitivity, specificity and limitations of each panel (Article 1); 2) the methodology for the extraction of DNA for its use in several downstream molecular processes was established: conventional PCR, Real-Time PCR, PCR-RFLP, MLPA, Sanger Sequencing and NGS in the Ion Torrent PGMTM platform; 3) The clinical use of the panels was established through: a) Genotyping of 48 patients with clinical and biochemical CLN2 (panel C) (Article 4), and of 3 other cases: 1 case of Niemann-Pick type B ( SMPD1) (panel B) and 2 cases of Danon's disease (LAMP2) (panel A) (Article 1), b) he differential diagnosis was made through the case of a patient with initial suspicion of Niemann-Pick C who, after molecular analysis, was diagnosed with Niemann-Pick type B using panel B (Article 1), c) the applicability of the gene panels for diagnostic confirmation in cases of neonatal screening was performed in 2 cases with altered results in neonatal screening: one case of Pompe (panel A) and another case of Gaucher (panel B) (Article 3 and Annex I) . Conclusion: The NGS approach using gene panels was able to identify different genetic alterations in the genes studied, including missense, nonsense, splicing variants, and small indels. These panels offer a screening strategy for the 24 genes associated with the selected LDs. This work was innovative by using NGS for the molecular analysis of genes associated with LDs in Brazil; it was developed in the Laboratory of Genetics and Molecular Biology of the Medical Genetics Service of the Hospital de Clinicas de Porto Alegre, known as a reference center in diagnosis and research of LDs, and that currently has the NGS panels as the main tool of molecular diagnosis

Detección de E8SJM, mutación más frecuente para la enfermedad por almacenamiento de ésteres de colesterol (CESD), en explantes de hígado de pacientes sometidos a trasplante hepático en el Hospital de Clínicas de Porto Alegre (HCPA)-Brasil

Publicación a texto completo no autorizada por el autorLa enfermedad por almacenamiento de ésteres de colesterol (CESD, Cholesteryl ester storage disease) es una enfermedad metabólica rara causada por la deficiencia de la actividad de la enzima lipasa ácida lisosomal, ocasionando la acumulación de ésteres de colesterol, particularmente en el hígado. Su presentación es bastante variable y su diagnóstico es un reto, cuando no es tratada a tiempo puede llevar a cirrosis, insuficiencia hepática y muerte. Además, existe un número de anomalías observadas en los pacientes con CESD que se sobreponen a diagnósticos más comunes, siendo probable que esté siendo subdiagnosticada. La mayoría de pacientes relatados hasta el momento, 60%, son portadores de la mutación E8SJM en el gen LIPA. El objetivo de la investigación fue detectar la variante patogénica E8SJM en los explantes de hígado de los pacientes sometidos a trasplante en el Hospital de Clínicas (HCPA) con diagnóstico clínico de esteatohepatitis no alcohólica y cirrosis criptogénica. De esta manera comprobaríamos si la CESD está siendo sub-diagnosticada y categorizada como otra enfermedad hepática entre los pacientes sometidos a trasplante. Se realizó el estudio con muestras parafinadas de explantes de hígado. De manera paralela también fue detectada en un banco de controles de amplificación, así como en pacientes con sospecha de CESD con ingreso al Servicio de Genética Médica del HCPA para análisis molecular del gen LIPA. La mutación E8SJM fue detectada por PCR en tiempo real utilizando una sonda personalizada, previa estandarización de todos los métodos. La mutación fue detectada en una paciente que ingresó vía redes de apoyo al diagnóstico de errores innatos del metabolismo con sospecha de DLAL. Este resultado fue confirmado por secuenciación por el método de Sanger y el genotipo fue establecido por secuenciación de nueva generación utilizando la plataforma Ion Torrent Personal Genome Machine®.Tesi

Evaluación de marcadores moleculares Ilps y Strs heterólogos en la anchoveta peruana Engraulis Ringens para estudios poblacionales

A pesar de la importancia económica de la anchoveta peruana, Engraulis ringens, en nuestro país, no existe información acerca de la variabilidad y estructura genético poblacional de esta especie, principalmente porque no se han identificado marcadores moleculares específicos Por esta razón, se evaluó la utilidad de cinco marcadores moleculares para la caracterización molecular de la anchoveta peruana: dos microsatélites, Ee10 y Ee2, reportados en la anchoveta europea E. encrasicolus, y tres marcadores intrónicos, basados en el polimorfismo en longitud: el intrón 3 de la cadena ligera de la miosina Mlc-3, el intrón 7 de la creatina quinasa Ck-7 y el intrón 5 de la hormona de crecimiendo Gh-5. Dos de ellos, el microsatélite Ee2, y el marcador intrónico Gh-5, no amplificaron en esta especie. Para el locus Ee10, se encontraron altas probabilidades de existencia de alelos nulos, 0.2005-0.7420, lo que explicaría el déficit significativo de heterocigotos encontrado. Basándose en la heterocigosidad, se reporta un nivel bajo de polimorfismo para Mlc-3 y un nivel alto para Ck-7. Los resultados muestran que sólo Ck-7 puede ser utilizado en futuros estudios poblacionales. Este marcador sugiere que las seis capturas colectadas a lo largo del litoral peruano, una del stock norte (Salaverry) y cinco del centro (Callao, Huacho, Supe y Pisco), constituyen una misma unidad reproductiva, desde un punto de vista genético. -- Palabras clave: Anchoveta peruana, marcador intrónico, marcador microsatélite, amplificación cruzada, EPIC-PCR, estudios genéticos--- Despite Engraulis ringens economic importance in our country, there is a lack of information about the variability and genetic population structure of this species, where no specific molecular markers have been identified. For this reason, five molecular markers were evaluated for the molecular characterization of the Peruvian anchovy: two microsatellites, Ee10 and Ee2, reported in the european anchovy E. encrasicolus, and three intronic length polymorphism markers (ILPs): myosin light chain intron 3 Mlc-3, creatin kinase intron 7 Ck-7 and the growth hormone intron 5 Gh-5. Two of them, the microsatellite Ee2 and the intronic marker Gh-5, did not amplify in this species. For Ee10 locus, there was a high probability of presence of null alleles, ranging from 0.2005 to 0.7420, which would explain the significant heterozygote deficiencies found. Based on the observed heterozygosity, a low polymorphism is reported for Mlc-3 and a high one for Ck-7. The results show that only Ck-7 can be used in future population studies. This marker suggests that the six collected sacks along the Peruvian coast, a northern stock (Salaverry) and five from the centre (Callao, Huacho, Supe and Pisco), constitute a single reproductive unit from a genetic point of view. -- Keywords: Peruvian anchovy, intronic marker, microsatellite marker, cross amplification, EPIC-PCR, genetic studies.Tesi

Precision medicine for lysosomal disorders

Precision medicine (PM) is an emerging approach for disease treatment and preventionthat accounts for the individual variability in the genes, environment, and lifestyle of each person.Lysosomal diseases (LDs) are a group of genetic metabolic disorders that include approximately70 monogenic conditions caused by a defect in lysosomal function. LDs may result from primarylysosomal enzyme deficiencies or impairments in membrane-associated proteins, lysosomal enzymeactivators, or modifiers that affect lysosomal function. LDs are heterogeneous disorders, and thephenotype of the affected individual depends on the type of substrate and where it accumulates,which may be impacted by the type of genetic change and residual enzymatic activity. LDs areindividually rare, with a combined incidence of approximately 1:4000 individuals. Specific therapiesare already available for several LDs, and many more are in development. Early identification mayenable disease course prediction and a specific intervention, which is very important for clinicaloutcome. Driven by advances in omics technology, PM aims to provide the most appropriatemanagement for each patient based on the disease susceptibility or treatment response predictionsfor specific subgroups. In this review, we focused on the emerging diagnostic technologies that mayhelp to optimize the management of each LD patient and the therapeutic options available, as well asin clinical developments that enable customized approaches to be selected for each subject, accordingto the principles of PM

A case report on the challenging diagnosis of neuronal ceroid lipofuscinosis type 2 (CLN2)

Neuronal ceroid lipofuscinoses (NCLs), also referred as “Batten disease”, are a group of thirteen rare genetic conditions, which are part of the lysosomal storage disorders. CLN type 2 (CLN2) is caused by the deficient activity of the tripeptidyl peptidase I (TPP1) enzyme, encoded by the TPP1 gene, most frequently leading to the classic late infantile phenotype. Nearly 140 CLN2- causing mutations have been described. In this case report, we describe the identification of a new disease-causing mutation and highlight the importance of appropriate laboratory investigation based on clinical suspicion. The collection of dried blood spots (DBS) on filter paper, which is a convenient sample, can be used to measure the TPP1 enzyme activity and detect CLN2-related mutations. Since the biochemical and genetic diagnoses are possible and as the disease progression is fast and the therapeutic window is short, the investigation of CLN2 should be always considered when this diagnostic hypothesis is raised in order to enable the patients to benefit from the specific pharmacological treatment

Phenotype-oriented NGS panels for mucopolysaccharidoses : validation and potential use in the diagnostic flowchart

Mucopolysaccharidosis (MPS) are a group of rare genetic disorders caused by deficiency in the activity of specific lysosomal enzymes required for the degradation of glycosaminoglycans (GAGs). A defect in the activity of these enzymes will result in the abnormal accumulation of GAGs inside the lysosomes of most cells, inducing progressive cellular damage and multiple organ failure. DNA samples from 70 patients with biochemical diagnosis of different MPSs genotypes confirmed by Sanger sequencing were used to evaluate a Next Generation Sequencing (NGS) protocol. Eleven genes related to MPSs were divided into three different panels according to the clinical phenotype. This strategy led to the identification of several pathogenic mutations distributed across all exons of MPSs-related genes. We were able to identify 96% of all gene variants previously identified by Sanger sequencing, showing high sensitivity in detecting different types of mutations. Furthermore, new variants were not identified, representing 100% specificity of the NGS protocol. The use of this NGS approach for genotype identification in MPSs is an attractive option for diagnosis of patients. In addition, the MPS diagnosis workflow could be divided in a two-tier approach: NGS as a first-tier followed by biochemical confirmation as a second-tier