Aspectos moleculares relacionados ao diagnóstico e progressão da Doença de Fabry

A Doença de Fabry (DF) é uma doença rara com herança ligada ao X causada pela deficiência de atividade da hidrolase lisossomal α-galactosidase A (α-GAL), codificada pelo gene GLA. Este defeito enzimático leva ao acúmulo de glicoesfingolipídios, principalmente globotriaosilceramida (Gb3) pelo corpo. Pacientes com a forma clássica apresentam baixa atividade residual da enzima com início dos sintomas na infância e desenvolvimento de sintomas cardíacos, cerebrovasculares e/ou renais. Pacientes com apresentações não clássicas tem atividade enzimática residual maior, início tardio dos sintomas e podem ser mono ou oligo-sintomáticos. Características marcantes da DF são alta variabilidade alélica e fenotípica. Dessa forma, o objetivo desta tese foi investigar aspectos moleculares relacionados ao diagnóstico e progressão da Doença de Fabry na população brasileira. Para triagem bioquímica de pacientes do sexo feminino em populações de risco, foi proposta uma metodologia de uso combinado de atividade de α-GAL em leucócitos e plasma. Com o uso de valores de referência gênero-específicos, esta metodologia amplamente acessível pode reduzir o número de amostras que necessitam de triagem genética em 35%. Com relação à triagem molecular, foi descrito um protocolo com a técnica de HRM (high resolution melting) para amostras de sangue coletadas em EDTA. Todas as variantes analisadas neste estudo resultaram em perfis distintos dos controles normais. Além disso, com exceção das variantes localizadas no éxon 7, também foi possível a identificação de todos os genótipos diferentes analisados. A análise molecular do gene GLA por sequenciamento de Sanger foi realizada em 408 pacientes brasileiros, provenientes de 213 famílias, com histórico familiar ou suspeita clínica de DF. Conforme o esperado para uma doença com mutações privadas, a maioria destas variantes foi encontrada em famílias únicas. Das 26 variantes identificadas em regiões codificantes, cerca de 80% eram do tipo missense, o que está de acordo com a literatura. Em seguida, a existência de genes modificadores do fenótipo cardíaco foi avaliada por análise de exoma de pacientes com ou sem doença cardíaca. A frequência da variante rs3188055 gene inositol polifosfato 5- fosfatase (INPP5F) foi significativamente maior no grupo com doença cardíaca. Portanto; INPP5F, envolvido na maturação e reciclagem dos endossomos e regulação negativa da 11 hipertrofia cardíaca, é um possível modulador da doença cardíaca em DF. Finalmente, a variabilidade fenotípica vista em mulheres com DF foi analisada com o uso de transcriptomas de células sanguíneas. Foram obtidos perfis de expressão distintivos para pacientes com diferentes manifestações: exclusivamente cardíacas, exclusivamente renais ou ambas. Os genes com expressão diferencial validados (DEFA1, A1B, A3; CEACAM8, PRKXP1, IFI44, IFI44L, IFIT1, HERC5, EIF2AK2 e RSAD2), representam possíveis vias especificas das manifestações analisadas, envolvidas com a variabilidade vista em DF, que podem auxiliar no manejo clínico dos pacientes.Fabry disease (FD) is a rare X-linked disorder resulting from deficient activity of the lysosomal hydrolase α-galactosidase A (α-GAL), encoded by the GLA gene. This deficiency leads to the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), throughout the body. Patients with the classical form of the disease have low residual enzymatic activity with childhood onset of symptoms and development of cardiac, cerebrovascular and/or renal complications. Patients with non-classical phenotypes have higher residual activity, later onset of symptoms and are mono or oligo-symptomatic. Striking features of FD are high allelic and phenotypic variability. Thus, the aim of this thesis was to investigate molecular aspects related to the diagnosis and progression of Fabry disease in the Brazilian population. For biochemical screening of female patients in risk populations, a methodology of combined use of α-GAL activity in leukocytes and plasma was proposed. With the use of gender-specific reference values, this widely accessible methodology can reduce the number of samples that require genetic screening by 35%. Regarding molecular screening, a protocol with high resolution melting (HRM) technique for blood samples collected in EDTA was described. All the variants analyzed in this study displayed different profiles from normal controls. In addition, with the exception of variants located in exon 7, it was also possible to identify all the different genotypes analyzed. The molecular analysis of the GLA gene by Sanger sequencing was performed in 408 Brazilian patients, from 213 families, with family history or clinical suspicion of FD. As expected for a disease with private mutations, most of these variants were found in single families. Of the 26 variants identified in coding regions, about 80% were missense, which is in agreement with the literature. Then, the existence of cardiac phenotype modifier genes was evaluated by exome analysis of patients with and without heart disease. The frequency of variant rs3188055 variant in Inositol Polyphosphate-5-Phosphatase F gene (INPP5F) was significantly higher in the group with heart disease. Therefore, INPP5F, which is involved in the maturation and recycling of endosomes and negative regulation of cardiac hypertrophy, is a possible modulator of cardiac disease in FD. Finally, the phenotypic variability seen in women with FD was analyzed with the use of transcriptomes from whole blood. Distinctive expression profiles were obtained for patients with different 13 manifestations: exclusively cardiac, exclusively renal or both. The validated genes with differential expression (DEFA1, A1B, A3, CEACAM8, PRKXP1, IFI44, IFI44L, IFIT1, HERC5, EIF2AK2 and RSAD2) indicate possible disease specific pathways involved in phenotypic variability seen in FD, which might aid in patient management

Effects of gene therapy on cardiovascular symptoms of lysosomal storage diseases

Lysosomal storage diseases (LSDs) are inherited conditions caused by impaired lysosomal function and consequent substrate storage, leading to a range of clinical manifestations, including cardiovascular disease. This may lead to significant symptoms and even cardiac failure, which is an important cause of death among patients. Currently available treatments do not completely correct cardiac involvement in the LSDs. Gene therapy has been tested as a therapeutic alternative with promising results for the heart disease. In this review, we present the results of different approaches of gene therapy for LSDs, mainly in animal models, and its effects in the heart, focusing on protocols with cardiac functional analysis

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

Genome-wide interrogation of structural variation reveals novel African-specific prostate cancer oncogenic drivers

ADDITIONAL FILE 1: FIGURE S1. Concordant SV call generation from Manta and GRIDSS. FIGURE S2. Summary of SVs in each type, compared to other studies. FIGURE S3. CIRCOS plot of hyper-SV mutated tumours. FIGURE S4. The spread of SV breakpoints and samples in each 1 Mbp genomic bin. FIGURE S5. TMPRSS2-ERG fusion with interstitial region retention. TABLE S1. Clinical and pathological characteristics of 180 prostate cancer patients included in this study. TABLE S2. Biallelic assessment of CDK12 in hyper-duplicated samples. TABLE S3. Biallelic assessment of BRCA2 in hyper-deleted samples.ADDITIONAL FILE 2: TABLE S4. Summary of gene fusions identified from SVs. ADDITIONAL FILE 3: TABLE S5. SV calls resulting in gene fusions.DATA AND MATERIALS AVAILABILITY : The datasets analysed in this study were obtained and accessible through Jaratlerdsiri et al [6], with sequence data deposited in the European GenomePhenome Archive (EGA; https://ega-archive.org) under overarching accession EGAS00001006425 and including the Southern African Prostate Cancer Study (SAPCS) Dataset (EGAD00001009067) and Garvan/St Vincent’s Prostate Cancer Database (EGAD00001009066). The computational code used to analyse SV subtypes, SV hotspots and gene fusions is available on GitHub [68].BACKGROUND : African ancestry is a significant risk factor for advanced prostate cancer (PCa). Mortality rates in sub- Saharan Africa are 2.5-fold greater than global averages. However, the region has largely been excluded from the benefits of whole genome interrogation studies. Additionally, while structural variation (SV) is highly prevalent, PCa genomic studies are still biased towards small variant interrogation. METHODS : Using whole genome sequencing and best practice workflows, we performed a comprehensive analysis of SVs for 180 (predominantly Gleason score ≥ 8) prostate tumours derived from 115 African, 61 European and four ancestrally admixed patients. We investigated the landscape and relationship of somatic SVs in driving ethnic disparity (African versus European), with a focus on African men from southern Africa. RESULTS : Duplication events showed the greatest ethnic disparity, with a 1.6- (relative frequency) to 2.5-fold (count) increase in African-derived tumours. Furthermore, we found duplication events to be associated with CDK12 inactivation and MYC copy number gain, and deletion events associated with SPOP mutation. Overall, African-derived tumours were 2-fold more likely to present with a hyper-SV subtype. In addition to hyper-duplication and deletion subtypes, we describe a new hyper-translocation subtype. While we confirm a lower TMPRSS2-ERG fusion-positive rate in tumours from African cases (10% versus 33%), novel African-specific PCa ETS family member and TMPRSS2 fusion partners were identified, including LINC01525, FBXO7, GTF3C2, NTNG1 and YPEL5. Notably, we found 74 somatic SV hotspots impacting 18 new candidate driver genes, with CADM2, LSAMP, PTPRD, PDE4D and PACRG having therapeutic implications for African patients. CONCLUSIONS : In this first African-inclusive SV study for high-risk PCa, we demonstrate the power of SV interrogation for the identification of novel subtypes, oncogenic drivers and therapeutic targets. Identifying a novel spectrum of SVs in tumours derived from African patients provides a mechanism that may contribute, at least in part, to the observed ethnic disparity in advanced PCa presentation in men of African ancestry.The Medical Health and Medical Research Council (NHMRC) of Australia, University of Sydney Bridging Grant, the USA. Department of Defense (DoD) Prostate Cancer Research Program (PCRP) Idea Development.https://genomemedicine.biomedcentral.comam2023School of Health Systems and Public Health (SHSPH

African-specific molecular taxonomy of prostate cancer

Data availability DNA-sequencing data have been deposited at the European Genome- Phenome Archive (EGA) under overarching accession EGAS00001006425 and including the Southern African Prostate Cancer Study (SAPCS) Dataset (EGAD00001009067 and Garvan/St Vincent’s Prostate Cancer Database EGAD00001009066). Academic researchers meeting the data-access policy criteria may apply for data access through the respective data access committees. CPGEA data are available through http://www.cpgea.com. PCAWG data are available at ICGC Data Portal (https://dcc.icgc.org/releases/PCAWG).Prostate cancer is characterized by considerable geo-ethnic disparity. African ancestry is a significant risk factor, with mortality rates across sub-Saharan Africa of 2.7-fold higher than global averages. The contributing genetic and non-genetic factors, and associated mutational processes, are unknown. Here, through whole-genome sequencing of treatment-naive prostate cancer samples from 183 ancestrally (African versus European) and globally distinct patients, we generate a large cancer genomics resource for sub-Saharan Africa, identifying around 2 million somatic variants. Significant African-ancestry-specific findings include an elevated tumour mutational burden, increased percentage of genome alteration, a greater number of predicted damaging mutations and a higher total of mutational signatures, and the driver genes NCOA2, STK19, DDX11L1, PCAT1 and SETBP1. Examining all somatic mutational types, we describe a molecular taxonomy for prostate cancer differentiated by ancestry and defined as global mutational subtypes (GMS). By further including Chinese Asian data, we confirm that GMS-B (copy-number gain) and GMS-D (mutationally noisy) are specific to African populations, GMS-A (mutationally quiet) is universal (all ethnicities) and the African–European-restricted subtype GMS-C (copy-number losses) predicts poor clinical outcomes. In addition to the clinical benefit of including individuals of African ancestry, our GMS subtypes reveal different evolutionary trajectories and mutational processes suggesting that both common genetic and environmental factors contribute to the disparity between ethnicities. Analogous to gene–environment interaction—defined here as a different effect of an environmental surrounding in people with different ancestries or vice versa—we anticipate that GMS subtypes act as a proxy for intrinsic and extrinsic mutational processes in cancers, promoting global inclusion in landmark studies.The National Health and Medical Research Council (NHMRC) of Australia, NHMRC Ideas Grants, University of Sydney Bridging Grant, the US Department of Defense (DoD) Prostate Cancer Research Program (PCRP) Idea Development Award TARGET Africa.http://www.nature.com/natuream2023School of Health Systems and Public Health (SHSPH

Efeitos da terapia de reposição enzimática com início tardio no modelo miurino de mucopolissacaridose do tipo I

A Mucopolissacaridose do tipo I (MPS I) é uma doença autossômica recessiva causada pela deficiência da hidrolase lisossomal α-L-iduronidase (IDUA, EC 3.2.1.76). Essa deficiência leva ao acúmulo progressivo dos glicosaminoglicanos (GAGs) heparan e dermatan sulfato nos tecidos com subsequente alteração da função celular e dano em múltiplos órgãos. Existem evidências na literatura de que a introdução precoce da Terapia de Reposição Enzimática (TRE) leva a um melhor prognóstico, principalmente para pacientes com a forma grave da doença (Síndrome de Hurler), prevenindo ou minimizando danos irreversíveis. Tendo em vista que a maioria dos pacientes brasileiros com MPS I é diagnosticada tardiamente e não recebe tratamento imediato, o objetivo deste estudo foi avaliar os efeitos da TRE na reversibilidade dos sintomas no modelo murino de MPS I. Animais com MPS I foram tratados dos 6 aos 8 meses com laronidase na dose de 1,2mg/kg a cada duas semanas e comparados com camundongos normais e MPS I não tratados de 8 meses. A TRE tardia foi efetiva na redução de GAGs urinários e viscerais. Apesar da normalização dos GAGs do miocárdio e da fração de encurtamento ventricular esquerda, a função cardíaca não foi completamente restaurada. A fração de ejeção ventricular esquerda e a razão entre aceleração e ejeção na artéria pulmonar dos camundongos tratados atingiram apenas níveis intermediários entre camundongos normais e não tratados. Não foram encontradas diferenças estatisticamente significativas na espessura da parede da aorta, mas as válvulas cardíacas foram significativamente reduzidas no grupo tratado. Uma grande variabilidade nos resultados dos testes comportamentais foi encontrada nos animais tratados. Esse achado não pode ser correlacionado com nenhuma outra variável como níveis de GAGs ou atividade de catepsina D no córtex cerebral, além da função cardíaca ou formação de anticorpos. Todos os animais que receberam laronidase desenvolveram anticorpos contra a enzima, sem que os níveis de anticorpos apresentassem correlação com os outros parâmetros analisados. Em conclusão, a administração da TRE tardia melhora diversos aspectos da doença e deve ser considerada sempre que possível.Mucopolysaccharidosys type I (MPS I) is a rare disorder caused by deficiency of the lysosomal hydrolase α-L-iduronidase (IDUA, EC 3.2.1.76). This deficiency leads to progressive storage of glycosaminoglycans (GAGs) heparan and dermatan sulphate, with subsequent disturbances in cell function and multiorgan damage. There is a consensus in the literature that early Enzyme Replacement Therapy (ERT) leads to a better outcome, particularly in patients with the severe form of the disease (Hurler syndrome), preventing or minimizing irreversible damage. Since most Brazilian patients are diagnosed late and don’t receive immediate treatment, the aim of this study was to evaluate the effects of late ERT on symptom reversibility in a MPS I murine model. We treated 10 MPS I mice from 6 to 8 months (ERT 6-8mo) with 1.2mg laronidase/kg every 2 weeks and compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). Late ERT was effective reducing urinary and visceral GAGs to normal levels. Although myocardium GAGs and left ventricular (LV) shortening fraction were normalized, cardiac function wasn’t completely restored. LV ejection fraction and acceleration/ejection ratio at the pulmonary valve reached intermediary levels between normal and untreated MPS I mice. While no significant results were found on aortic wall width, heart valves were significantly smaller in the ERT 6-8mo than in untreated mice. A wide variability was found on the behavior tests of treated animals. No correlation was found between this finding and any other variable, such as GAG levels, cerebral cortex cathepsin D activity, heart function or antibody formation. All animals treated with laronidase developed antibodies against the enzyme but no correlation was found with other parameters analyzed. In conclusion, late ERT improves many aspects of the disease and should be considered whenever possible

Diagnóstico molecular de doença de Fabry em amostras de sangue

A Doença de Fabry (DF) é uma doença lisossômica de depósito ligada ao cromossomo X. É causada pela deficiência da enzima -Galactosidase A, codificada pelo gene GLA localizado na região Xq21.33-Xq22. A progressão da doença leva a danos vasculares, renais e em diversos outros tecidos. Os pacientes podem apresentar a forma clássica ou as variantes renal, cardíaca e neurológica. Homens afetados geralmente apresentam atividade muito reduzida da enzima. Entretanto, devido à inativação aleatória do X, mulheres portadoras apresentam uma grande variabilidade na expressão clínica, podendo ser assintomáticas ou apresentar formas moderadas ou graves da doença. Logo, o diagnóstico bioquímico de mulheres é, na maioria das vezes, inconclusivo. Portanto, o diagnóstico molecular é fundamental para tratamento e aconselhamento genético. A DF apresenta alta heterogeneidade alélica e a maioria das mutações é privada. O objetivo principal desse estudo foi analisar o gene GLA em 301 amostras de pacientes latinoamericanos, originárias do Brasil (n=277), México (n=14) e Colômbia (n=10). Em 51 pacientes foram encontrados quatro polimorfismos não patogênicos: c.-12G>A (n=13), c.370-77_81delCAGCC (n=10), c.-10C>T (n=6) e p.A292D (n=3). Associações entre eles também foram encontradas; sendo a mais comum c.-10C>T em combinação com c.370-77_81delCAGCC (n=9). Ao total foram diagnosticados 50 pacientes com 12 mutações patogênicas já descritas: c.30-32delG, c.195-1G>C, p.R112S, p.R118C, p.M187T, p.C202Y, c.718_719delAA, p.V269M, p.D313Y, p.R342Q , p.R356W e p.R342X. Em 14 pacientes de uma família brasileira, foi encontrada a mutação nova c.467C>A (p.A156D), predita como possivelmente patogênica. A atividade enzimática em heterozigotas dessa família ficou abaixo do normal em plasma e muito abaixo do normal em leucócitos. Comparando-se a sensibilidade e especificidade do diagnóstico bioquímico em papel filtro de mulheres (n=70, 15 heterozigotas), leucócitos e plasma (n=19, 11 heterozigotas) com a Curva ROC, atividade em plasma se mostrou muito mais eficiente, com valores de 91% e 100%, respectivamente. Esse ensaio também foi o único que se mostrou capaz de não gerar falso-positivos e gerou apenas 27% de falso-negativos