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Objective Li-Fraumeni syndrome (LFS) is a pan-cancer predisposition syndrome caused by germline pathogenic variants in the gene TP53. The interpretation of TP53 variants in clinical scenarios outside the classic LFS criteria may be challenging. Here, we report a patient affected by 2 primary cancers at later ages, who harbored a likely pathogenic TP53 at low allele frequency detected in a blood sample. Methods The Molecular Tumor Board committee at our institution revisited the case of a patient who was enrolled in a research protocol for the investigation of genetic conditions associated with neuroendocrine tumors. Clinical, familial, and molecular data were reviewed. The patient received germline testing using a next generation sequencing multi-gene panel and was incidentally found to harbor a TP53 likely pathogenic variant, with 22% of variant allele fraction. Additional samples, including a second blood sample, oral swab, and saliva, were collected for DNA analysis. A new TP53 sequencing round was performed with the attempt to distinguish between a true constitutional germline variant and a somatically acquired variant due to aberrant clonal expansion of bone marrow precursors. Results Patient’s personal and familial history of cancer did not meet classic nor Chompret LFS criteria. Environmental risk factors for cancer were identified, such as alcohol abuse and tobacco exposure. The TP53 variant initially found in the next-generation sequencing was confirmed by Sanger sequencing in the previous DNA sample extracted from blood for the first analysis and in a second blood sample collected 6 years later. The TP53 variant was not detected in the DNA extracted from the oral swab and saliva samples. Conclusion Considering the low TP53 variant allele fraction in blood, absence of variant detection in oral swab and saliva samples, the lack of LFS clinical criteria, and history of exposure to environmental risk factors for cancer, the main hypothesis for this case was aberrant clonal expansion due to clonal hematopoiesis. Oncologists should interpret TP53 findings during germline testing with caution.

Funder: VTCT Foundation Fellowship, BACKGROUND: Auriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants have been identified in three genes involved in the EDN1-DLX5/6 pathway (PLCB4, GNAI3 and EDN1) and some cases remain unsolved. Here we studied a large unsolved four-generation family. METHODS: We performed linkage analysis, resequencing and Capture-C to investigate the causative variant of this family. To test the pathogenicity of the CNV found, we modelled the disease in patient craniofacial progenitor cells, including induced pluripotent cell (iPSC)-derived neural crest and mesenchymal cells. RESULTS: This study highlights a fourth locus causative of ARCND, represented by a tandem duplication of 430 kb in a candidate region on chromosome 7 defined by linkage analysis. This duplication segregates with the disease in the family (LOD score=2.88) and includes HDAC9, which is located over 200 kb telomeric to the top candidate gene TWIST1. Notably, Capture-C analysis revealed multiple cis interactions between the TWIST1 promoter and possible regulatory elements within the duplicated region. Modelling of the disease revealed an increased expression of HDAC9 and its neighbouring gene, TWIST1, in neural crest cells. We also identified decreased migration of iPSC-derived neural crest cells together with dysregulation of osteogenic differentiation in iPSC-affected mesenchymal stem cells. CONCLUSION: Our findings support the hypothesis that the 430 kb duplication is causative of the ARCND phenotype in this family and that deregulation of TWIST1 expression during craniofacial development can contribute to the phenotype.

BACKGROUND: Optimal therapy for high-risk non-muscle invasive bladder cancer (NMIBC) includes intravesical instillation of Bacillus Calmette-Guérin (BCG). However, about 25-45% of patients do not benefit from BCG immunotherapy, and there is no biomarker to guide therapy. Also, many questions regarding BCG mechanisms of action remain unanswered. OBJECTIVE: To identify genomic biomarkers and characterize the underlying mechanism of benefit from BCG in NMIBC. PATIENTS AND METHODS: Pre-treatment archival index-tumors of 35 patients with NMIBC treated with BCG were analyzed by whole-exome sequencing (WES). Tumor mutation burden (TMB) and neoantigen load (NAL) were correlated with BCG response rate (RR) and recurrence-free survival (RFS). The presence of deleterious mutations in DNA damage response (DDR) genes was also compared between BCG-responsive (BCG-R, N = 17) and unresponsive (BCG-UR, N = 18) subgroups. RESULTS: TMB and NAL were higher in BCG-R compared to BCG-UR patients (median TMB 4.9 vs. 2.8 mutations/Mb, P = 0.017 and median NAL 100 vs. 65 neoantigens, P = 0.032). Improved RR and RFS were observed in patients with high vs. low TMB (RR 71% vs. 28%, P = 0.011 and mRFS 38.0 vs. 15.0 months, P = 0.009) and with high vs. low NAL (RR 71% vs. 28%, P = 0.011 and mRFS 36.0 vs. 18.5 months, P = 0.016). The presence of deleterious mutations in DDR genes was associated with improved RFS (mRFS 35.5 vs. 11.0 months, P = 0.017). CONCLUSIONS: In our cohort, improved outcomes after BCG immunotherapy were observed in patients with high TMB, high NAL and deleterious mutations in DDR genes. BCG may induce tumor-specific immune response by enhancing the recognition of neoantigens.

Extracellular vesicles (EV) are comprised of vesicles budding from cell membranes and smaller intracellular vesicles shed by cells. EV play a role in remodeling the tumor microenvironment (TME) and support tumor progression. Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein with a carboxypeptidase function, frequently associated with poor clinical prognosis in prostate cancer (PCa). We previously identified an oncogenic PSMA signaling function in prostate cancer. Others demonstrated that EV isolated from the plasma of patients with high-grade PCa carry PSMA, but so far no pathophysiological effect has been associated with PSMA-bearing EV. Here we demonstrate that EV from PCa cells are able to transfer PSMA and its functionality to cells in the TME. The consequence of that EV-mediated PSMA transfer is an acute to long-term increased secretion of vascular endothelial growth factor-A (VEGF-A), angiogenin, pro-angiogenic and pro-lymphangiogenic mediators and increased 4E binding protein 1 (4EBP-1) phosphorylation in tumors. We compare EV from PCa cells with or without PSMA expression to address the role of PSMA-bearing EV in promoting pro-tumoral changes in the TME using classical molecular biology and novel molecular imaging approaches.

Glioblastoma (GBM), supported by a subpopulation of glioblastoma stem cells (GSCs), stands among the most harmful tumors. Its invasive and aggressive behavior has been correlated with poor therapy outcomes and reduced patient survival, which remains difficult to model and treat due to its cellular and molecular heterogeneity. GSCs are responsible for the malignant features of this cancer, including increased recurrence rates and resistance to therapy. Efforts have been employed to understand the molecular and cellular mechanisms underlying GSCs biology. The cellular prion protein (PrPC), encoded by the PRNP gene, is a key player in GBM biology, and our group has proposed PrPC as a scaffold protein that integrates signaling platforms involved in GSCs maintenance. A deeper understanding of the intracellular pathways modulated by PrPC is essential to identify novel targets for GBM treatment. We have analyzed bulk-RNA sequencing (RNA-seq) data from patient-derived xenografts (PDX) and in vitro preclinical experimental models. RNA-seq analyses identified differentially expressed genes (DEGs) associated with adhesion and migration signaling pathways in PDX cells with high PRNP expression (PRNP−HIGH). Morphological characterization of PDX samples with PRNP−HIGH levels showed an increase in morphologically undifferentiated cell groups, that are more commonly found in younger GBM patients. Furthermore, molecular profiling using Exome data showed that groups PRNP−HIGH present enhanced global patterns of gene mutations. Due to the importance of experimental models to translate basic science into clinical applications, we are establishing emerging models to study PrPC function in GBM biology: glioblastoma organoids (GBO) from patient’s surgically resected GBM tumors; and brain organoids derived from human-induced pluripotent stem cells. We noticed a morphological and molecular reproducibility of the cytoarchitecture of brain organoids and GBO, similar to that described in the literature, indicating consistency in 3D structures, despite the variability between patients. Furthermore, brain organoids will be co-cultured with GSCs, providing platforms for studying the interaction between tumor cells and brain parenchyma. The establishment of these models will improve our comprehension of the role of PrPC in GSCs maintenance and provide additional tools to understand GBM biology and predict tumor progression and response to treatment. Citation Format: Maria Clara da Silva Souza, Barbara P. Coelho, Camila F. Fernandes, Tiago G. Dos Santos, Stefan Knorst, Cibele Masotti, Maria Rita S. Passos-Bueno, Marilene H. Lopes. PDX, mini-brain co-cultures and patient-derived tumor organoids: Preclinical models to study the prion protein in glioblastoma biology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4666.

PURPOSE Li-Fraumeni syndrome (LFS) is rare in the worldwide population, but it is highly prevalent in the Brazilian population because of a founder mutation, TP53 p.R337H, accounting for 0.3% of south and southeastern population. Clinical criteria for LFS may not identify all individuals at risk of carrying the Brazilian founder mutation because of its lower penetrance and variable expressivity. This variant is rarely described in databases of somatic mutations. Somatic findings in tumor molecular profiling may give insight to identify individuals who might be carriers of LFS and allow the adoption of risk reduction strategies for cancer. MATERIALS AND METHODS We determined the frequency of the TP53 p.R337H variant in tumor genomic profiling from 755 consecutive Brazilian patients with pan-cancer. This is a retrospective cohort from January 2013 to March 2020 at a tertiary care center in Brazil. RESULTS The TP53 p.R337H variant was found in 2% (15 of 755) of the samples. The mutation allele frequency ranged from 30% to 91.7%. A total of seven patients were referred for genetic counseling and germline testing after tumor genomic profiling results were disclosed. All the patients who proceeded with germline testing (6 of 6) confirmed the diagnosis of LFS. Family history was available in 12 cases. Nine patients (9 of 12) did not meet LFS clinical criteria. CONCLUSION The identification of the TP53 p.R337H variant in tumor genomic profiling should be a predictive finding of LFS in the Brazilian population and should prompt testing for germline status confirmation.

Auriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants have been identified in three genes involved in the EDN1-DLX5/6 pathway (We performed linkage analysis, resequencing and Capture-C to investigate the causative variant of this family. To test the pathogenicity of the CNV found, we modelled the disease in patient craniofacial progenitor cells, including induced pluripotent cell (iPSC)-derived neural crest and mesenchymal cells.This study highlights a fourth locus causative of ARCND, represented by a tandem duplication of 430 kb in a candidate region on chromosome 7 defined by linkage analysis. This duplication segregates with the disease in the family (LOD score=2.88) and includesOur findings support the hypothesis that the 430 kb duplication is causative of the ARCND phenotype in this family and that deregulation of

I.ABSTRACTScalable, cost-effective screening methods are an essential tool to control SARS-CoV-2 spread. We have developed a straight saliva-based, RNA extraction-free, RT-LAMP test that is comparable to current nasopharyngeal swab RT-PCR tests in both sensitivity and specificity. Using a 2-step readout of fluorescence and melting-point curve analysis, the test is scalable to more than 30,000 tests per day with average turnaround time of less than 3 hours. The test was validated using samples from 244 symptomatic patients, and showed sensitivity of 78.9% (vs. 85.5% for nasopharyngeal swabs RT-PCR) and specificity of 100% (vs. 100% for nasopharyngeal swabs RT-PCR). Our method is therefore accurate, robust, time and cost effective and therefore can be used for screening of SARS-CoV-2.

About 70% of breast cancers (BC) express estrogen receptor α (ER) and most of these are sensitive to endocrine therapies (ET). However, a significant fraction of patients with ER-positive (ER+) BC who benefit from adjuvant ET recur with metastatic disease after prolonged exposure to these therapies. Even patients with ER+ metastatic BC who respond to ET will eventually progress with antiestrogen-resistant hormone-independent disease. Activating mutations in the estrogen receptor gene ESR1 have been described in advanced ER+ BC resistant to ET, particularly in metastatic patients exposed to therapy with aromatase inhibitors (AI). ESR1 mutations frequently occur in a hotspot region within the ligand-binding domain of ER, constitutively activating the receptor in a ligand-independent manner and thus explaining the acquired resistance to hormonal therapy. Recent studies have reported the detection of ESR1 mutations in codons 536, 537, and 538 in circulating tumor DNA (ctDNA) of metastatic BC patients, ensuring the establishment of a causal relationship among ESR1 mutations and acquired resistance to AI therapy. Thereby we evaluated the presence of ESR1 mutations in plasma cell-free DNA (cfDNA) of metastatic ER-positive BC patients who progressed on AI therapy. Among patients with no ESR1 mutations detected, we questioned the presence of any other mutated gene that could be related to acquired resistance to ET. To answer these questions, we first investigated the presence of ESR1 recurrent mutations (L536R, Y537C/N/S, D538G) by Droplet Digital PCR (ddPCR) in ctDNA of 24 ER+ metastatic BC patients after acquired resistance to therapy with AI. ESR1 mutations were detected in 25% (6/24) of the patients, with ESR1 D538G and Y537N being the most frequent, and four patients showed polyclonal disease with more than one ESR1 mutation. The longest interval between the end of aromatase inhibitor treatment and the detection of ESR1 mutations was 2.3 years, corroborating with some studies that showed that these mutations remain readily detectable in ctDNA many months and years after stopping therapy. cfDNA from patients with no ESR1 mutations detected were analyzed by targeted cfDNA sequencing (cfDNA seq) of a 151-cancer gene panel. We were able to detect mutations in genes already known to be associated to ET resistance such as PIK3CA and AKT. We found one patient with AKT E17K mutation and four patients (4/12, 33%) with detectable PIK3CA-mutated ctDNA, with three patients with E545K mutation and one with H1047L mutation. Mutations in PIK3CA are frequently observed in BC and have been suggested as a predictive biomarker for PI3K-selective inhibitor treatment, alpelisib. Based in our results, we can conclude that detection of tumor genetic alterations in plasma cfDNA may have important clinical implications for personalized medicine, including monitoring of recurrence and therapy resistance and therapeutic guidance. Citation Format: Cibele Masotti, Franciele H. Knebel, Rudinei D. M. Linck, Julianne Lima, Andrea K. Shimada, João Victor Alessi, Bruna M. Zucchetti, Dimitrios Kleftogiannis, Louise Barber, Marco Gerlinger, Artur Katz, Max S. Mano, Anamaria A. Camargo, Fabiana Bettoni. Detection of ESR1 mutations in plasma cell-free DNA from metastatic ER-positive breast cancer patients resistant to hormone therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A19.

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.

Recent reports have demonstrated that a significant proportion of human genes display allelic differential expression (ADE). ADE is associated with phenotypic variability and may contribute to complex genetic diseases. Here, we present a computational analysis of ADE using allele-specific serial analysis of gene expression (SAGE) tags representing 1295 human genes. We identified 472 genes for which unequal representation (>3-fold) of allele-specific SAGE tags was observed in at least one SAGE library, suggesting the occurrence of ADE. For 235 out of these 472 genes, the difference in the expression level between both allele-specific SAGE tags was statistically significant (p < 0.05). Eleven candidate genes were then subjected to experimental validation and ADE was confirmed for 8 out of these 11 genes. Our results suggest that at least 25% of the human genes display ADE and that allele-specific SAGE tags can be efficiently used for the identification of such genes.

Neste trabalho, estudamos duas malformações craniofaciais mendelianas, decorrentes de um distúrbio do desenvolvimento dos primeiro e segundo arcos faríngeos: a síndrome de Treacher Collins (STC) e a síndrome Aurículo-condilar (SAC). A identificação de genes e de mecanismos moleculares associados a essas condições, além de contribuir para a compreensão do desenvolvimento das estruturas derivadas desses arcos faríngeos, é fundamental para o desenvolvimento do diagnóstico molecular, uma ferramenta importante para diagnóstico diferencial e aconselhamento genético. Contribuímos para uma melhor caracterização clínica da SAC com a descrição de uma nova família com 11 afetados. Após excluirmos quatro genes/regiões candidatas para síndromes de 1º e 2º arcos faríngeos, realizamos estudos de ligação usando marcadores polimórficos ao longo do genoma. Mapeamos o primeiro lócus associado à SAC, 1p21.1-q23.3 (lod score=3.0), e nossos dados sugerem que há heterogeneidade genética para essa patologia. Com relação ao estudo da STC, realizamos uma extensa revisão da nomenclatura das mutações patogênicas descritas na literatura, além de investigar mecanismos mutacionais atípicos na STC, corroborando a hipótese de que mutações nos exons que sofrem splicing alternativo são capazes de gerar o fenótipo da síndrome. Demos continuidade à caracterização do espectro de mutações no gene TCOF1 e investigamos a correlação genótipo-fenótipo numa amostra de 58 pacientes com STC. A análise dos dados de polimorfismos da região codificadora do gene permitiu que fizéssemos inferências sobre o regime de seleção ao qual o TCOF1 está submetido, e os resultados sugeriram que o gene TCOF1 está sob seleção purificadora, que atua sobre mutações fracamente deletérias. Também inferimos a fase para um conjunto de polimorfismos da região codificadora, verificando se havia associação de algum haplótipo à gravidade do quadro clínico ou à predisposição para a doença. Dada a observação de ausência de correlação haplótipo/genótipo-fenótipo, testamos a hipótese de que variações nos níveis de expressão do alelo normal poderiam ser responsáveis pela variabilidade clínica observada nos pacientes portadores da STC. Para tanto, iniciamos o estudo funcional das regiões regulatórias do gene TCOF1 , inclusive, de regiões distantes do promotor mínimo, preditas como enhancers. Identificamos polimorfismos em sua região promotora, sendo um deles capaz de diminuir os níveis de transcrição e de afetar a ligação do fator de transcrição YY1 ao promotor do TCOF1 . Caracterizamos a ação de YY1 como repressora in vitro. Testamos também a hipótese de haploinsuficiência como mecanismo associado à STC. Quantificamos os níveis de transcritos do TCOF1 em indivíduos afetados e normais e observamos uma diferença significativa. Nosso trabalho corrobora a hipótese de haploinsuficiência e mostra pela primeira vez que pacientes têm degradação de transcritos. Também investigamos a possibilidade de os níveis de transcritos estarem correlacionados à variabilidade fenotípica. Comparamos os dados de expressão de cada paciente à freqüência de nove sinais clínicos principais da STC, mas nenhuma correlação foi observada. Investigamos o padrão de metilação da ilha CpG do gene TCOF1 em pacientes e em controles, com o intuito de verificar se diferentes níveis de metilação inter-individual estariam associados à grande variação na expressão gênica. Demonstramos que a metilação da ilha CpG não é o mecanismo regulatório por trás dessa ampla variação de expressão do TCOF1 In the present study, we investigate two craniofacial mendelian disorders, resulting from abnormalities in the development of the first and second pharyngeal arches: the Treacher Collins Syndrome (TCS ) and the auriculo condylar syndrome (ACS). The identification of genes and molecular mechanisms associated to these conditions, in addition to contributing to the understanding of the development of structures derived from these pharyngeal arches, is fundamental for the development of molecular diagnostics, an important tool for differential diagnosis and genetic counseling. We contributed to a better clinical characterization of ACS with a description of a new family with 11 affected individuals. After excluding four candidate genes/regions for syndromes of the 1st and 2nd pharyngeal arches, we carried out linkage studies using polymorphic markers throughout the genome. We mapped the first locus associated to ACS, 1p21.1-q23.3 (lod score=3.0), and our data suggest genetic heterogeneity exists for this pathology. With respect to the study of TCS , we carried out an extensive review of the nomenclature for the pathogenic mutations described in the literature, in addition to investigating atypical mutation mechanisms in TCS , corroborating the hypothesis that mutations in the exons that undergo alternative splicing can result in the TCS phenotype. We continued the characterization of the mutation spectrum for TCOF1 and we investigated the genotype-phenotype correlation in a sample of 58 patients with TCS . The analysis of polymorphisms in the coding region allowed us to make inferences about the selective regime experienced by TCOF1 , and our results suggested that TCOF1 is under purifying selection, which acts upon weakly deleterious mutations. We also inferred the phase for a set of polymorphisms in the coding region, testing whether there was association between any haplotype and the severity of the phenotype or the susceptibility to the disease. Given the observation of no correlation between haplotype/genotype and phenotype, we tested the hypothesis that variation in the levels of expression of the normal allele could be responsible for the clinical variability observed in TCS patients. To do this, we started a functional study of the TCOF1 regulatory regions, including regions distant from the minimal promoter, predicted to be enhancers. We identified polymorphisms in the promoter region, one of which reduced the levels of transcription and affected the binding of the YY1 transcription factor to the TCOF1 promoter. Using an in vitro assay we characterized YY1 as a repressor. We also tested the hypothesis of haploinsufficiency as a mechanism associated to TCS . We quantified the levels of TCOF1 transcripts in normal and affected individuals and found a significant difference. Our study corroborates the haploinsufficiency hypothesis and for the first time shows that patients have transcript degradation. We also investigated the possibility that transcripts levels are correlated to phenotypic variability. We compared the expression data for each patient with the frequency of nine clinical signs of TCS , but no correlation was found. We investigated the pattern of methylation on the CpG island of TCOF1 in patients and controls, in order to test whether different levels of methylation among individuals were associated to the great variation in gene expression. We demonstrated that methylation of the CpG island is not the regulatory mechanisms underlying the broad variation in TCOF1 expression.

Intratumoral heterogeneity (ITH) is a phenomenon where a tumor, over space and time, branches into a set of genetically distinct clones, which have unique somatic branch mutations, although sharing several somatic trunk mutations. Clonal populations expand or regress in response to selective pressures exerted by the tumor microenvironment. Therefore, ITH has already been linked to several cancer features such as the development of metastases and resistance against a broad range of therapies. Through whole exome sequencing, we can access the number of reads covering the alleles of somatic mutations in tumor samples. Thus, mutant-allele frequency (MAF), i.e., the proportion of reads reporting the mutant allele over all reads mapped to the locus, is informative about the number of clones within a tumor bearing this genetic variant. However, MAF estimations can be impaired by tumor purity, copy number alteration, or sampling approaches. It is proposed that the analysis of multiple samples of a tumor improve accuracy in quantifying clone populations, but single sample is cheaper and more feasible for the clinicians. Mutant-Allele Tumor Heterogeneity (MATH) is a method capable of trustworthily scoring ITH in single samples, as it is based on the range of the MAF distribution of a tumor. The higher the MATH score, the more heterogeneous is a tumor. The goal of this project is to evaluate the ITH levels of 6,390 tumor samples of 20 distinct tumor types, for which genomic data is publicly available through GDAC Firehose repository. We accessed the MAF files and filtered somatic variants: (i) selected only single nucleotide variants, (ii) mutant alleles reported by at least three reads, (iii) total coverage equal to or higher than 30 reads. We estimated MATH scores using R (v3.2.4). The distribution of MATH scores across distinct tumor types exhibited a wide variability (ranging from 12 to 128). Uterine corpus endometrial carcinoma had the lowest MATH scores, whereas prostate adenocarcinoma and testicular germ cell tumors presented the highest MATH scores. The wide ITH variability among nine distinct tumor types has already been assessed in previous studies by using another scoring methodology. High MATH score was significantly associated with decreased overall survival in head and neck squamous cell carcinoma and predicted the development of metastasis in individuals with colorectal cancer. We will further explore the correlation between MATH scores and clinical data to all the 20 tumors here described by exploring the corresponding public clinical data from the cBioPortal for Cancer Genomics. Should MATH score prove to be an optimal scoring method for single biopsies, capable of predicting patient's prognosis, drug response, or disease outcome, it will be a valuable tool for clinicians for a broad spectrum of cancer treatments. Citation Format: Cibele Masotti, Filipe F. Santos, Anamaria A. Camargo, Pedro A.F. Galante. Measuring intratumoral heterogeneity with a mutant-allele frequency score across distinct cancer types [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B65.

Response to neoadjuvant chemoradiotherapy (nCRT) varies substantially among patients with locally advanced rectal cancer, but key determinants of treatment response are not fully understood. Intratumoral genetic heterogeneity (ITGH) is a common feature of solid tumors and has been proposed to drive disease progression and treatment response. Others and we have recently demonstrated that primary nontreated rectal tumors exhibit significant ITGH, but the effect of nCRT in shaping the clonal architecture of rectal cancer has not yet been addressed. In the present work, we further expanded the analysis of ITGH in rectal cancer by analyzing whole-exome sequencing (WES) and clinical data from 79 primary nontreated rectal cancers obtained from The Cancer Genome Atlas. We show that primary rectal tumors exhibit a remarkable variability in the degree of ITH. We also analyzed the effect of nCRT on ITGH and monitored tumor cell subpopulation dynamics after treatment through WES of paired tumor samples collected at diagnosis (pretreatment) and at surgery following nCRT (post-treatment) from seven patients. We show that treatment exposure does not introduce novel somatic mutations or significantly alter tissue genetic heterogeneity. Instead, nCRT acts as a potent selective pressure, shaping the mutational landscape of rectal tumors and allowing the selection and expansion of tumor cell subpopulations, which are more prone to resist nCRT. Our results shed light on the highly heterogeneous and dynamic mutational landscape of rectal tumors and indicate that ITGH may directly affect response to nCRT. Citation Format: Cibele Masotti, Fabiana Bettoni, Bruna R. Correa, Angelita Habr-Gama, Joaquim Gama-Rodrigues, Maria Viana, Bruna B. Vailati, Guilherme P. Sao Juliao, Laura M. Fernandez, Pedro A. F. Galante, Rodrigo O. Perez, Anamaria A. Camargo. Intratumoral genetic heterogeneity in rectal cancer and response to neoadjuvant chemoradiotherapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B62.

We wish to draw the attention of cancer geneticists to a particular genetic variant of the Prolactin-Induced Protein (PIP) gene that may be an important predisposing factor to cancer because of its high frequency and significant association with cancer, as determined in this study. In an initial copy number variation (CNV) screen for germ-line deletions in 123 Brazilian cancer patients selected as high risk either because of early age of onset (pediatric cancer) or a positive family history (TP53-negative Li–Fraumeni and APC/MUTYH-negative Familial Adenomatous Polyposis patients), a previously undescribed microdeletion was discovered in four patients. The deletion carried in these patients was apparently identical (Figure 1), with a size of 69 kb and similar base-pair position in chromosome region 7q34. All four deletions were validated by qPCR and found to harbor only the PIP and TAS2R39 (taste receptor type 2 member 39) genes. TAS2R39 is a member of ∼30 TAS2R bitter taste receptors,1 several of which are known to exhibit variation in copy number. It is unlikely that TAS2R39 has a role in tumorigenesis, and will not be considered further in this submission. We here refer to the deletion encompassing the PIP gene as PIP-Sao Paulo, following the convention of adding the place of discovery to the gene name. This deletion was not detected in a control group of 260 non-related individuals from the Sao Paulo urban area that had attended our genetic service for reasons other than cancer (normal relatives of patients with intellectual disabilities). The difference in deletion frequency between patients and controls was significant at the 0.01 level (Fisher's exact test). Another example of the PIP-Sao Paulo deletion was serendipitously detected in a TP53-mutated patient who was not part of these cohorts, but had been investigated by aCGH for presenting with an atypically severe course of cancer that commenced at 4 years of age with the diagnosis of a rhabdomyosarcoma, followed by choroid plexus tumor (7 years of age), liposarcoma and osteochondroma (10 years of age), and finally passed away at 11 years. This patient was not included in the statistics of the patient–control comparison; however, a possible interaction between the TP53 mutation and PIP-Sao Paulo deletion may have contributed to the severity of the cancer progression. Figure 1 PIP-Sao Paulo microdeletion at 7q34, detected by array-CGH in cancer patients. Array-CGH profile of a chromosome region at 7q34 (microarray platform Agilent 180K—Agilent Technologies, Santa Clara, CA, USA), showing heterozygous losses ... Following our initial observations, we interrogated the 1000 Genomes Project database and found a comparable deletion to be present in 13 out of 1097 individuals: 9 of European and 4 of Latin-American origin (see Supplementary Table 1). Manual review of the sequence data in this region indicated that 7 out of the 13 individuals had sufficient sequence depth to determine that they shared identical breakpoints. From this information, we designed PCR primers and were able to amplify across the breakpoints in all the five Brazilian deletion cases. Subsequent sequencing of the PCR fragments (see Supplementary Figure 1) demonstrated that all identified PIP-Sao Paulo deletions were identical to the deletions present in the 1000 Genomes Project data set. In a replicate study, we determined by breakpoint PCR the frequency of PIP deletions in an independent Brazilian cancer group (219 individuals) that had either presented with more than one primary cancer prior to 60 years of age (166 individuals) or were probands of hereditary melanoma families (53 individuals with no CDKN2A or CDK4 mutations). This identified a further 6 individuals, who had had cancer and carried the PIP deletion (2.6%), as opposed to 10 out of 847 individuals (1.2%) from a second control group. As in the initial study, the deletion frequency in the replicate study in cancer patients was higher than that in controls, although this was not significant at the 5% level (P=0.11). However, when cancer and control samples from both studies were pooled, statistical significance was attained (P=0.04, Fisher's exact test). Importantly, heterogeneity tests demonstrated that no significant differences existed at the 0.05 level between the two control and two patient samples, respectively. We conclude that the relatively frequent germ-line deletion of the PIP gene has more than a two-fold increase in frequency in the Brazilian cancer patients compared with the Brazilian controls. The clinical phenotype, mutation status, type of tumor and age of diagnosis of all patients carrying the PIP-Sao Paulo deletion are summarized in Table 1, demonstrating that several types of cancer are involved in this study and that no single type or group of cancers predominates. Presence of the PIP-Sao Paulo deletion was investigated in paraffin blocks from several different primary tumors taken from four of the deleted patients. In all samples, except for one from patient 7, the PIP-Sao Paulo deletion was detected (Supplementary Figure 1); additionally, at least one exon of the PIP gene was amplified from each of the tested samples, indicating that one allele was retained, similar to blood. Unfortunately, DNA quality did not allow sequence screening for point mutations or other deletions that may have occurred in the remaining allele. We conclude that if the PIP-Sao Paulo deletion is directly responsible for tumor induction, this has not occurred by induction of homozygosity of the deletion itself in the cases studied. Table 1 Characteristics of the PIP-Sao Paulo deletion patients: mutation status, clinical phenotype, type of tumor and age at diagnosis To date, several publications have reported association between PIP expression and tumor progression, particularly for prostate and breast cancers.2, 3 The gene has been implicated in multiple functions, including apoptosis, cell proliferation and migration,4 but mutations have only been studied in tumor cell lines and never investigated as germ-line mutations. According to the TCGA database (http://cancergenome.nih.gov/), PIP gains in tumors are more frequent than deletions; however, data in different type of tumors are very heterogeneous and difficult to interpret. We have no specific insights into the mechanisms through which the PIP-Sao Paulo deletion could be oncogenic. It is evident from the foregoing that the PIP-Sao Paulo deletion is not specific to Brazil. Inspection of the HapMap3 database shows that the haplotype adjacent to the deletion (Table 2) is either absent or extremely rare in African populations. Interestingly, although about ∼25% of the 1094 individuals sequenced in data set 1 of the 1000 Genome Project were of African origin, none of the 13 individuals carrying the deletion were African and, except for one Colombian and two Mexican individuals from Los Angeles, CA, USA, all others were of European origin. Ethnic certainty of Brazilian cancer patients and controls is obscured by miscegenation and ethnic diversity; however, they are most likely to be mainly Caucasians, given the relatively high Caucasian composition of Sao Paulo, and through hospital attendance preferences and other socioeconomic differences. It is probable that PIP-Sao Paulo arose in an European population as a founder mutation that was subsequently exported to the New World. Table 2 SNPs, genomic positions (Hg19) and the common haplotype deduced from the proximal region of the patients with 7q34 deletion The crucial question of whether the PIP-Sao Paulo deletion constitutes a widespread cancer risk can only be answered by similar investigations in other countries, which we hope will be stimulated by this report. However, maintenance of a deletion variant associated with cancer at such high frequency is surprising and probably implies that, if the cancer association is confirmed by independent studies, PIP-Sao Paulo appears to have a relatively low cancer penetrance rate so that many carriers either do not manifest cancer or do so at later age, following transmission of the deletion to their offspring, similar to the well-documented Brazilian TP53 variant R337H4. Whereas R337H has an estimated population frequency of ∼0.3% in Southern Brazil and exhibits an ∼8% penetrance rate in families with adrenocortical cancer,5 PIP-Sao Paulo is practically an order of magnitude more frequent (estimated at ∼2% in European populations from the 1000 Genome Project data set) and appears as a very frequent factor associated with increased cancer risk. Further, if the PIP-Sao Paulo deletion frequency is at least two-fold higher in European cancer patients than in controls, as in the Sao Paulo study, then even allowing for a low penetrance rate, there would be an increased cancer risk for Europeans. In the case of PIP-Sao Paulo, it is the high frequency and not the penetrance that constitutes the main risk parameter. This completely unanticipated aspect deserves a rapid, coordinated response to confirm whether indeed such an increased risk exists, and if so, to establish its magnitude and specificity. Rapid and cost-effective detection of the deletion can be performed using either PCR across the breakpoints, or copy number estimates by RT-PCR, MLPA and so on. The exact genome location of PIP-Sao Paulo is chr7: 142824847-142893913 (Genome build GRCh37/hg19).

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate is a relatively common craniofacial defect with multifactorial inheritance. The association of the rs987525 single nucleotide variant, located in a gene desert at 8q24.21 region, has been consistently replicated in European populations. We performed a structured association approach combined with transcriptional analysis of the MYC gene to dissect the role of rs987525 in oral clefting susceptibility in the ethnically admixed Brazilian population. METHODS: We performed the association study conditioned on the individual ancestry proportions in a sample of 563 patients and 336 controls, and in an independent sample of 221 patients and 261 controls. The correlation between rs987525 genotypes and MYC transcriptional levels in orbicularis oris muscle mesenchymal stem cells was also investigated in 42 patients and 4 controls. RESULTS: We found a significant association in the larger sample (p 5 0.0016; OR 5 1.80 [95% confidence interval {CI}, 1.21–2.69], for heterozygous genotype, and 2.71 [95% CI, 1.47–4.96] for homozygous genotype). We did not find a significant correlation between rs987525 genotypes and MYC transcriptional levels (p 5 0.14; r 52 0.22, Spearman Correlation). CONCLUSIONS: We present a positive association of rs987525 in the Brazilian population for the first time, and it is likely that the European contribution to our population is driving this association. We also cannot discard a role of rs987515 in MYC regulation, because this locus behaves as an expression quantitative locus of MYC in another tissue. Birth Defects Research (Part A) 94:464–468, 2012. 2012 Wiley Periodicals, Inc.

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies. Electronic supplementary material The online version of this article (doi:10.1007/s12015-010-9197-3) contains supplementary material, which is available to authorized users.

Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial malformation caused by null mutations in the TCOF1 gene. High inter and intra familial clinical variability, ranging from mild malar hypoplasia to perinatal death due to airway collapse is observed, but, to date, no genotype-phenotype correlation has been reported. Considering haploinsufficiency as the molecular mechanism underlying the disease, we have hypothesized that mutations in the promoter region of the gene, which has never been previously characterized, in trans with a pathogenic mutation, could modulate the phenotype. Therefore, the aims of the present study were to determine the TCOF1 gene's core promoter and to identify mutations in this region that could contribute to the phenotypic variation observed in this syndrome. We have delimitated the minimal promoter to a region of less than 150 bp, with 63% of identity among 5 different species. We screened 1.2 kbp of the TCOF1 5' flanking sequence in the DNA obtained from 21 patients and 51 controls and identified four new single nucleotide polymorphisms (SNPs), one of which (-346C>T), was proved to be functional, as it decreased the promoter activity by 38%. Electrophoretic mobility shift assay (EMSA) analysis demonstrated that the -346T allele impairs DNA-binding to the YY1 transcription factor. This promoter variant represents a candidate allele to explain the clinical variability in patients bearing TCS.

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26–27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p

Abstract 1908 Introduction: MAGE-C1/CT7 encodes for a cancer/testis antigen (CTA) frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. The expression of this CTA is restricted to malignant plasma cells and a positive correlation between MAGEC1/CT7 expression and advanced stage has been demonstrated for MM. It has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function of this protein in the pathophysiology of MM is not yet understood. Objectives: (1) To clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle regulation in myeloma cell line SKO-007 and (2) to evaluate the impact of silencing MAGE-C1/CT7 on cells treated with bortezomib. Material and Methods: Short hairpin RNA (shRNA) specific for MAGE-C1/CT7 was inserted in the pRETROSUPER(pRS) retroviral vector. The pRS-shRNA-MAGE-C1/CT7 was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time PCR (RQ-PCR) and western blot. Functional studies included cell proliferation, cell cycle analysis using propidium iodide, and analysis of apoptosis using annexin V staining. Results: SKO-007 MM cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. SKO-007 cells were divided into three derivatives: empty vector (pRS) and ineffective shRNA (antisense strand deleted – GC bases) [both used as controls for all the experiments] and inhibited (shMAGE-C1/CT7). MAGE-C1/CT7 mRNA expression was ∼5 times lower in inhibited cell line than control cells by RQ-PCR. Western blot showed 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. We used empty vector, ineffective shRNA and inhibited cells to determine whether inhibition of MAGE-C1/CT7 was associated with cell cycle dysregulation. We detected differences between inhibited cells and both controls regarding the proportion of myeloma cells in the G2/M phase (p Conclusions: MAGE-C1/CT7 antigen inhibition did not change cell proliferation and DNA synthesis in SKO-007 cells. However, we found that MAGE-C1/CT7 plays in cell cycle regulation, protecting SKO-007 cells against bortezomib-induced apoptosis. Therefore, MAGE-C1/CT7 silencing by shRNA could be a strategy for future therapies in MM, i.e. in combination with proteasome inhibitors. [Supported by CNPq and LICR] Disclosures: No relevant conflicts of interest to declare.

Abstract 4888 Introduction MAGEC1/CT7 gene encodes for a cancer testis (CT) antigen frequently expressed in multiple myeloma (MM) and may be a potential target for immunotherapy in this still incurable disease. This CT gene expression is restricted to malignant plasma cells and may be related with tumor progression, since it seems to play a role as a “gatekeeper” gene for other CT antigens and can be associated with a more aggressive phenotype. However, the exact function of this protein for tumor biology is not yet understood. Objective The aim of this study is to elucidate the role of MAGEC1/CT7 in the control of cellular proliferation, DNA synthesis and invasive potential of Sko-007 myeloma cell line. Material and Methods We used a short hairpin RNA (shRNA) specific for MAGEC1/CT7 gene that was previously inserted in the pRS (pRETROSUPER) retroviral vector. The pRS-shRNA-MAGEC1/CT7 construct was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced and selected for stable expression of shRNA-MAGEC1/CT7. Expression of MAGEC1/CT7 was analyzed by Western Blot and Real Time PCR (RQ-PCR). Functional studies included cell proliferation, 3H thymidine incorporation and invasion potential, using matrigel-coated transwell filters. Results Sko-007 was chosen to perform the initial functional studies since the basal MAGEC1/CT7 gene expression level was higher in this cell line when compared with the other myeloma cell lines (U266 and SK-MM-2). Sko-007wt (wild type) cell was divided into three derivatives, Sko-007ev (empty vector), Sko-007shMAGEC1/CT7Δ (antisense strand deleted – GC bases) and Sko-007shMAGEC1/CT7. MAGEC1/CT7 expression was approximately five times down regulated in Sko-007shMAGEC1/CT7 when compared with Sko-007wt, Sko-007ev, and Sko-007shMAGEC1/CT7Δ cells by RQ-PCR. Sko-007shMAGE1/CT7 had an 80% decrease in MAGEC1/CT7 protein expression when compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ by Western Blot. Our preliminary functional assays did not show any change in cell proliferation and DNA synthesis when Sko-007shMAGEC1/CT7 was compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ. Indeed, Sko-007 cell line has invasive potential, but no change was observed in Sko-007shMAGEC1/CT7 or other cells (Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ). Conclusions Expression of MAGEC1/CT7 mRNA could be inhibited by the shRNA strategy, but the concomitant reduction of its protein level did not seem to have impact in proliferation, DNA synthesis or in invasive potential of Sko-007 MM cell line (Supported CNPq and LICR São Paulo Branch). Disclosures No relevant conflicts of interest to declare.

Background Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the TCOF1 gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if TCOF1 expression levels are decreased in post-embryonic human cells. Methods We have estimated TCOF1 transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively. Results All the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of TCOF1 is 18-31% lower in patients than in controls (p < 0.05), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells. Conclusions This is the first study to report decreased expression levels of TCOF1 in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of TCOF1 expression. Further, considering that TCOF1 deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.

Os transtornos do espectro autista (TEA) e a deficiência intelectual (DI) podem ser encontrados de forma isolada ou em combinação. Juntos, eles afetam cerca de 3% da população e, exceto quando associados à síndromes, ambos não possuem biomarcadores. Geneticamente, um dos fatores que dificulta a identificação de possíveis biomarcadores é o aspecto heterogêneo que essas doenças possuem. Isto é, diferentes genes podem estar alterados em diferentes pacientes, sendo ainda desconhecido quais seriam exatamente esses genes. Entretanto, se a presença de variantes genéticas responsáveis pela exibição desses fenótipos é bastante variada, por outro lado, é possível que tais alterações em níveis funcionais, e.g., transcrição gênica, sejam mais homogêneas entre os pacientes. Com efeito, vários estudos independentes, incluindo um de nosso grupo recém submetido, têm consistentemente encontrado uma rede de genes co-expressos desregulada, cuja função está relacionada a sinapse de neurônios de pacientes com TEA, indicando que o perfil de expressão desses genes poderia ser utilizado como biomarcador para o TEA. Todavia, ainda não se tem conhecimento se há outros perfis de desregulação dessa e de outras redes de co-expressão que possam ser utilizados para diferenciar DI e TEA. Deste modo, com o propósito avaliar as semelhanças e diferenças nos perfis de expressão dessas duas condições, o presente projeto pretende comparar os perfis de expressão gênica de células neuronais de indivíduos com TEA isolado, TEA+DI e DI isolado, visando identificar a existência de módulos de co-expressão cuja desregulação é exclusiva de uma ou outra condição. Para isso usaremos dados de transcriptoma públicos oriundos de tecido post-mortem. Um dos potenciais deste estudo é a contribuição para o desenvolvimento de ferramentas diagnósticas para estas duas condições, favorecendo assim uma intervenção terapêutica precoce e direcionada. Found isolated from each other or in combination, autism spectrum disorders (ASD) and intellectual deficiency (ID), affect around 3% of the population and, except when associated with a syndrome, both of them do not have a molecular biomarker. From a genetic point of view, one of the factors that makes the identification of possible biomarkers difficult, is the genetic heterogeneity associated with these diseases, i.e., different genes can be altered in different patients, being its total identification still unexplored. Though the presence of genetic variants associated with these phenotypes is still quite variable, it is possible that such alterations might result in functional alterations, e.g., at the genetic transcription level, that are more consistent among patients. Indeed, many independent studies, including one recently submitted to publication by our group, have consistently found a co-expression gene network as dysregulated in neurons from ASD patients, whose function is related to synapse, suggesting that the expression profile of these genes could be used as a biomarker for ASD. Nonetheless, there is still no knowledge regarding other possible dysregulation profiles related to this network or to any other co-expression gene network that could be used to differentiate ID and ASD. In this sense, we aim to evaluate the similarities and differences in the expression profiles of these two conditions. To achieve this goal the present study intends to compare the gene expression profile of neuronal cells from patients with ASD only, ASD + ID and ID only, to identify the existence of co-expressed gene networks, also called modules, whose dysregulation might be both exclusive or common to these conditions, by using publicly available transcriptomic datasets. In this case, we are particularly interested in datasets from post-mortem brain tissue.

As fissuras orofaciais não-sindrômicas (FO-NS) correspondem a 70% de todos os casos de FO, possuem etiologia complexa e pouco compreendida, sendo consideradas de herança multifatorial com forte influência de fatores genéticos e ambientais. Apesar de estudos de análise de ligação e associação apontarem vários loci de susceptibilidade às FO-NS, o componente genético ainda não está totalmente explicado. Fatores ambientais também possuem um importante papel na etiologia das FO, e alguns já foram replicados em várias populações. Fatores como exposição materna ao álcool, drogas, tabaco, medicamentos, desnutrição e baixo nível socioeconômico são alguns dos fatores já associados a esta condição. As infecções periodontais são comuns em mulheres grávidas e estão associadas a parto prematuro, baixo peso fetal e, mais recentemente, foram reportadas como fator de risco aumentado para FO-NS nos fetos. Adicionalmente, o avanço das tecnologias de sequenciamento do DNA melhorou exponencialmente a compreensão do microbioma humano e sua influência no estado de saúde e doença, e, mais especificamente, o conhecimento sobre o impacto do microbioma na gravidez. O objetivo deste projeto foi identificar novos fatores etiológicos genéticos e ambientais das FO-NS. Para isso, primeiramente, sequenciamos 68 genes candidatos a FO por sequenciamento de nova geração em 193 indivíduos com FO-NS familial. Nós encontramos enriquecimento significativo de variantes raras e patogênicas de perda de função nos indivíduos com FO-NS e observamos que essas variantes estão em genes intolerantes a esse tipo de mutação. Também reportamos novas variantes raras do tipo perda de função no gene ARHGAP29 e sua importância na susceptibilidade as FO-NS familiais. Além disso, sugerimos o uso de um ponto de corte baseado no escore pLI do banco de dados ExAC como parâmetro para priorizar variantes em estudos de FO-NS familiares, assumindo modelo de herança mono ou oligogênico. Adicionalmente, estudamos o microbioma oral de mães de crianças com FO-NS e mães de crianças sem malformações, utilizando o sequenciamento da subunidade 16S do rRNA das bactérias com o objetivo de verificar diferenças consistentes na composição do microbioma oral de mães de crianças com FO-NS, levando em consideração a presença ou não de doenças infecciosas periodontais maternas. A casuística foi composta de 6 mães de recém-nascidos de até 1 mês que apresentaram FO-NS ao nascimento e mães de crianças sem qualquer malformação congênita. As análises de alfa e beta diversidades não demonstraram diferença significativa na composição do microbioma oral de mães de crianças com FO-NS e mães de crianças controle, contudo observamos que o grupo com infecções periodontais possui a diversidade taxonômica mais abundante do que o grupo hígido. Em resumo, nesse estudo piloto não foi possível identificar alterações no microbioma oral como um fator etiológico das FO-NS. Novas análises em uma casuística maior são necessárias para a confirmação desse achado The non-syndromic orofacial clefts (nsOFC) correspond to 70% of all OFC cases, have complex etiology and are poorly understood, being considered multifactorial inheritance with a strong influence of genetic and environmental factors. Although linkage and association analysis studies point to several nsOFC susceptibility loci, the genetic component is not yet fully explained. Environmental factors also play an important role in OFC etiology, and some have been replicated in several populations. Factors such as maternal exposure to alcohol, drugs, tobacco, drugs, malnutrition and low socioeconomic status are some of the factors already associated with this condition. Periodontal infections are common in pregnant women and are associated with preterm birth, low birth weight and, more recently, have been reported as an increased risk factor for nsOFC in fetuses. Additionally, the advancement of DNA sequencing technologies has exponentially improved the understanding of the human microbiome and its influence on health and disease status, and, more specifically, knowledge about the impact of the microbiome on pregnancy. The objective of this project was to identify new genetic and environmental etiological factors of nsOFC. For this, we first sequenced 68 candidate genes by next generation sequencing in 193 individuals with familial nsOFC. We found significant enrichment of rare and pathogenic loss of function variants in individuals with nsOFC and we observed that these variants were in genes intolerant to this type of mutation. We also reported new rare loss-of-function variants in the ARHGAP29 gene and its importance in the liability of familial nsOFC. In addition, we suggested the use of a cutoff point based on the ExAC database pLI score as a parameter to prioritize variants in familial nsOFC studies, assuming a mono or oligogenic inheritance model. In addition, we studied the oral microbiome of 6 mothers of newborns up to 1-month-old with nsOFC and 6 mothers of newborns without congenital malformations using the 16S rRNA sequencing in order to verify consistent differences in the composition of the oral microbiome of mothers of children with nsOFC, taking into account the presence or absence of maternal periodontal infectious diseases. The analysis of alpha and beta diversities did not show a significant difference in the composition of the oral microbiome of mothers of nsOFC children and mothers of control children, however, we observed that the group with periodontal infectious diseases has more abundant taxonomic diversity than the healthy group. In summary, in this pilot study, it was not possible to identify alterations in the oral microbiome as an etiological factor of FO-NS. New analyzes in a larger cohort are necessary to confirm this finding

O Transtorno do Espectro Autista (TEA) é um transtorno neuropsiquiátrico grave caracterizado por comprometimento da capacidade de interação social e comunicação e pela presença de interesses, atividades e comportamentos repetitivos e restritos. O TEA apresenta alta heterogeneidade genética e pode ser enquadrado em diferentes modelos de herança, o que torna difícil apontar os fatores genéticos associados ao transtorno e compreender de que forma eles interagem e ocasionam, em última instância, sua manifestação clínica. Ainda, fenômenos como penetrância incompleta e discordância entre gêmeos monozigóticos são frequentemente observados em famílias com indivíduos afetados, tornando ainda mais complexo o entendimento do envolvimento de cada um dos fatores genéticos na etiologia do TEA. Uma forma de contornar esses problemas é a utilização de abordagens transcriptômicas no estudo desse transtorno. Nesse contexto, a busca por variantes patogênicas deixa de ser o principal foco do estudo e a atenção volta-se para a investigação de vias de sinalização e processos biológicos que estejam desregulados durante o processo de diferenciação neuronal e que sejam compartilhados pelos indivíduos afetados. Usando como principal ferramenta o sequenciamento do transcriptoma de células progenitoras neurais (NPC) e neurônios derivados de células-tronco pluripotentes induzidas (iPSC), identificamos uma série de alterações transcricionais importantes entre pacientes e controles, sugerindo que, a despeito da grande heterogeneidade genética associada ao transtorno, essas alterações se refletem em processos biológicos comuns. Nossos resultados são consistentes com os obtidos em trabalhos anteriores realizados com outros modelos de estudo e as linhagens celulares geradas a partir de nosso protocolo parecem refletir de forma fidedigna o perfil de expressão do cérebro fetal. Ainda, em um caso particular incluído neste estudo, investigamos as consequências transcricionais e funcionais de uma duplicação na região 17p13.3 - alteração previamente implicada em outros casos de autismo - nas células neuronais deste paciente, contribuindo para a compreensão do papel desta duplicação na patofisiologia do TEA. Dessa forma, nosso trabalho reforça a validade do uso de células neurais derivadas de iPSC e de abordagens transcricionais para o estudo de transtornos do neurodesenvolvimento Autism Spectrum Disorder (ASD) is a severe neuropsychiatry disorder characterized by impaired social interaction and communication, restricted interests and repetitive behaviors. ASD is highly genetically heterogeneous and can be caused by different inheritance models, which hampers the identification of the genetic factors associated to the disorder. Incomplete penetrance and discordance between monozygotic twins are often observed in families of affected individuals, making it even more complex to understand the involvement of genetic factors in ASD. Applying transcriptomic approaches to study this disorder is an interesting way to overcome these problems. In this context, searching for pathogenic variants is no longer the study\'s main focus. Instead, one aims to investigate signaling pathways and biological process that are deregulated during neuronal differentiation and shared by affected individuals. Based on the transcriptome sequencing of neural progenitor cells (NPC) and neurons derived from induced pluripotent stem cells (iPSC), we were able to identify a series of important transcriptional alterations between patients and controls, which suggest that despite the great genetic heterogeneity associated to ASD, those alterations are reflected in common biological processes. Our results are consistent with those obtained in previous studies performed with other models and the lineages generated by our protocol seem to reliably reflect fetal brain expression profile. Moreover, in a particular case included in this study, we investigated transcriptional and functional consequences of a duplication located at 17p13.3 - an alteration previously found in other autism cases - in the neuronal cells of this patient, contributing towards a better comprehension of the role of this duplication in ASD pathophysiology. Hence, our work reinforces the validity of the use of neural cell derived from iPSC and transcriptional approaches in the studies of neurodevelopmental disorders