Gianna Carvalheira

Genética do autismo

Autism is a neuropsychiatric disorder with profound family and social consequences. An extraordinary number of genetical-clinical, cytogenetics and molecular studies were done in recent years. A multiloci epistatic model involved in the causation of autism have emerged from these studies.

microRNA-106b-mediated down-regulation of C1orf24 expression induces apoptosis and suppresses invasion of thyroid cancer.

We previously showed that C1orf24 expression is increased in thyroid carcinomas. Nonetheless, the mechanism underlying C1orf24 deregulation is not fully understood. It has been widely demonstrated that microRNAs are involved in post-transcriptional gene regulation in several diseases, including cancer. Using in silico prediction approach, five microRNAs that bind to the 3′-untranslated region (3′-UTR) of C1orf24 were identified. The expression of two selected microRNAs (miR-17-5p, miR-106b) and the expression of C1orf24 were tested in 48 benign and malignant thyroid lesions and in five thyroid carcinoma cell lines. miR-106b was down-regulated in thyroid cancer specimens and thyroid carcinoma cell lines, while C1orf24 expression was markedly increased. To demonstrate that miR-106b reduces C1orf24 expression, follicular (WRO) and papillary (TPC1) thyroid carcinoma cell lines were transiently transfected with miR-106b mimic. Ectopic expression of the miR-106b mimic significantly inhibits C1orf24 mRNA and protein expression in both WRO and TPC1 cells. Dual-luciferase report assays demonstrated that miR-106b directly targets C1orf24 by binding its 3′- UTR. Moreover, miR-106b-mediated down-regulation of C1orf24 expression increased apoptosis and inhibited migration. We additionally demonstrated that siRNA against C1orf24 significantly decreased its expression, inhibited cell migration and cell cycle progression while induced apoptosis. In summary, our findings not only provide new insights into molecular mechanism associated with C1orf24 overexpression in thyroid carcinomas but also show that C1orf24 might increase proliferation and cell migration. Thus, decreasing C1orf24 levels, by restoring miR-106b function, may have therapeutic implications.

X-linked intellectual disability related genes disrupted by balanced X-autosome translocations

Detailed molecular characterization of chromosomal rearrangements involving X‐chromosome has been a key strategy in identifying X‐linked intellectual disability‐causing genes. We fine‐mapped the breakpoints in four women with balanced X‐autosome translocations and variable phenotypes, in order to investigate the corresponding genetic contribution to intellectual disability. We addressed the impact of the gene interruptions in transcription and discussed the consequences of their functional impairment in neurodevelopment. Three patients presented with cognitive impairment, reinforcing the association between the disrupted genes (TSPAN7—MRX58, KIAA2022—MRX98, and IL1RAPL1—MRX21/34) and intellectual disability. While gene expression analysis showed absence of TSPAN7 and KIAA2022 expression in the patients, the unexpected expression of IL1RAPL1 suggested a fusion transcript ZNF611‐IL1RAPL1 under the control of the ZNF611 promoter, gene disrupted at the autosomal breakpoint. The X‐chromosomal breakpoint definition in the fourth patient, a woman with normal intellectual abilities, revealed disruption of the ZDHHC15 gene (MRX91). The expression assays did not detect ZDHHC15 gene expression in the patient, thus questioning its involvement in intellectual disability. Revealing the disruption of an X‐linked intellectual disability‐related gene in patients with balanced X‐autosome translocation is a useful tool for a better characterization of critical genes in neurodevelopment.

Genetic mechanisms leading to primary amenorrhea in balanced X-autosome translocations

OBJECTIVE To map the X-chromosome and autosome breakpoints in women with balanced X-autosome translocations and primary amenorrhea, searching candidate genomic loci for female infertility. DESIGN Retrospective and case-control study. SETTING University-based research laboratory. PATIENT(S) Three women with balanced X-autosome translocation and primary amenorrhea. INTERVENTION(S) Conventional cytogenetic methods, genomic array, array painting, fluorescence in situ hybridization, and quantitative reverse transcription-polymerase chain reaction. MAIN OUTCOME MEASURE(S) Karyotype, copy number variation, breakpoint mapping, and gene expression levels. RESULT(S) All patients presented with breakpoints in the Xq13q21 region. In two patients, the X-chromosome breakpoint disrupted coding sequences (KIAA2022 and ZDHHC15 genes). Although both gene disruptions caused absence of transcription in peripheral blood, there is no evidence that supports the involvement of these genes with ovarian function. The ZDHHC15 gene belongs to a conserved syntenic region that encompasses the FGF16 gene, which plays a role in female germ line development. The break in the FGF16 syntenic block may have disrupted the interaction between the FGF16 promoter and its cis-regulatory element. In the third patient, although both breakpoints are intergenic, a gene that plays a role in the DAX1 pathway (FHL2 gene) flanks distally the autosome breakpoint. The FHL2 gene may be subject to position effect due to the attachment of an autosome segment in Xq21 region. CONCLUSION(S) The etiology of primary amenorrhea in balanced X-autosome translocation patients may underlie more complex mechanisms than interruption of specific X-linked candidate genes, such as position effect. The fine mapping of the rearrangement breakpoints may be a tool for identifying genetic pathogenic mechanisms for primary amenorrhea.

AGK‐BRAF gene fusion is a recurrent event in sporadic pediatric thyroid carcinoma

Thyroid cancer is the fastest increasing cancer worldwide in all age groups. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer in both adults and children. PTC genomic landscape has been extensively studied in adults, but information regarding sporadic pediatric patients is lacking. Although BRAF V600E mutation is highly prevalent in adults, this mutation is uncommon in pediatric cases. As adult and pediatric PTC is a mitogen‐activated protein kinase‐driven cancer, this altered pathway might be activated by different genetic events. The aim of this study was to investigate the occurrence of AGK‐BRAF fusion gene, recently described in radiation‐exposed pediatric PTC, in a cohort of exclusively sporadic pediatric PTC. The series consisted of 30 pediatric PTC younger than 18 years of age at the time of diagnosis and 15 matched lymph node metastases (LNM). Primary tumors and matched LNM were screened for the presence of the AGK‐BRAF fusion transcript by RT‐PCR. To confirm the identity of the amplified products, randomly selected samples positive for the presence of the fusion transcripts were sequenced. Moreover, BRAF dual‐color, break‐apart probes confirmed BRAF rearrangement. Overall, the AGK‐BRAF fusion gene was detected in 10% (3/30) of primary tumors. For one of these cases, paired LNM was also available, which also shows the presence of AGK‐BRAF fusion gene. This study described, for the first time, the presence of AGK‐BRAF in sporadic pediatric PTC. Understanding the molecular events underlying pediatric PTC may improve preoperative diagnosis, allow molecular prognostication and define a therapeutic approach toward sporadic PTC patients.

APO A-V-1131T-->C polymorphism frequency and its association with morbidity in a Brazilian elderly population.

Abstract Identification of genetic polymorphisms as risk factors for complex diseases affecting older people can be relevant for their prevention, diagnosis and management. The –1131T→C polymorphism of the apolipoprotein A-V gene (APO A-V) is tightly linked to lipid metabolism and has been associated with increased triglyceride levels and familial dyslipidemia. The aims of this study were to analyze the allele and genotype frequencies of this polymorphism in a Brazilian elderly population and to investigate any association between the polymorphism and major morbidities affecting elderly people. This polymorphism was investigated in 371 individuals, aged 66–97years, in a Brazilian Elderly Longitudinal Population Study. Major morbidities investigated were: cerebrovascular diseases (CVD); myocardial infarction (MI); type 2 diabetes; hypertension; obesity; dementia; depression; and neoplasia. DNA was isolated and amplified by PCR and its products were digested with restriction enzyme Tru1I. T and C allele frequencies were 0.842 and 0.158, respectively. Our population showed allele frequencies that were similar to European and Afro-American and different from Asiatic populations. Genotype distributions were not within Hardy-Weinberg equilibrium only for the obesity subject sample. On the other hand, a significant association between the C allele and obesity in the presence of CVD×depression interaction was observed. Logistic analysis showed no association of the polymorphism with each morbidity group. Therefore, the C allele in elderly Brazilian subjects may represent a risk factor for these morbidity interactions, which may lead to better comprehension of their pathophysiology.

Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) as a novel strategy for identification of the skewed X inactivation pattern in balanced and unbalanced X-rearrangements

X-chromosome inactivation occurs randomly in normal female cells. However, the inactivation can be skewed in patients with alterations in X-chromosome. In balanced X-autosome translocations, normal X is preferentially inactivated, while in unbalanced X alterations, the aberrant X is usually inactivated. Here, we present a novel strategy to verify the skewed X inactivation pattern through the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into cells, in 11 patients: five carriers of balanced X-autosome translocations and six of unbalanced X-chromosome alterations. Since EdU is a labeled nucleoside analog of thymidine, its incorporation during DNA synthesis can reveal late replication regions and the inactive X-chromosome. All EdU findings were validated by the human androgen receptor gene (HUMARA) assay. The late replication regions were easily and quickly visualized in all cells, where inactive Xs are marked with strong green fluorescence. It was observed that the normal X-chromosome was preferentially inactivated in patients with balanced X-autosome translocations; while the aberrant X-chromosome was inactivated in most cells from patients with unbalanced alterations. By performing the fluorescence-based EdU assay, the differences between the active and inactive X-chromosomes are more easily recognizable than by classic cytogenetic methods. Furthermore, EdU incorporation allows the observation of the late replication regions in autosomal segments present in X derivatives from X-autosome translocations. Therefore, EdU assay permits an accurate and efficient cytogenetic evaluation of the X inactivation pattern with a low-cost, easy to perform and highly reproducible technique.

19q13.33→qter trisomy in a girl with intellectual impairment and seizures

Rearrangements in chromosome 19 are rare. Among the 35 patients with partial 19q trisomy described, only six have a breakpoint defined by array. The 19q duplication results in a variable phenotype, including dysmorphisms, intellectual disability and seizure. In a female patient, although G-banding at 550 band-resolution was normal, multiplex ligation-dependent probe amplification (MLPA) technique and genomic array showed a 10.6 Mb terminal duplication of chromosome 19q13. Fluorescent in situ hybridization (FISH) revealed that the duplicated region was attached to the short arm of chromosome 21 and silver staining showed four small acrocentrics with nucleolar organization region (NOR) activity, suggesting that the breakpoint in chromosome 21 was at p13. This is the first de novo translocation between 19q13.33 and 21p13 described in liveborn. The chromosome 19 is known to be rich in coding and non-coding regions, and chromosomal rearrangements involving this chromosome are very harmful. Furthermore, the 19q13.33→qter region is dense in pseudogenes and microRNAs, which are potent regulators of gene expression. The trisomic level of this region may contribute to deregulation of global gene expression, and consequently, may lead to abnormal development on the carriers of these rearrangements.

Hydrocephaly, penoscrotal transposition, and digital anomalies associated with de novo pseudodicentric rearranged chromosome 13 characterized by classical cytogenetic methods and mBAND analysis

We report on a patient with hydrocephaly, penoscrotal transposition, oligodactyly, and minor anomalies. Comprehensive cytogenetic studies involving both classical cytogenetic methods and mBAND analysis were used to show a stable dicentric rearranged chromosome 13 that result in a 46,XY,psu dic(13;13)(13pter → 13q32::13q11 → 13pter) de novo karyotype. This aberration probably arose as a consequence of unequal sister chromatid breakage repair events. This report is the first to describe all of the most severe features associated with partial monosomy in one patient. Moreover, the delineation of monosomy 13q32 → qter in this patient facilitates identification of the developmental genes responsible for the clinical features within this region of chromosome 13.

DDIT3, STT3A (ITM1), ARG2 and FAM129A (Niban, C1orf24) in diagnosing thyroid carcinoma: variables that may affect the performance of this antibody-based test and promise

DDIT3, STT3A (ITM1), ARG2 and FAM129A (Niban, C1orf24) in diagnosing thyroid carcinoma: variables that may affect the performance of this antibody-based test and promise