Proceedings of the 26th Annual Meeting of the Portuguese Society of Human Genetics (SPGH – Sociedade Portuguesa de Genética Humana) Coimbra, 17–19 November 2022

ORAL PRESENTATIONS Basics Research OPI-1 DISRUPTION OF REGULATORY ELEMENTS IN THE CDH1 VICINITY AS A POTENTIAL HDGC CAUSE, INCREASED PENETRANCE AND EARLY ONSET CANCER São José C.*1,2,3,4, Garcia-Pelaez J.*1,2,3,4, André A.1,2 Ferreira M.1,2,5 Tusié M.T.6 Sommer A.7 te Paske I.8 Caldas C.9,10,11 Senz J.12 Tischkowitz M.13 Solve-RD DITF-GENTURIS, Solve-RD CNV-GENTURIS working group, de Voer R.M.8, Demidov G.14 Laurie S.15 Huntsman D.12,16,17 Hoogerbrugge N.8, Oliveira C.1,2,4,18 1i3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal;2IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal;3Doctoral Programme in Biomedicine, Faculty of medicine, University of Porto, Porto, Portugal;4Full Member of the European Reference Network on Genetic Tumor Risk Syndromes (ERN GENTURIS);5Dept. Computer Science Faculty of Science, University of Porto;6Department of Molecular Biology, Instituto Nacional de Ciencias Médicas y Nutrición, Salvador Zubirán, México City, México;7Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany;8Radboud Institute for Molecular Life Sciences, Department of Human Genetics, Radboud university medical center, Nijmegen, Netherlands;9Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, UK;10Department of Oncology, University of Cambridge, Cambridge, UK;11CRUK Cambridge Centre, Cambridge Experimental Cancer Medicine Centre (ECMC) and NIHR Cambridge Biomedical Research Centre, University of Cambridge and Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK;12Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada;13University of Cambridge, Medical Genetics, Cambridge, United Kingdom;14Institute of Medical Genetics and Applied Genomics, Tübingen, Germany;15The Barcelona Institute of Science and Technology, CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona, Spain;16Centre for Translational and Applied Genomics (CTAG), BC Cancer Agency, Vancouver, British Columbia, Canada;17Genetic Pathology Evaluation Centre, University of British Columbia and Vancouver General;18FMUP – Faculty of Medicine of the University of Porto, Porto, Portugal.*Equal contribution – Project ID No 739547 Hereditary diffuse gastric cancer (HDGC) predisposes for diffuse gastric cancer (DGC) and lobular breast cancer (LBC). CDH1 and CTNNA1 single nucleotide variants and copy number variants (CNVs) are the main HDGC triggers, explaining <50% of cases. We used whole exome/genome sequencing (WES/WGS), CNV analysis and MLPA to identify the missing heritability in HDGC families. CRISPR-Cas9, 4C-seq, RT-PCR, flow cytometry and RNA-seq were used to characterize potential causing mechanisms in cell lines. We found and validated a 39bp CDH1-TANGO6 intergenic deletion (WGS) in a HDGC family, and a highly penetrant CDH1-TANGO6 116kb deletion (WES) in a 2nd HDGC family with extremely early onset DGC. 4C-seq showed chromatin interactions between the CDH1 promoter and the CDH1-TANGO6 intergenic region affected in both families, suggesting cross regulation between regulatory elements. We hypothesized that the intergenic CNV causes HDGC in the 1st family, and the CDH1-TANGO6 intergenic region with the CNV aggravates disease presentation in the 2nd family. By CRISPR-Cas9, we mimicked each deletion, in parallel with CDH1 or TANGO6 CNV portions independently, all in homozygosity. CDH1 mRNA downregulation relative to the wild-type, was 3.5-fold for deletion of CDH1-TANGO6, 1.5-fold for the intergenic region, 1.6-fold for CDH1 deletion alone, and unchanged for the TANGO6 deletion. Deletions of both CDH1-TANGO6 and the intergenic region induced downregulation of CDH1-associated pathways, namely cell-cell junction, cadherin binding, cell substrate junction, and mitosis and nucleosome organization pathways. Herein we identify a novel regulatory region contributing as much as the CDH1 coding region for CDH1 downregulation. Deletion of this intergenic region alone may cause HDGC, and justify the extreme phenotype in the 2nd family when the deletion extends into the CDH1 coding portion. Ongoing enhancer activity in mouse embryos will add on to these findings. Funding: 1) Solve-RD project, grant agreement 779257 (European Union’s Horizon 2020 research and innovation programme); 2) FEDER/COMPETE,“PTDC/ BTM-TEC/ 30164/ 2017”, “PTDC/BTM-TEC/6706/2020”,“22184”; 3) FCT Fellowship “SFRH/BD/140796/2018”. OPI-2 DDIT4 AND TRIM13 TRANSCRIPT LEVELS ARE BLOOD-BASED BIOMARKERS OF EARLY STAGES OF MACHADO-JOSEPH DISEASE/SPINOCEREBELLAR ATAXIA TYPE 3 Ana F. Ferreira, Mafalda Raposo, João Vasconcelos, Teresa Kay, Emily D. Shaw, Conceição Bettencourt, Maria Luiza Saraiva-Pereira, Laura Bannach Jardim, Maria do Carmo Costa, Manuela Lima Universidade dos Açores Corresponding author email: [email protected] Machado-Joseph disease/Spinocerebellar ataxia type 3 (MJD) is a rare late-onset polyglutamine (polyQ) neurodegenerative disease. MJD is characterized by a long preclinical phase, preceding disease onset, during which some unspecific clinical manifestations, brain abnormalities and molecular alterations are known to be already present. MJD is caused by the expansion of a polyQ-encoding CAG repeat in the ATXN3 gene encoding the ataxin-3 protein that above a pathological threshold initiates a cascade of pathogenic events, such as transcriptional impairment. Importantly, some of these molecular alterations have the potential to be used as disease biomarkers, in the context of the ongoing and emergent clinical trials for MJD. The identification of biomarkers for early stages of MJD is particularly important as current clinical scales are insufficient to rigorously measure the efficacy of disease-modifying therapeutic agents, more so in the preclinical phase. Pursuing the overarching goal to identify novel blood-based transcriptional biomarkers for MJD and building on previously reported as well as novel data from a cross-sectional blood-based whole genome microarray of MJD carriers and controls, we identified DDIT4 (down-regulated) as well as TRIM13 and P2RY13 (upregulated) as being consistently dysregulated in MJD subjects from different cohorts. Our results showed that DDIT4 and TRIM13 are reliable blood-based transcriptional biomarkers for MJD, in particular for the early stages of the disease. Additionally, their respective protein abundance was also found altered in brains of MJD patients, linking these transcriptional changes with MJD pathogenesis. Our results further show that the MJD mechanism(s) in which TRIM13 and P2RY13 proteins are involved could be preserved in the central nervous system and the periphery of patients. In conclusion, DDIT4 and TRIM13 are blood-based transcriptional biomarkers for the early stages of MJD and, thus, we anticipate that they can be tested in a clinical setup to determine their value to complement clinical scales for the accurate monitoring of disease progression and measurement of the efficacy of therapeutics in MJD carriers. OPI-3 DAB1-ANTISENSE EXPRESSION AT THE SPINOCEREBELLAR ATAXIA TYPE 37 (SCA37) LOCUS AND DISEASE Catarina C. Rodrigues1,2,3 Ana F. Castro1,2,4 Ana S. Figueiredo1,2,4 Hugo Marcelino2,5 Joana R. Loureiro1,2 José Bessa2,5,* Isabel Silveira 2,* 1Genetics of Cognitive Dysfunction Laboratory, IBMC – Instituto de Biologia Celular e Molecular, Porto, Portugal;23S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal;3Escola de Ciências, Universidade do Minho, Braga, Portugal;4ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal;5Vertebrate Development and Regeneration Laboratory, IBMC - Instituto de Biologia Celular e Molecular, Porto, Portugal; *Equal contribution Corresponding author email: [email protected] Introduction: Spinocerebellar ataxias (SCAs) are neurodegenerative diseases, characterized by progressive speech, gait and limb incoordination. We discovered that SCA37 is caused by a noncoding (ATTTC)n insertion into a polymorphic (ATTTT)n in a 5’-UTR intron of the neurodevelopmental gene DAB1. The RNA transcribed from this mutation is toxic, leading to loss of Purkinje cells. Many repeat expansion diseases are bidirectionally transcribed. In DAB1-antisense strand, the (AAAAT)n is in a primate-specific AluJb middle poly(A), near a CpG island and a transcription factor binding sites, raising the hypothesis of a transcriptional regulatory role for the SCA37 locus. Methods and Result: sWe generated zebrafish transgenic lines with the DAB1-antisense repeat and its flanking region, named Fragment T (FragT), fused with a GFP sequence. This FragT was able to drive GFP expression to the muscle. After generation of transgenic lines with fragments of FragT, Frag1 (upstream the repeat), Frag2 (AluJb with and of Frag1 and the to GFP This that in the in DAB1-antisense strand, are of the its and are in we their brain expression in transgenic lines and promoter activity in cell lines. By we that FragT and were also able to drive GFP expression to the and and By in and with the and with FragT, Frag1 or we a in activity for FragT and for discovered a promoter the SCA37 to the The promoter able to drive pathogenic repeat expression was into the zebrafish suggesting regulatory elements. MACHADO-JOSEPH DISEASE IN OF André de - for and Biology, University of - for and University of for University of for University of of University of Introduction: Machado-Joseph disease (MJD) the of polyglutamine (polyQ) as the of by expansion of the CAG the ATXN3 MJD is characterized by the of that with mechanisms and in induced can MJD disease mechanisms in a context are by the to we a genome to a CAG the ATXN3 by and lines that MJD from were with the by of a ATXN3 gene was by sequencing and validated by lines were into by to characterize MJD disease mechanisms and We found of in with and a of from lines with and by the of were were from lines. of MJD phenotype is In novel in of MJD to therapeutic and a to CAG for cell of by the the 2020 and FCT for Science and - and as well as the - Research by the European and IN DISEASE Ana R. of Medical Sciences, Institute of University of Institute of Research Institute, University of Medicine, In of protein is as a central in the of neurodegenerative are particularly on and of mRNA and in have a in of these are are molecular that are by to and mRNA In the in that and loss of have in and being with the protein the of in disease is After analysis of late-onset the for found that a is in the is also in and and its expression with and suggesting a between and Additionally, with from the mutation that in the with protein expression and data that of in and that can Research by the Foundation for Science and and and the European Research OF A OF A IN Joana Mafalda Ana Isabel Maria João de e de de e de Introduction: and are a leading cause of and in the particularly in the of disease in a by and of and clinical A medical of for Genetics from to In were to The main clinical for was by disease single system and disease In a a of In is ongoing and were clinical a were the in with a of sequencing (WES) was in of which as a and as a of sequencing and The molecular between and A of were Additionally, a during that clinical Genomic and clinical in patients, as for early and Our important data for further to the of patients. of No THE OF THE AND THE IN Teresa Ana of Medical Genetics, de de de S. – de Molecular Instituto de Molecular, of de de Introduction: loss and can to Genetic of loss a in the of early to a molecular is to clinical and to We a on a of with in the the Department of between and causes were were to which a of to Our variants on the of the and were between and of We identified variants in different and in of these reported in the No variants were identified in In the further are ongoing to potential we to in which variants were identified in and We the results of the to sequencing for to and also the in The of in to be for Our into the variants to in the and the identification of novel which may to the of the of THE OF DISEASE AND AND EXPRESSION Ana João and Sciences, University of - Research and Laboratory, of and Institute of University of - of Medicine, University of Minho, Braga, Institute of Faculty of Medicine, University of is a factor for disease and and the mechanisms by which and to alterations the was to the of for and and gene expression changes on Methods This was on between and from a by a of and data was were of of and the analysis and data was the were with and the was in involved in system regulation of and of changes were for to cell and regulation of Additionally, between and and transcriptional that to involved in and system was into the of the mechanisms the between disease and and and gene expression changes to This was by FCT and the 2020 and The research is by FCT HDGC THE CDH1 João Joana Faculty of Medicine of the University of of Biomedical Abel Salazar, University of Faculty of of the University of e de São - University of - University of Faculty of Medicine and Biomedical of the University of Hereditary diffuse gastric cancer (HDGC) caused by CDH1 or variants predisposes to early onset diffuse gastric (DGC) and lobular breast cancer (LBC). disease is and clinical presentation or of onset suggesting a role for We to characterize the genome of CDH1 carriers and affected from carriers in a of the CDH1 We to a of as or of the in carriers of this pathogenic and for this disease. from were for the CDH1 were for and with that previously in and single nucleotide variants with the of different analysis was to and on the data from each We found carriers of the CDH1 families. a of variants were from the data on all patients, with of these by both and The each was we found that the number of rare variants in between affected and of the rare variants are found in regulatory and in this intergenic and were found to be to the This a in the of for and patients. 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Isabel S. – Instituto de Investigação e Inovação em Saúde, Porto, – Institute of Molecular Pathology and of the University of Porto, Porto, Portugal; 3 Programme in Molecular and Applied to from Institute of Biomedical Abel University of Porto, Porto, Portugal; de de and in Department of University Department of Genetics, University Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, for Hereditary Tumor University Bonn, Bonn, Member of the European Reference Network on Genetic Tumor Risk Syndromes (ERN – Project ID No Department of Medicine University Bonn, Bonn, Department of Genetics, Hereditary Cancer Medical Department and Institute of Oncology, Barcelona, of Human Genetics, Medical University of and University of of Medicine, Centre, Cancer Genetics Cancer Molecular Research Cancer Department of Genetics, Cancer Institute, Genetics, Department of Barcelona, de y University of Barcelona Barcelona, of Medical and Sciences, of Department of Medicine, Cancer of Medicine, University of Cancer Cancer Institute, The Department of Oncology, Cancer Institute, The IBMC – Instituto de Biologia Molecular e Porto, Portugal; – Faculty of Medicine of the University of Porto, Porto, author Introduction: pathogenic variants are a rare cause of Hereditary Cancer and CTNNA1 variants cause The disease and with CTNNA1 is and in are to and we clinical data from CTNNA1 and to be We a and to CTNNA1 in different with rare CTNNA1 and all and Cancer of were In by and cancer BC or Tumor early between and of in and caused in the Human expression in the a are in CTNNA1 and rare in extends in and in by in as the is for CTNNA1 Project AND João João Ana de de São Porto, de de São Porto, de Patologia e Imunologia Molecular da Universidade do Porto, Porto, - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, de de São Porto, Universidade do Porto, Porto, de da Universidade do Porto, Porto, Introduction: are a of characterized by and of are caused by variants in the or of A was to for the 1st was the of a The showed a with and to the to the analysis a with which the with and of all of a long of each and with and of with and and with A was variants of in found to be in is a novel that is to by and is rare in a previously to be pathogenic by characterize their we have RNA which that the of and to of both a results are with and the of a to a Our the clinical and molecular which AS THE ONSET OF Maria João Genetics de do e de do e Introduction: is caused by loss of is characterized by loss in progressive in early early onset and of We a family with affected and and the A was to and early onset loss limb and a novel pathogenic in the of Genetic of the family the of 3 - 3 and of a The to loss was and were The were and both of the and of the by to The with and 3 were for the early of to can be more of the is for and be the of the monitoring of and loss and in is as early may their This family the of of loss and its on – Isabel João João Ana de de de de de de Instituto de de de Patologia e Imunologia Molecular da Universidade do Porto, de Investigação e Inovação em da Universidade do Porto, de da Universidade do Porto, Porto, de Instituto de de Introduction: The between type type and is are such as rare of with and or in the of Additionally, or from is leading to with a a on pathological relative and to and a and and a of a on the of a was on with of and is A was on a a pathogenic with a of The was to be this to the of In the to a The as a of a In of is in in in this the of molecular in which is for clinical and are ongoing clinical trials for in patients, reported in the AND Mafalda de de de de Introduction: A in the gene to the variants in leading to were to a different namely with or of in to in A with and and was sequencing identified a pathogenic in from A was for and to and and sequencing identified a pathogenic in The to was by the further was showed a in and polymorphic a do have a phenotype with gene The a which is that is a transcriptional of the The a with the the of when the phenotype is with the Additionally, this the and and of OF A – AND Mafalda Joana Ana Genetics e de e de of Genetics, Faculty of Medicine, University of - Universidade de of Introduction: Genomics and is identify and of the in is a novel – caused by in a gene with have reported and we cases. 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F. André C. Genetics e de Genetics de of Sciences, Universidade da Introduction: of RNA transcription the of a the gene promoter region, transcription such as for of the the binding protein factor variants in are to with and from families, have of a family with affected to the previously reported and of the The was from were in the the of as and brain showed that to and 3 with and and with or with and showed sequencing 3 identified novel variants of clinical in and in The for a and the was with clinical with so and This was to have the Our as well as the type of variants identified to the The of the variants in the family, with a affected and the in the to this We are to in to this have previously research ATXN3 IN SPINOCEREBELLAR ATAXIA TYPE MACHADO-JOSEPH AND OF IN THE AND Mafalda Ana F. Ana João Teresa European Spinocerebellar ataxia type Machado-Joseph disease group, Maria do Carmo Manuela de Biologia Molecular e Celular Instituto de Investigação e Inovação em Universidade do Porto, Porto, de Ciências e Universidade dos of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, for University of Tübingen, Medicine, University of de do de do da do de D. Tübingen, Tübingen, of Medicine, University of Spinocerebellar ataxia type 3 disease is polyglutamine disease. The is known to and are the and regulation of ATXN3 the abundance of in and disease and the of In this we the abundance of the ATXN3 RNA-seq from and of subjects and controls, and the a for ATXN3 were in protein coding and and abundance were in the in as a role of ATXN3 in this that is affected with cell loss in the in the was a long RNA the with the abundance in the was the that is into ataxin-3 protein In the abundance of and encoding ataxin-3 3 and in subjects and was with expression 3 was in more in was in the more in the abundance of the was in the and in the of subjects and controls, and were in of in into the of ATXN3 AS in the and which may to the of ATXN3 contributing to a of pathogenesis. IN MACHADO-JOSEPH DISEASE de and Catarina Oliveira - for and Biology, University of for University of - for and of University of of Science and Technology, University of University of contribution Introduction: Machado-Joseph disease (MJD) is characterized by of the CAG in the into polyglutamine the ataxin-3 This protein causes and in brain this and their have as the mechanisms by which these In the we in and in MJD, as well as the of in MJD We the of and in MJD transgenic with from were by were by of a cell ATXN3 with from to was by or MJD of and and was able to these number was found and of in The of these is we that to MJD in This and in MJD we found that to MJD in and alterations in in their therapeutic in in MJD. by

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