Exploring the frequency of a TP53 polyadenylation signal variant in tumor DNA from patients diagnosed with lung adenocarcinomas, sarcomas and uterine leiomyomas.

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.

Functional pri-miR-34b/c rs4938723 and KRAS 3'UTR rs61764370 SNPs: Novel phenotype modifiers in Li-Fraumeni Syndrome?

PURPOSE: Li-Fraumeni Syndrome (LFS) is a rare cancer predisposing condition caused by germline pathogenic TP53 variants, in which core tumors comprise sarcomas, breast, brain and adrenocortical neoplasms. Clinical manifestations are highly variable in carriers of the Brazilian germline founder variant TP53 p.R337H, possibly due to the influence of modifier genes such as miRNA genes involved in the regulation of the p53 pathway. Herein, we investigated the potential phenotypic effects of two miRNA-related functional SNPs, pri-miR-34b/c rs4938723 and 3'UTR KRAS rs61764370, in a cohort of 273 LFS patients from Southern and Southeastern Brazil. METHODS: The genotyping of selected SNPs was performed by TaqMan® allelic discrimination and subsequently custom TaqMan® genotyping results were confirmed by Sanger sequencing in all SNP-positive LFS patients. RESULTS: Although the KRAS SNP showed no effect as a phenotype modulator, the rs4938723 CC genotype was significantly associated with development of LFS non-core tumors (first tumor diagnosis) in p.R337H carriers (p = 0.039). Non-core tumors were also more frequently diagnosed in carriers of germline TP53 DNA binding domain variants harboring the rs4938723 C variant allele. Previous studies described pri-miR-34b/c rs4938723 C as a risk allele for sporadic occurrence of thyroid and prostate cancers (non-core tumors of the LFS spectrum). CONCLUSION: With this study, we presented additional evidence about the importance of analyzing miRNA genes that could indirectly regulate p53 expression, and, therefore, may modulate the LFS phenotype, such as those of the miR-34 family.

Challenges in periodic revision of genetic testing results: Comparison of the main classification guidelines and report of a retrospective analysis involving BRCA1/BRCA2 variants of uncertain significance.

In the context of cancer predisposition syndromes, it is widely known that the correct interpretation of germline variants identified in multigene panel testing is essential for adequate genetic counseling and clinical decision making, in which variants of uncertain significance (VUS) are not considered actionable findings. Thus, their periodic re-evaluation using appropriate guidelines is notably important. In the present study, we compared the performance of the main variant classification guidelines (ACMG, Sherloc and ENIGMA) in variant reassessment, using as input a BRCA1/2 VUS case series (retrospective analysis) from Brazil, an ethnically diverse and admixed country with substantial challenges in VUS reclassification. As main findings, two of the 15 VUS analyzed were reclassified as likely pathogenic by the 3 guidelines, BRCA1 c.4987-3C > G (rs397509213) and BRCA2 c.7868A > G (rs80359012). Moreover, challenges in variant classification and reassessment are described and additional in silico data about structural impact of the variant BRCA2 c.7868A > G are provided. We hypothesize that the establishment of a framework to reassess VUS could improve this process in health centers that have not yet implemented this practice. Results of this study underscore that periodic monitoring of the functional, clinical, and bioinformatics data of a VUS by a multidisciplinary team are of utmost importance in clinical practice. When there is a specific guideline for a given gene, such as ENIGMA for BRCA1/2, it should be considered the first option for variant assessment. Finally, recruitment of VUS carriers and their relatives to participate in variant segregation studies and publication of VUS reclassification results in the international scientific literature should be encouraged.

Contribution of miR-124 rs531564 polymorphism to the occurrence of congenital Zika syndrome.

Zika virus (ZIKV) cause Congenital Zika Syndrome (CZS) in individuals exposed during pregnancy. Studies have shown that ZIKV infection positively regulates the miR-124 expression in neural cells, which leads to a decrease of TFRC, a gene targeted of this miRNA. Both miR-124 and TFRC exhibit a pivotal role in nervous system development. Therefore, in this study we aimed to investigate whether genetic variants that affect the expression of these genes could act together with ZIKV to increase the risk of individuals developing CZS. TFRC rs406271 and MIR-124-1 rs531564 polymorphisms were genotyped, using TaqMan® Genotyping Assays, in a sample of children who were exposed to ZIKV during pregnancy, of whom 40 were born with CZS and 48 without congenital anomalies. We identified that individuals with CZS presented a higher frequency of CG genotype of rs531564 polymorphism in MIR-124-1 (p=0.048), which is associated with increased expression of miR-124. Since ZIKV also upregulates the expression of this miRNA, the presence of CG genotype in individuals exposed to the virus could lead to a scenario of overexpression of miR-124 in the brain. Since teratogenesis is a multifactorial event, this genetic finding could partly explain why such individuals are more susceptible to CZS, considering both the downregulation of important neurodevelopment genes, as well as deregulation of the neurogenesis process. Thus, we provide preliminary evidence about a possible genetic risk factor to CZS and highlight the importance of analyzing functional polymorphisms related to epigenetic modulators of neurodevelopment genes in the context of ZIKV teratogenesis.

Feature selection reveal peripheral blood parameter's changes between COVID-19 infections patients from Brazil and Ecuador.

The investigation of conventional complete blood-count (CBC) data for classifying the SARS-CoV-2 infection status became a topic of interest, particularly as a complementary laboratory tool in developing and third-world countries that financially struggled to test their population. Although hematological parameters in COVID-19-affected individuals from Asian and USA populations are available, there are no descriptions of comparative analyses of CBC findings between COVID-19 positive and negative cases from Latin American countries. In this sense, machine learning techniques have been employed to examine CBC data and aid in screening patients suspected of SARS-CoV-2 infection. In this work, we used machine learning to compare CBC data between two highly genetically distinguished Latin American countries: Brazil and Ecuador. We notice a clear distribution pattern of positive and negative cases between the two countries. Interestingly, almost all red blood cell count parameters were divergent. For males, neutrophils and lymphocytes are distinct between Brazil and Ecuador, while eosinophils are distinguished for females. Finally, neutrophils, lymphocytes, and monocytes displayed a particular distribution for both genders. Therefore, our findings demonstrate that the same set of CBC features relevant to one population is unlikely to apply to another. This is the first study to compare CBC data from two genetically distinct Latin American countries.

Clinical and molecular characterization of patients fulfilling Chompret criteria for Li-Fraumeni syndrome in Southern Brazil.

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome caused by pathogenic germline variants in the TP53 gene, characterized by a predisposition to the development of a broad spectrum of tumors at an early age. The core tumors related to LFS are bone and soft tissue sarcomas, premenopausal breast cancer, brain tumors, adrenocortical carcinomas (ACC), and leukemias. The revised Chompret criteria has been widely used to establish clinical suspicion and support TP53 germline variant testing and LFS diagnosis. Information on TP53 germline pathogenic variant (PV) prevalence when using Chompret criteria in South America and especially in Brazil is scarce. Therefore, the aim of this study was to characterize patients that fulfilled these specific criteria in southern Brazil, a region known for its high population frequency of a founder TP53 variant c.1010G>A (p.Arg337His), as known as R337H. TP53 germline testing of 191 cancer-affected and independent probands with LFS phenotype identified a heterozygous pathogenic/likely pathogenic variant in 26 (13.6%) probands, both in the DNA binding domain (group A) and in the oligomerization domain (group B) of the gene. Of the 26 carriers, 18 (69.23%) were R337H heterozygotes. Median age at diagnosis of the first tumor in groups A and B differed significantly in this cohort: 22 and 2 years, respectively (P = 0.009). The present study shows the clinical heterogeneity of LFS, highlights particularities of the R337H variant and underscores the need for larger collaborative studies to better define LFS prevalence, clinical spectrum and penetrance of different germline TP53 pathogenic variants.

A novel DMD intronic alteration: a potentially disease-causing variant of an intermediate muscular dystrophy phenotype.

Pathogenic germline variants in DMD gene, which encodes the well-known cytoskeletal protein named dystrophin, are associated with a wide range of dystrophinopathies disorders, such as Duchenne muscular dystrophy (DMD, severe form), Becker muscular dystrophy (BMD, mild form) and intermediate muscular dystrophy (IMD). Muscle biopsy, immunohistochemistry, molecular (multiplex ligation-dependent probe amplification (MLPA)/next-generation sequencing (NGS) and Sanger methods) and in silico analyses were performed in order to identify alterations in DMD gene and protein in a patient with a clinical manifestation and with high creatine kinase levels. Herein, we described a previously unreported intronic variant in DMD and reduced dystrophin staining in the muscle biopsy. This novel DMD variant allele, c.9649+4A>T that was located in a splice donor site within intron 66. Sanger sequencing analysis from maternal DNA showed the presence of both variant c.9649+4A>T and wild-type (WT) DMD alleles. Different computational tools suggested that this nucleotide change might affect splicing through a WT donor site disruption, occurring in an evolutionarily conserved region. Indeed, we observed that this novel variant, could explain the reduced dystrophin protein levels and discontinuous sarcolemmal staining in muscle biopsy, which suggests that c.9649+4A>T allele may be re-classified as pathogenic in the future. Our data show that the c.9649+4A>T intronic sequence variant in the DMD gene may be associated with an IMD phenotype and our findings reinforce the importance of a more precise diagnosis combining muscle biopsy, molecular techniques and comprehensive in silico approaches in the clinical cases with negative results for conventional genetic analysis.

Prevalence of the Brazilian TP53 Founder c.1010G>A (p.Arg337His) in Lung Adenocarcinoma: Is Genotyping Warranted in All Brazilian Patients?

In Southern and Southeastern Brazil, there is a germline pathogenic variant with incomplete penetrance located in the oligomerization domain of TP53, c.1010G>A (p.Arg337His). Due to a founder effect, the variant is present in 0.3% of the general population of the region. Recently, this variant was identified in 4.4 and 8.9% of two apparently unselected, single center case series of Brazilian lung adenocarcinoma (LUAD) patients from the Southeastern and Central regions of the country, respectively. In the present study, our aim was to examine TP53 c.1010G>A allele and genotype frequencies in LUAD samples obtained from patients diagnosed in Southern Brazil. A total of 586 LUAD samples (tumor DNA) recruited from multiple centers in the region were tested, and the mutant allele was identified using TaqMan® assays in seven cases (7/586, 1.2%) which were submitted to next generation sequencing analyses for confirmation. Somatic EGFR mutations were more frequent in TP53 c.1010G>A carriers than in non-carriers (57.1 vs. 17.6%, respectively). Further studies are needed to confirm if TP53 c.1010G>A is a driver in LUAD carcinogenesis and to verify if there is a combined effect of EGFR and germline TP53 c.1010G>A. Although variant frequency was higher than observed in the general population, it is less than previously reported in LUAD patients from other Brazilian regions. Additional data, producing regional allele frequency information in larger series of patients and including cost-effectiveness analyses, are necessary to determine if TP53 c.1010G>A screening in all Brazilian LUAD patients is justified.

MIR605 rs2043556 is associated with the occurrence of multiple primary tumors in TP53 p.(Arg337His) mutation carriers.

Li-Fraumeni and Li-Fraumeni-like (LFS/LFL) Syndrome are cancer predisposition syndromes caused by germline pathogenic variants in TP53 and are associated with an increased risk of multiple early-onset cancers. In Southern and Southeastern Brazil, a germline founder variant with partial penetrance located in the oligomerization domain of TP53, c.1010G>A p.(Arg337His, commonly known as R337H), has been detected in 0.3% of the general population. Recently, the functional MIR605 variant rs2043556 (A>G) has been identified as a novel LFS phenotype modifier in families with germline TP53 DNA binding variants. In this study, our goal was to verify MIR605 rs2043556 allele frequencies and further explore its possible effects on the phenotype of 238 Brazilian individuals carrying TP53 p.(Arg337His). The MIR605 rs2043556 G allele was detected in 136 (57.1%) individuals, including 25 homozygotes (10.5%), and although it had been previously associated with an earlier mean age of tumor onset, this effect was not observed in this study (p = 0.8). However, in p.(Arg337His) mutation carriers, the GG genotype was significantly associated with the occurrence of multiple primary tumors (p = 0.005). We provide further evidence of MIR605 rs2043556 G allele's effect as a phenotype modulator in carriers of germline TP53 pathogenic variants.

TP53 variants of uncertain significance: increasing challenges in variant interpretation and genetic counseling.

Li-Fraumeni syndrome (LFS) and Li-Fraumeni Like (LFL) are autosomal dominant cancer predisposition syndromes caused by pathogenic germline variants in the TP53 gene. Recent studies have shown that the incorporation of next-generation sequencing by using multigene panels in clinical practice has resulted in the frequent identification of variants of uncertain significance (VUS). Given that there is no established medical management for VUS carriers, the identification of these variants may cause confusion and anxiety for both patients and practitioners. Herein, we aimed to verify VUS frequency and review VUS classification and interpretation in 1844 patients submitted for comprehensive germline TP53 testing independent of clinical criteria. Variant characterization was done assessing clinical information whenever available, variant frequency in population databases, pathogenicity predictions using in silico tools and previous functional studies. All variants were classified based on the guidelines proposed by the American College of Medical Genetics and Genomics (2015) and by the Sherloc framework (2017). Of the twelve VUS (0.65%) identified in TP53, two were classified as likely pathogenic and two were classified as likely benign after re-evaluation, potentially resulting in significant management modification for the proband and relatives. This report cases highlights the challenges and impact of TP53 variant interpretation especially when there is no clear LFS/LFL phenotype.

A comprehensive analysis of core polyadenylation sequences and regulation by microRNAs in a set of cancer predisposition genes.

Two core polyadenylation elements (CPE) located in the 3' untranslated region of eukaryotic pre-mRNAs play an essential role in their processing: the polyadenylation signal (PAS) AAUAAA and the cleavage site (CS), preferentially a CA dinucleotide. Herein, we characterized PAS and CS sequences in a set of cancer predisposition genes (CPGs) and performed an in silico investigation of microRNAs (miRNAs) regulation to identify potential tumor-suppressive and oncogenic miRNAs. NCBI and alternative polyadenylation databases were queried to characterize CPE sequences in 117 CPGs, including 81 and 17 known tumor suppressor genes and oncogenes, respectively. miRNA-mediated regulation analysis was performed using predicted and validated data sources. Based on NCBI analyses, we did not find an established PAS in 21 CPGs, and verified that the majority of PAS already described (74.4%) had the canonical sequence AAUAAA. Interestingly, "AA" dinucleotide was the most common CS (37.5%) associated with this set of genes. Approximately 90% of CPGs exhibited evidence of alternative polyadenylation (more than one functional PAS). Finally, the mir-192 family was significantly overrepresented as regulator of tumor suppressor genes (P < 0.01), which suggests a potential oncogenic function. Overall, this study provides a landscape of CPE in CPGs, which might be useful in development of future molecular analyses covering these frequently neglected regulatory sequences.

Targeting Histone Deacetylase Activity to Arrest Cell Growth and Promote Neural Differentiation in Ewing Sarcoma.

There is an urgent need for advances in the treatment of Ewing sarcoma (EWS), an aggressive childhood tumor with possible neuroectodermal origin. Inhibition of histone deacetylases (HDAC) can revert aberrant epigenetic states and reduce growth in different experimental cancer types. Here, we investigated whether the potent HDAC inhibitor, sodium butyrate (NaB), has the ability to reprogram EWS cells towards a more differentiated state and affect their growth and survival. Exposure of two EWS cell lines to NaB resulted in rapid and potent inhibition of HDAC activity (1 h, IC50 1.5 mM) and a significant arrest of cell cycle progression (72 h, IC50 0.68-0.76 mM), marked by G0/G1 accumulation. Delayed cell proliferation and reduced colony formation ability were observed in EWS cells after long-term culture. NaB-induced effects included suppression of cell proliferation accompanied by reduced transcriptional expression of the EWS-FLI1 fusion oncogene, decreased expression of key survival and pluripotency-associated genes, and re-expression of the differentiation neuronal marker ßIII-tubulin. Finally, NaB reduced c-MYC levels and impaired survival in putative EWS cancer stem cells. Our findings support the use of HDAC inhibition as a strategy to impair cell growth and survival and to reprogram EWS tumors towards differentiation. These results are consistent with our previous studies indicating that HDis can inhibit the growth and modulate differentiation of cells from other types of childhood pediatric tumors possibly originating from neural stem cells.

p53 signaling pathway polymorphisms, cancer risk and tumor phenotype in TP53 R337H mutation carriers.

Li-Fraumeni and Li-Fraumeni-like syndrome (LFS/LFL) are clinically heterogeneous cancer predisposition syndromes characterized by diagnosis of early-onset and often multiple cancers with variable tumor patterns and incomplete penetrance. To date, the genetic modifiers described in LFS/LFL have been shown to map to either TP53 or its main negative regulator, MDM2. Additionally, all studies were focused on families with different TP53 germline mutations. Hence, in this study we explored the effect of the most studied polymorphisms of p53 pathway genes on clinical manifestations of individuals carrying the founder TP53 mutation R337H (n = 136) and controls (n = 186). Cancer-affected carriers had been diagnosed either with adrenocortical carcinoma (ACC, n = 29) or breast cancer (BC, n = 43). Allelic discrimation using TaqMan assay was used for genotyping MDM2 SNP 309 (rs2279744) as well as MDM4 (rs1563828) and USP7 (rs1529916) polymorphisms. We found significantly higher MDM2 SNP 309 GG genotype and G allele frequencies in the LFS cohort than in controls. Furthermore, median age at first diagnosis was earlier in MDM2 SNP309 GG carriers when compared to other genotypes for both cancers (ACC: age 1 vs. 2 years; BC: age 35 vs. 43 years, respectively), although not statistically different. The allelic and genotypic frequencies for all SNPs did not differ between cancer affected and unaffected carriers, neither between patients with ACC or BC. In conclusion, our results suggest that MDM2 SNP 309 may contribute to the LFL phenotype and also to an earlier age at diagnosis of ACC and BC cancer in carriers of the R337H founder mutation.

Gastrin-Releasing Peptide Receptor Knockdown Induces Senescence in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor, characterized by excessive cell proliferation, resistance to apoptosis, and invasiveness. Due to resistance to currently available treatment options, the prognosis for patients with GBM is very dismal. The activation of gastrin-releasing peptide receptors (GRPR) stimulates GBM cell proliferation, whereas GRPR antagonists induce antiproliferative effects in in vitro and in vivo experimental models of GBM. However, the role of GRPR in regulating other aspects of GBM cell function related to tumor progression remains poorly understood, and previous studies have not used RNA interference techniques as tools to examine GRPR function in GBM. Here, we found that stable GRPR knockdown by a lentiviral vector using a short hairpin interfering RNA sequence in human A172 GBM cells resulted in increased cell size and altered cell cycle dynamics consistent with cell senescence. These changes were accompanied by increases in the content of p53, p21, and p16, activation of epidermal growth factor receptors (EGFR), and a reduction in p38 content. These results increase our understanding of GRPR regulation of GBM cells and further support that GRPR may be a relevant therapeutic target in GBM.

Combined Treatments with a Retinoid Receptor Agonist and Epigenetic Modulators in Human Neuroblastoma Cells.

Neuroblastoma (NB) is the most common extracranial solid childhood tumor accounting for around 15% of pediatric cancer deaths and most probably originates from a failure in the development of embryonic neural crest cells. Retinoids can inhibit the proliferation and stimulate differentiation of NB cells. In addition, epigenetic events involving changes in chromatin structure and DNA methylation can mediate the effects of retinoids; hence, the scope of this study is to investigate the use of retinoids and epigenetic drugs in NB cell lines. Here, we demonstrate that the combination of retinoid all trans-retinoic acid (ATRA) with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase is more effective in impairing the proliferation of human SH-SY5Y and SK-N-BE(2) NB cells than any drug given alone. Treatments also induced differential changes on the messenger RNA (mRNA) expression of retinoid receptor subtypes and reduced the protein content of c-Myc, the neuronal markers NeuN and ß-3 tubulin, and the oncoprotein Bmi1. These results suggest that the combination of retinoids with epigenetic modulators is more effective in reducing NB growth than treatment with single drugs.