Abstract The coronavirus disease 2019 (COVID-19) mortality rates varied among the states of Brazil during the course of the pandemics. The human leukocyte antigen (HLA) is a critical component of the antigen presentation pathway. Individuals with different HLA genotypes may trigger different immune responses against pathogens, which could culminate in different COVID-19 responses. HLA genotypes are variable, especially in the highly admixed Brazilian population. In this ecological study, we aimed to investigate the correlation between HLA haplotypes and the different regional distribution of COVID-19 mortality in Brazil. HLA data was obtained from 4,148,713 individuals registered in The Brazilian Voluntary Bone Marrow Donors Registry. COVID-19 data was retrieved from epidemiological bulletins issued by State Health Secretariats via Brazil’s Ministry of Health from February/2020 to July/2022. We found a positive significant correlation between the HLA-A*01~B*08~DRB1*03 haplotype and COVID-19 mortality rates when we analyzed data from 26 states and the Federal District. This result indicates that the HLA-A*01~B*08~DRB1*03 haplotype may represent an additional risk factor for dying due to COVID-19. This haplotype should be further studied in other populations for a better understanding of the variation in COVID-19 outcomes across the world. 201 COVID19 COVID 19 (COVID-19 pandemics (HLA pathway pathogens COVID-1 variable population study 4148713 4 148 713 4,148,71 Registry Brazils s February2020 February 2020 February/202 July2022 July 2022 July/2022 HLAA*01~B*08~DRB1*03 HLAA01B08DRB103 HLAABDRB A*01~B*08~DRB1*03 A 01 B 08 DRB1 03 DRB HLA-A*01~B*08~DRB1*0 2 District COVID19. 19. world 20 COVID1 1 (COVID-1 COVID- 414871 14 71 4,148,7 February202 202 February/20 July202 July/202 HLAA HLAA*01~B*08~DRB1*0 HLAA01B08DRB10 A01B08DRB103 ABDRB A*01~B*08~DRB1*0 0 HLA-A*01~B*08~DRB1* (COVID- 41487 7 4,148, February20 February/2 July20 July/20 HLAA*01~B*08~DRB1* HLAA01B08DRB1 A01B08DRB10 A*01~B*08~DRB1* HLA-A*01~B*08~DRB1 (COVID 4148 4,148 February2 February/ July2 July/2 HLAA*01~B*08~DRB1 HLAA01B08DRB A01B08DRB1 A*01~B*08~DRB1 HLA-A*01~B*08~DRB 414 4,14 July/ HLAA*01~B*08~DRB A01B08DRB A*01~B*08~DRB 41 4,1 4,

Abstract Tuberous Sclerosis Complex (TSC) is caused by loss of function germline variants in the TSC1 or TSC2 tumor suppressor genes. Genetic testing for the detection of pathogenic variants in either TSC1 or TSC2 was implemented as a diagnostic criterion for TSC. However, TSC molecular diagnosis can be challenging due to the absence of variant hotspots and the high number of variants described. This review aimed to perform an overview of TSC1/2 variants submitted in the ClinVar database. Variants of uncertain significance (VUS), missense and single nucleotide variants were the most frequent in clinical significance (37-40%), molecular consequence (37%-39%) and variation type (82%-83%) categories in ClinVar in TSC1 and TSC2 variants, respectively. Frameshift and nonsense VUS have potential for pathogenic reclassification if further functional and segregation studies were performed. Indeed, there were few functional assays deposited in the database and literature. In addition, we did not observe hotspots for variation and many variants presented conflicting submissions regarding clinical significance. This study underscored the importance of disseminating molecular diagnostic results in a public database to render the information largely accessible and promote accurate diagnosis. We encourage the performance of functional studies evaluating the pathogenicity of TSC1/2 variants. (TSC genes However described TSC12 2 TSC1/ VUS, , (VUS) 3740%, 3740 37 40% 40 (37-40%) 37%39% 3739 37% 39% 39 (37%-39% 82%83% 8283 82% 83% 82 83 (82%-83% respectively performed Indeed literature addition (VUS 3740% 374 3 4 (37-40% 37%39 373 (37%-39 82%83 828 8 (82%-83 (37-40 37%3 (37%-3 82%8 (82%-8 (37-4 (37%- (82%- (37- (37% (82% (37 (82 (3 (8 (

Abstract The TP53 3’UTR variant rs78378222 A>C has been detected in different tumor types as a somatic alteration that reduces p53 expression through modification of polyadenylation and miRNA regulation. Its prevalence is not yet known in all tumors. Herein, we examine tumor tissue prevalence of rs7837822 in Brazilian cohorts of patients from south and southeast regions diagnosed with lung adenocarcinoma (LUAD, n=586), sarcoma (SARC, n=188) and uterine leiomyoma (ULM, n=41). The minor allele (C) was identified in heterozygosity in 6/586 LUAD tumors (prevalence = 1.02 %) and none of the SARC and ULM samples. Additionally, next generation sequencing analysis revealed that all variant-positive tumors (n=4) with sample availability had additional pathogenic or likely pathogenic somatic variants in the TP53 coding regions. Among them, 3/4 (75 %) had the same pathogenic or likely pathogenic sequence variant (allele frequency <0.05 in tumor DNA) namely c.751A>C (p.Ile251Leu). Our results indicate a low somatic prevalence of rs78378222 in LUAD, ULM and SARC tumors from Brazilian patients, which suggests that no further analysis of this variant in the specific studied regions of Brazil is warranted. However, these findings should not exclude tumor molecular testing of this TP53 3’UTR functional variant for different populations. TP TP5 3UTR UTR 3 rs AC A C p p5 regulation Herein rs783782 (LUAD n=586, n586 n n=586 , 586 n=586) SARC, (SARC n=188 n188 188 ULM, (ULM n=41. n41 n=41 . 41 n=41) (C 6586 6 6/58 102 1 02 1.0 % samples Additionally variantpositive positive n=4 n4 4 (n=4 them 34 3/ 75 (7 005 0 05 <0.0 DNA c751AC cAC c 751A p.Ile251Leu. pIle251Leu pIleLeu p.Ile251Leu Ile251Leu Ile Leu (p.Ile251Leu) warranted However populations rs78378 n58 n=58 58 n=18 n18 18 658 6/5 10 1. n= (n= 7 ( 00 <0. pIle IleLeu (p.Ile251Leu rs7837 n5 n=5 5 n=1 n1 65 6/ (n <0 rs783 < rs78 rs7

Abstract Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder caused by germline mutations in TSC1 or TSC2 genes, which leads to the hyperactivation of the mTORC1 pathway, an important negative regulator of autophagy. This leads to the development of hamartomas in multiple organs. The variability in symptoms presents a challenge for the development of completely effective treatments for TSC. One option is the treatment with mTORC1 inhibitors, which are targeted to block cell growth and restore autophagy. However, the therapeutic effect of rapamycin seems to be more efficient in the early stages of hamartoma development, an effect that seems to be associated with the paradoxical role of autophagy in tumor establishment. Under normal conditions, autophagy is directly inhibited by mTORC1. In situations of bioenergetics stress, mTORC1 releases the Ulk1 complex and initiates the autophagy process. In this way, autophagy promotes the survival of established tumors by supplying metabolic precursors during nutrient deprivation; paradoxically, excessive autophagy has been associated with cell death in some situations. In spite of its paradoxical role, autophagy is an alternative therapeutic strategy that could be explored in TSC. This review compiles the findings related to autophagy and the new therapeutic strategies targeting this pathway in TSC.

Abstract Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.

RESUMO Introdução: O desenvolvimento de anticorpos (inibidores) contra o fator exógeno é a principal complicação do tratamento de hemofilias. Tanto fatores genéticos quanto não genéticos estão relacionados com o surgimento dos inibidores. Entre os fatores genéticos, o tipo de mutação que originou a doença é um dos mais importantes. Os objetivos do presente estudo foram estabelecer a prevalência das inversões nos íntrons 1 e 22 do gene do fator VIII em pacientes com hemofilia A grave, correlacionando-a com o desenvolvimento de inibidores, bem como comparar os resultados encontrados com dados da literatura mundial. Método: Foram analisados pacientes hemofílicos A graves não aparentados quanto à presença da inversão no íntron 1 (n = 77) e da inversão no íntron 22 (n = 39), utilizando a técnica de reação em cadeia dapolimerase (PCR). A detecção do inibidor foi realizada pelo teste de mistura; a sua quantificação, pelo método de Bethesda. Resultados: As prevalências das inversões nos íntrons 1 e 22 foram de 2,6% e 41%, respectivamente. Nenhum paciente com a inversão no íntron 1 apresentou inibidores, enquanto 26,3% dos pacientes com a inversão no íntron 22 desenvolveram os anticorpos. Conclusão: O número reduzido de pacientes com a inversão no íntron 1 não permitiu a aplicação de teste estatístico para a correlação com o risco de desenvolvimento de inibidores. A inversão no íntron 22 do gene do fator VIII não se associou ao maior risco de desenvolvimento de inibidores na amostra analisada (p = 1).

ABSTRACT Objectives: The development of antibodies (inhibitors) against exogenous factors is the main complication in the treatment of hemophilia. Both genetic and non-genetic factors are related to inhibitor development. Among the genetic factors, the type of mutation that caused the disease is one of the most important. The objectives of the present study were to establish the prevalence of inversions in introns 1 and 22 of the factor VIII gene in patients with severe hemophilia A, correlating these with inhibitor development, and to compare the results with data from the literature. Method: Unrelated severe hemophilia A patients were analyzed for the presence of inversions in intron 1 (n = 77) and intron 22 (n = 39) by polymerase chain reaction (PCR). Detection of the inhibitor was performed by the mixing test and its quantification was performed by the Bethesda method. Results: The prevalence of inversions in introns 1 and 22 was 2.6% and 41%, respectively. No patient with inversions in intron 1 had inhibitors, whereas 26.3% of patients with inversions in intron 22 developed inhibitors. Conclusion: Due to the small number of patients with inversions in intron 1, it was not possible to perform a statistical test for the correlation with risk of inhibitor development. Inversions in intron 22 of the factor VIII gene were not associated with an increased risk of inhibitor development in the analyzed samples (p = 1).

RESUMEN Introducción: El desarrollo de anticuerpos (inhibidores) contra elfactor exógeno es la principal complicación del tratamiento de hemofilias. Tanto factores genéticos como no genéticos están relacionados con la aparición de los inhibidores. Entre los factores genéticos, el tipo de mutación que originó la enfermedad es uno de los más importantes. El objetivo de este estudio fue establecer laprevalencia de las inversiones en los intrones 1 y 22 del gen del factor VIII en pacientes con hemofilia A severa, relacionándola con el desarrollo de inhibidores, así como comparar los resultados encontrados con datos de la literatura en el mundo. Método: Pacientes con hemofilia A severa no emparentados fueron analizados cuanto a la presencia de inversión en el intrón 1 (n = 77) y de la inversión en el intrón 22 (n = 39), usando la técnica de reacción en cadena de lapolimerasa. La detección del inhibidor fue realizada por el estudio de mezclas; su cuantificación, por el método Bethesda. Resultados: La prevalencia de las inversiones en los intrones 1 y 22 fueron 2,6% y 41%, respectivamente. Ningúnpaciente con la inversión en el intrón 1 presentó inhibidores, mientras 26,3% de los pacientes con la inversión en el intrón 22 desarrollaron anticuerpos. Conclusión: El pequeno número de pacientes con inversión en el intrón 1 no permitió la aplicación de la prueba estadística para correlación con el riesgo de desarrollo de inhibidores. La inversión en el intrón 22 del gen del factor VIII no se asoció a un mayor riesgo de desarrollo de inhibidores en la muestra analizada (p = 1).

Abstract Tuberous sclerosis complex is an autosomal dominant disorder characterized by skin manifestations and formation of multiple tumors in different organs, mainly in the central nervous system. Tuberous sclerosis is caused by the mutation of one of two tumor suppressor genes, TSC1 or TSC2. Currently, the development of novel techniques and great advances in high-throughput genetic analysis made mutation screening of the TSC1 and TSC2 genes more widely available. Extensive studies of the TSC1 and TSC2 genes in patients with TSC worldwide have revealed a wide spectrum of mutations. Consequently, the discovery of the underlying genetic defects in TSC has furthered our understanding of this complex genetic disorder, and genotype-phenotype correlations are becoming possible, although there are still only a few clearly established correlations. This review focuses on the main symptoms and genetic alterations described in TSC patients from 13 countries in three continents, as well as on genotype-phenotype correlations established to date. The determination of genotype-phenotype correlations may contribute to the establishment of successful personalized treatment for TSC.