The complexity of identification of pathogenic variants

Introduction: Natural occurring genomic variant, from single nucleotide to balanced, unbalanced and complex rearrangements, spanning large chromosomal regions, has been reported to cause human pathologies. As such, we present cases with neurodevelopmental disorder, infertility, and recurrent miscarriage, which reflect the complexity of the identification of pathogenic variants, considering the variation spectrum, the underlying pathogenic mechanisms, and the heterogeneous clinical presentations. Methods: Long and small insert genomic sequencing (GS) was applied to four cases. Variants were identified from GS data mapped against the reference human genome and confirmed through Sanger sequencing. Results were interpreted using SVInterpreter, Exomiser, genotype-phenotype correlation and convergent genomic data analysis. Results: Although the first case is a carrier of a t(17;19)(p13.1;p13.3)mat, disrupting GSG1L2, and of a presumably paternally inherited dup(2)(q14.3q21.1), encompassing the autosomal dominant (AD) phenotype-associated PROC and HS6ST1 genes, the identified novel frameshift c.4442del, p.(Gly1481Valfs*21) variant of CHD4, was considered the disease-causing variant, since the proband’s phenotype fits the CHD4-associated Sifrim-Hitz-Weiss syndrome (Da Silva et al., 2022). Cases 2 and 3 were both reported with infertility, and carriers of t(5;9)(q31.3;p13) and t(4;21)(p14;q21.3), respectively. Our study revealed that the phenotype most plausibly resulted from a chromosomal position effect over YIPF5 and SPATC1L. The last case, presented intellectual disability and recurrent miscarriage, associated to t(7;22)(p13;q13.1). The 7p13 breakpoint disrupts the brain specific CAMK2B, causing AD mental retardation 54 (OMIM #617799), whereas increased meiotic segregation of der(22), during gametogenesis, most likely explain the reported miscarriage (David et al., 2023). Conclusions: These cases highlight the intricacy of pathogenic mechanisms leading to human disorders, the necessity for identification and evaluation of the “full” spectrum of genomic and genetic variants, of comparative reverse phenotyping, including patients with pathogenic variants affecting the same genes. Finally, highlight the need of introducing a more precise genomic medicine in clinical practice. This research was supported by FCT—Fundação para a Ciência e a Tecnologia, Research Grant HMSP-ICT/0016/2013 of the Harvard Medical School—Portugal Program.Study supported by Fundação para a Ciência e Tecnologia (HMSP-ICT/0016/2013).info:eu-repo/semantics/publishedVersio

SVInterpreter: A Comprehensive Topologically Associated Domain-Based Clinical Outcome Prediction Tool for Balanced and Unbalanced Structural Variants

With the advent of genomic sequencing, a number of balanced and unbalanced structural variants (SVs) can be detected per individual. Mainly due to incompleteness and the scattered nature of the available annotation data of the human genome, manual interpretation of the SV’s clinical significance is laborious and cumbersome. Since bioinformatic tools developed for this task are limited, a comprehensive tool to assist clinical outcome prediction of SVs is warranted. Herein, we present SVInterpreter, a free Web application, which analyzes both balanced and unbalanced SVs using topologically associated domains (TADs) as genome units. Among others, gene-associated data (as function and dosage sensitivity), phenotype similarity scores, and copy number variants (CNVs) scoring metrics are retrieved for an informed SV interpretation. For evaluation, we retrospectively applied SVInterpreter to 97 balanced (translocations and inversions) and 125 unbalanced (deletions, duplications, and insertions) previously published SVs, and 145 SVs identified from 20 clinical samples. Our results showed the ability of SVInterpreter to support the evaluation of SVs by (1) confirming more than half of the predictions of the original studies, (2) decreasing 40% of the variants of uncertain significance, and (3) indicating several potential position effect events. To our knowledge, SVInterpreter is the most comprehensive TAD-based tool to identify the possible disease-causing candidate genes and to assist prediction of the clinical outcome of SVs. SVInterpreter is available at http://dgrctools-insa.min-saude.pt/cgi-bin/SVInterpreter.py.This research was supported by national funds through FCT—Fundação para a Ciência e a Tecnologia, Research Grant HMSP-ICT/0016/2013 of the Harvard Medical School—Portugal Program in Translational Research and Informationinfo:eu-repo/semantics/publishedVersio

SVInterpreter: a web-based tool for structural variants inspection and identification of possible disease-causing candidate genes

Introduction: With the advent of genomic sequencing, the identification of structural variants (SVs) is no longer a challenge, being possible to detect an average of 5 K SVs by individual. Contrarily, the annotation of the genome is incomplete, and the data is scattered along different databases, making SV manual evaluation complicated and time-consuming. Also, the available tools are limited on their scope. Thus, to address the need of a comprehensive application to assist evaluation of clinical outcome of SVs, we developed Structural Variant Interpreter (SVInterpreter). Methods: SVInterpreter is a free Python-CGI developed Web application able to analyze SVs using Topologically Associated Domains as genome units, within which genome browsers data, medically actionable genes, virtual gene panels and HPO similarity results, among other information, is retrieved. Results: We started by re-analysing 220 published SVs, of which about 50% were previously classified as VUS. SVInterpreter corroborated the previous classification in about 84% of the SVs. In about 5% of the SVs, SVInterpreter gave indication of possible position effect, through phenotype similarity, disrupted chromatin loops or genome wide association studies. Then, we show the applicability of SVInterpreter on the clinical setting, by inspecting 15 cases analysed by chromosomal microarray or genome sequencing. Conclusions: To our knowledge, SVInterpreter is the most comprehensive TAD based tool to assist prediction of clinical outcome of SVs. Based on gathered information, identification of possible disease-causing candidate genes and SVs is easily achievable. SVInterpreter is available at http://dgrctools-insa.min-saude.pt/cgi-bin/SVInterpreter.pyinfo:eu-repo/semantics/publishedVersio

Analysis of RNA-seq data from the interaction of Coffea spp. - Colletotrichum kahawae

Tese de mestrado em Bioinformática e Biologia Computacional (Bioinformática), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014O café e um dos produtos mais comercializados no mundo, com extrema importância económica e social, influenciando milhões de pessoas que dependem direta ou indiretamente desta industria. No entanto, a cultura do café e extremamente afetada por agentes patogénicos, nomeadamente fungos. Colletotrichum kahawae Waller and Bridge e um desses agentes, sendo responsável pela antracnose dos frutos verdes do cafeeiro, conhecida como “Coffee Berry Disease”. Esta doença afeta a espécie Coffea arabica L., a espécie de maior importância no mercado, apresentando os maiores volumes de produção. Atualmente, a antracnose dos frutos verdes do cafeeiro incide sobretudo em zonas de alta altitude, encontrando-se confinada ao continente africano. Contudo tal não significa que não se possa dispersar para outras zonas de cultivo onde as condições de desenvolvimento, tanto para a planta como para o fungo, sejam favoráveis. Foram desenvolvidas várias estratégias de melhoramento para o combate a doença, levando ao desenvolvimento de algumas variedades resistentes no Quénia. Apesar de já serem atualmente conhecidos vários genótipos com um caracter de resistência a esta doença, as bases genéticas e moleculares da mesma são ainda desconhecidas. Com o intuito de compreender as bases subjacentes ao processo de resistência, recorreu-se a sequenciação comparativa do transcriptoma de dois genótipos de cafeeiro, um susceptível (Caturra) e outro resistente (Catimor 88) durante as primeiras horas de interacção de C. kahawae, através da plataforma Illumina. A análise destes dados visou a identificação de genes diferencialmente expressos, envolvidos na resistência da planta a doença. Os dados desta sequenciação foram previamente analisados pela empresa ARK genomics (UK), embora utilizando softwares e parâmetros padronizados, normalmente aplicados para todo o tipo de analises deste género, desde bactérias a plantas. Com o objetivo de melhorar e aprofundar a analise, foi desenvolvida uma nova analise customizada, que aqui se apresenta, em comparação com a analise anterior. Varias ferramentas e abordagens foram aplicadas nesta nova analise, tendo em conta a inexistência de um genoma de referencia. Neste trabalho foi possível identificar vários problemas e cuidados a ter desde o tratamento das “reads”, ate ao cálculo de diferenças de expressão, bem como simples diferenças entre softwares. Neste novo estudo de expressão teve-se ainda em conta análises comparativas a diferentes níveis que não tinham sido efetuadas na analise anterior. A anotação de “unigenes” diferencialmente expressos indica uma tendência para categorias funcionais diretamente relacionadas com a produção de energia, envolvida no crescimento e desenvolvimento da planta, e com processos ja identificados como envolvidos na resposta de defesa a agentes patogénicos tais como o metabolismo de açúcares ou a biossíntese de fenilalanina e fenilpropanoides. De um modo geral, os objetivos deste trabalho foram cumpridos, tendo-se desenvolvido uma linha de análise que permitiu uma melhor e mais adequada exploração dos dados gerados por sequenciação de transcriptoma. Espera-se assim que os resultados obtidos venha a contribuir para o aumento do conhecimento científico sobre a resposta de defesa por parte da planta, gerando informações uteis para o estabelecimento de programas de melhoramento que apoiem a produção sustentável de uma cultura tao relevante a nível económico e social. Por outro lado, espera-se que este trabalho mostre a necessidade de uma analise cuidada de dados de “next generation sequencing”, em especial dados resultantes da sequenciação de RNA, tecnologia ainda bastante recente e sem um processo universalmente aceite para a analise correta dos dados gerados.Coffee is one of the most traded products in the world, with extremely social and economic importance, and millions of people who depend directly or indirectly on it. Coffee berry disease (CBD), caused by the fungus Colletotrichum kahawae Waller & Bridge, is considered the biggest threat to Arabica coffee production in Africa at high altitude. In Coffea arabica L. plantations, CBD can cause up to 20-50% of crop losses, reaching 80% in years of severe epidemics if chemical control is not applied. In order to control this disease, several coffee improvement strategies were developed which leaded to the selection of few hybrid commercial resistant varieties in Kenya. Therefore, breeding for coffee resistance remains a powerful strategy to fight CBD, in an economic and sustainable manner. With the purpose of gaining some insights on coffee resistance process, a RNA Illumina sequencing approach was used to characterize the transcriptional profile of two coffee genotypes, respectively susceptible (Caturra) and resistant (Catimor 88) to C. kahawae, during the early stages of the infection process. The differential expression analysis of this data aimed to identify genes putatively involved in the resistance process. Although a previous analysis was made by the sequencing company ARK genomics (UK), this was only based on non-specific methods generally applied to a wide range of organisms. To improve the analysis and consequently the results obtained, a new approach was taken aiming to produce a more customized workflow. Comparatively with the previous analysis, the present approach showed some improvement regarding the transcriptome assembly quality and size, or the level of confidence of the differential expression results, despite the CPU and RAM limitations. It was possible to account for additional comparative analyses for the differential expression assessment and to identify the enriched functional categories representing the differential expressed unigenes. Regarding the biological results, the resistant genotype showed a high effective response to the infection while the susceptible genotype showed an early stress-leaded response by the infection. The KOG and KEGG annotation of the differential expressed unigenes, was able to identify two main domains: plant development and defense response. It is expected that the results obtained here will contribute to increase the scientific knowledge on the plant defense response , generating useful information able to guide the establishment of breeding programs that support sustainable production. Moreover, it is expected that this study show the necessity of careful analysis of next generation sequencing data, especially when dealing with recent methods like RNA-seq, for which there is no clear consensus about the best analysis practices

Sustainable management of peel waste in the small-scale orange juice industries: A Colombian case study

Appropriate waste management in emerging economies like Colombia should be an asset for the overall sustainability. In the Orange Peel Waste case, incineration and Anaerobic Digestion are challenging solutions for the orange juice agro-industrial sector for avoiding the landfill, which is the conventional practice. However, these alternatives should be assessed in order to determine their feasibility. This paper aims to understand if incineration and Anaerobic Digestion are potential alternatives to landfill form a techno-economic and environmentally perspective. To this aim, a comparative Life Cycle Assessment was carried out in four scenarios. In the first scenario coal and landfill are used as source of energy and landfill disposal in the actual orange juice process. In the second scenario, the peels are incinerated to avoid landfill and reduce the need for coal. The third scenario includes the valorization of the peels by means of Anaerobic Digestion which produces biogas for the plant energy requirements. In the fourth scenario, apart from the energy from biogas, the digestate becomes a fertilizer for use in the orange crops. The results revealed that scenario III and IV are environmentally friendly options compared to Scenario I, but they incur higher costs than Scenario II. Carbon footprint found that 1.55 kg of CO2 are saving when coal substitution is reduced from 0.493 kg in SI to 0.279 kg in SII. Therefore, Scenario II is more suitable for the Colombian socioeconomic reality since Scenario II is not only techno-environmentally achievable, but also economically feasible. The methodology used in this case study could be applied to other countries or small and medium scale technologies and could also be useful for the scientific community, enterprises and policy-makers.The authors wish to acknowledge the financial support of the Fondo Regional de Tecnología Agropecuaria FONTAGRO [Contract:ATN/RF 16111RG, 2016] and of the Departamento Administrativo de Ciencia, Tecnología e Innovación, Doctorados Nacionales [Contract:727, 2015]. Also this article is the result of the work developed through the "Programa de investigación reconstrucción del tejido social en zonas de pos-conflicto en Colombia" [SIGP 57579] withthe research project "Competencias empresariales y de innovación para el desarrollo económico y la inclusión productiva de las regiones afectadas por el conflicto colombiano" [SIGP 58907]. Finally, the authors would like to express their appreciation to FLP Procesados Company for providing the data for the case study

Identification of Asparagopsis armata‐associated bacteria and characterization of their bioactive potential

Macroalgae‐associated bacteria have already proved to be an interesting source of compounds with therapeutic potential. Accordingly, the main aim of this study was to characterize Asparagopsis armata‐associated bacteria community and evaluate their capacity to produce substances with antitumor and antimicrobial potential. Bacteria were selected according to their phenotype and isolated by the streak plate technique. The identification was carried out by the RNA ribosomal 16s gene amplification through PCR techniques. The antimicrobial activities were evaluated against seven microorganisms (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae, Candida albicans) by following their growth through spectrophotometric readings. Antitumor activities were evaluated in vitro on human cell lines derived from hepatocellular (HepG‐2) and breast carcinoma (MCF‐7) using the MTT method. The present work identified a total of 21 bacteria belonging to the genus Vibrio, Staphylococcus, Shewanella, Alteromonadaceae, Bacillus, Cobetia, and Photobacterium, with Vibrio being the most abundant (42.86%). The extract of Shewanella sp. ASP 26 bacterial strain induced the highest antimicrobial activity, namely against Bacillus subtilis and Staphylococcus aureus with an IC50 of 151.1 and 346.8 μg/mL, respectively. These bacteria (Shewanella sp.) were also the ones with highest antitumor potential, demonstrating antiproliferative activity on HepG‐2 cells. Asparagopsis armata‐associated bacteria revealed to be a potential source of compounds with antitumor and antibacterial activity.info:eu-repo/semantics/publishedVersio

A Novel Frameshift CHD4 Variant Leading to Sifrim-Hitz-Weiss Syndrome in a Proband with a Subclinical Familial t(17;19) and a Large dup(2)(q14.3q21.1)

This article belongs to the Section Molecular Genetics and Genetic Diseases.The genetic complexity of neurodevelopmental disorders (NDD), combined with a heterogeneous clinical presentation, makes accurate assessment of their molecular bases and pathogenic mechanisms challenging. Our purpose is to reveal the pathogenic variant underlying a complex NDD through identification of the "full" spectrum of structural genomic and genetic variants. Therefore, clinical phenotyping and identification of variants by genome and exome sequencing, together with comprehensive assessment of these and affected candidate genes, were carried out. A maternally-inherited familial translocation [t(17;19)(p13.1;p13.3)mat] disrupting the GSG1 like 2 gene (GSG1L2), a 3.2 Mb dup(2)(q14.3q21.1) encompassing the autosomal dominant OMIM phenotype-associated PROC and HS6ST1 gene, and a novel frameshift c.4442del, p.(Gly1481Valfs*21) variant within exon 30 of the Chromodomain helicase DNA binding protein 4 (CHD4) have been identified. Considering the pathogenic potential of each variant and the proband's phenotype, we conclude that this case basically fits the Sifrim-Hitz-Weiss syndrome or CHD4-associated neurodevelopmental phenotype. Finally, our data highlight the need for identification of the "full" spectrum of structural genomic and genetic variants and of reverse comparative phenotyping, including unrelated patients with variants in same genes, for improved genomic healthcare of patients with NDD.If this article is accepted for publication, Open Access publication will be funded by UMIB—Unidade Multidisciplinar de Investigação Biomédica, ICBAS—Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal/ITR—Laboratory for Integrative and Trans lational Research in Population Health, Porto, Portugal (https://umib.icbas.up.pt/, accessed on 14 December 2022), both supported by FCT—Fundação para a Ciência e a Tecnologia in the frameworks of UIDP/00215/2020; LA/P/0064/2020. This research was supported by national funds through FCT—Fundação para a Ciência e a Tecnologia, Research Grant HMSP-ICT/0016/2013 of the Harvard Medical School—Portugal Program in Translational Research and Information.info:eu-repo/semantics/publishedVersio

Identification of OAF and PVRL1 as candidate genes for an ocular anomaly characterized by Peters anomaly type 2 and ectopia lentis

Keratolenticular dysgenesis (KLD) and ectopia lends are congenital eye defects. The aim of this study is the identification of molecular genetic alterations responsible for those ocular anomalies with neurologic impairment in an individual with a de novo balanced chromosome translocation t(11;18)(q23.3;q11.2)dn. Disruption of OAF, the human orthologue of the Drosophila oaf, by the 11q23.3 breakpoint results in reduced expression of this transcriptional regulator. Furthermore, four most likely nonfunctional chimeric transcripts comprising up to OAF exon 3, derived from the der(11) allele, have also been identified. This locus has been implicated by publicly available genome-wide association data in corneal disease and corneal topography. The expression of the poliovirus receptor-related 1(PVRL1) or nectin cell adhesion molecule 1 (NECTIN1), a paralogue of nectin cell adhesion molecule 3 (PVRL3) associated with congenital ocular defects, situated 500 kb upstream from 11q23.3 breakpoint, is increased. The 18q11.2 breakpoint is localized between cutaneous T-cell lymphoma-associated antigen 1(CTAGE1) and retinoblastoma binding protein 8 (RBBP8) genes. Genomic imbalance that could contribute to the observed phenotype was excluded. Analysis of gene expression datasets throughout normal murine ocular lens embryogenesis suggests that OAF expression is significantly enriched in the lens from early stages of development through adulthood, whereas PVRL1 is lens-enriched until E12.5 and then down-regulated. This contrasts with the observation that the proposita's lymphoblastoid cell lines exhibit low OAF and high PVRL1 expression as compared to control, which offers further support that the alterations described above are most likely responsible for the clinical phenotype. Finally, gene interaction topology data for PVRL1 also agree with our proposal that disruption of OAF by the translocation breakpoint and misregulation of PVRL1 due to a position effect contribute to the observed ocular and neurological phenotype.Peer reviewe

Antioxidant and Antimicrobial Potential of the Bifurcaria bifurcata Epiphytic Bacteria

This article belongs to the Special Issue Selected Papers from the 14th International Symposium on Marine Natural ProductsSurface-associated marine bacteria are an interesting source of new secondary metabolites. The aim of this study was the isolation and identification of epiphytic bacteria from the marine brown alga, Bifurcaria bifurcata, and the evaluation of the antioxidant and antimicrobial activity of bacteria extracts. The identification of epiphytic bacteria was determined by 16S rRNA gene sequencing. Bacteria extracts were obtained with methanol and dichloromethane (1:1) extraction. The antioxidant activity of extracts was performed by quantification of total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Antimicrobial activities were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans. A total of 39 Bifurcaria bifurcata-associated bacteria were isolated and 33 were identified as Vibrio sp. (48.72%), Alteromonas sp. (12.82%), Shewanella sp. (12.26%), Serratia sp. (2.56%), Citricoccus sp. (2.56%), Cellulophaga sp. (2.56%), Ruegeria sp. (2.56%) and Staphylococcus sp. (2.56%). Six (15.38%) of the 39 bacteria Bifurcaria bifurcata-associated bacteria presented less than a 90% Basic Local Alignment Search Tool (BLAST) match, and some of those could be new. The highest antioxidant activity and antimicrobial activity (against B. subtilis) was exhibited by strain 16 (Shewanella sp.). Several strains also presented high antimicrobial activity against S. aureus, mainly belonging to Alteromonas sp. and Vibrio sp. There were no positive results against fungi and Gram-negative bacteria. Bifurcaria bifurcata epiphytic bacteria were revealed to be excellent sources of natural antioxidant and antimicrobial compounds