NIBAN1, Exploring its Roles in Cell Survival Under Stress Context.

Cell survival must quickly activate specific mechanisms that enable to detect changes in the cellular microenvironment. The impact of these cell alteration has direct consequences on cellular homeostasis. Cellular stress, as well as its regulation and implication, has been studied in different pathologies. In this sense, the alteration in NIBAN1 expression seems to act in response to different cellular disturbances. Over the years, the knowledge of NIBAN1 functions has improved, demonstrating its important cell roles, favoring the cell survival under stress context. In response to the disturbances, NIBAN1 seems to be involved in the decision-making process between cell survival and death. The increase in NIBAN1 expression has been related to cellular mechanisms that seek to minimize the damage caused to cellular homeostasis. In this review, the main biological insights attributed to the NIBAN1 gene in different cellular contexts and its role as a mediator of cellular stress are discussed.

Intelligence Quotient Variability in Klinefelter Syndrome Is Associated With GTPBP6 Expression Under Regulation of X-Chromosome Inactivation Pattern.

Klinefelter syndrome (KS) displays a broad dysmorphological, endocrinological, and neuropsychological clinical spectrum. We hypothesized that the neurocognitive dysfunction present in KS relies on an imbalance in X-chromosome gene expression. Thus, the X-chromosome inactivation (XCI) pattern and neurocognitive X-linked gene expression were tested and correlated with intelligence quotient (IQ) scores. We evaluated 11 KS patients by (a) IQ assessment, (b) analyzing the XCI patterns using both HUMARA and ZDHHC15 gene assays, and (c) blood RT-qPCR to investigate seven X-linked genes related to neurocognitive development (GTPBP6, EIF2S3, ITM2A, HUWE1, KDM5C, GDI1, and VAMP7) and XIST in comparison with 14 (male and female) controls. Considering IQ 80 as the standard minimum reference, we verified that the variability in IQ scores in KS patients seemed to be associated with the XCI pattern. Seven individuals in the KS group presented a random X-inactivation (RXI) and lower average IQ than the four individuals who presented a skewed X-inactivation (SXI) pattern. The evaluation of gene expression showed higher GTPBP6 expression in KS patients with RXI than in controls (p = 0.0059). Interestingly, the expression of GTPBP6 in KS patients with SXI did not differ from that observed in controls. Therefore, our data suggest for the first time that GTPBP6 expression is negatively associated with full-scale IQ under the regulation of the type of XCI pattern. The SXI pattern may regulate GTPBP6 expression, thereby dampening the impairment in cognitive performance and playing a role in intelligence variability in individuals with KS, which warrants further mechanistic investigations.

The Natural History of a Man With Ovotesticular 46,XX DSD Caused by a Novel 3-Mb 15q26.2 Deletion Containing NR2F2 Gene.

Gonadal sex determination is a complex genetic process by which an embryonic primordium is driven to form an ovary or a testis, which requires a delicate dosage balance involving many genes. Disruption in this molecular pathway can lead to differences of sex development (DSD). Although some genetic mechanisms leading to 46,XY DSD have been elucidated, little is known about copy-number variation (CNV) causing testicular or ovotesticular 46,XX DSD. We describe a 20-year natural history of a man with SRY-negative 46,XX who was born with atypical male external genitalia, aortic coarctation, and bilateral blepharophimosis-ptosis. The molecular study identified a de novo heterozygous 3-Mb 15q26.2 deletion, a gene-poor locus containing NR2F2, which encodes the nuclear receptor COUP-TFII that is highly expressed in ovary and cardiac arteries. Immunohistochemistry confirmed the low COUP-TFII expression on his ovotestis tissue. Monosomy of 15q26.2, encompassing the NR2F2 gene, may act as a Z-factor regulating the male sex determination negatively. This finding supports a novel type of CNV resulting in DSD in an individual who developed male puberty spontaneously.

AGK-BRAF is associated with distant metastasis and younger age in pediatric papillary thyroid carcinoma.

BACKGROUND: The incidence of thyroid carcinoma has increased in most populations, including pediatric patients. The increase is almost exclusively due to an increase in the incidence of papillary thyroid carcinoma (PTC). Genetic alterations leading to mitogen-activated protein kinase (MAPK) pathway activation are highly prevalent in PTC, with BRAF V600E mutation being the most common event in adult PTC. Although a lower prevalence of BRAF V600E had been reported among pediatric patients, a higher prevalence of BRAF fusion has been identified in both radiation-exposed and sporadic pediatric PTC. However, little is known about the prognostic implications of BRAF fusions in pediatric PTC. PROCEDURE: In this study, we investigated the prevalence of BRAF alterations (AGK-BRAF fusion and BRAF V600E mutation) in a large set of predominantly sporadic pediatric PTC cases and correlate with clinicopathological features. Somatic AGK-BRAF fusion was investigated by RT-PCR and confirmed by FISH break-apart. The BRAF V600E mutation was screened using Sanger sequencing. RESULTS: AGK-BRAF fusion, found in 19% of pediatric PTC patients, was associated with distant metastasis and younger age. Conversely, the BRAF V600E, found in 15% of pediatric PTC patients, was correlated with older age and larger tumor size. CONCLUSION: Collectively, our results advance knowledge concerning genetic bases of pediatric thyroid carcinoma, with potential implications for diagnosis, prognosis, and therapeutic approaches.

Downregulation of genes outside the deleted region in individuals with 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome (22q11.2DS) is caused by recurrent hemizygous deletions of chromosome 22q11.2. The phenotype of the syndrome is complex and varies widely among individuals. Little is known about the role of the different genes located in 22q11.2, and we hypothesized that genetic risk factors lying elsewhere in the genome might contribute to the phenotype. Here, we present the whole-genome gene expression data of 11 patients with approximately 3 Mb deletions. Apart from the hemizygous genes mapped to the 22q11.2 region, the TUBA8 and GNAZ genes, neighboring the deleted interval but in normal copy number, showed altered expression. When genes mapped to other chromosomes were considered in the gene expression analysis, a genome-wide dysregulation was observed, with increased or decreased expression levels. The enriched pathways of these genes were related to immune response, a deficiency that is frequently observed in 22q11.2DS patients. We also used the hypothesis-free weighted gene co-expression network analysis (WGCNA), which revealed the co-expression gene network modules with clear connection to mechanisms associated with 22q11.2DS such as immune response and schizophrenia. These findings, combined with the traditional gene expression profile, can be used for the identification of potential pathways and genes not previously considered to be related to the 22q11.2 deletion syndrome.

FAM129A regulates autophagy in thyroid carcinomas in an oncogene-dependent manner.

We previously proposed that high expression of FAM129A can be used as a thyroid carcinoma biomarker in preoperative diagnostic exams of thyroid nodules. Here, we identify that FAM129A expression is increased under nutrient and growth factor depletion in a normal thyroid cell line (PCCL3), overlapping with increased expression of autophagy-related protein and inhibition of AKT/mTOR/p70S6K. Supplementation of insulin, TSH and serum to the medium was able to reduce the expression of both FAM129A and autophagy-related protein and reestablish the AKT/mTOR/p70S6K axis. To determine the direct role of FAM129A on autophagy, FAM129A was transfected into PCCL3 cells. Its overexpression induced autophagic vesicles formation, evidenced by transmission electron microscopy. Co-expression of FAM129A and mCherry-EGFP-LC3B in PCCL3 showed an increased yellow puncta formation, suggesting that FAM129Ainduces autophagy. To further confirm its role on autophagy, we knockdown FAM129A in two thyroid carcinoma cell lines (TPC1 and FTC-236). Unexpectedly, FAM129A silencing increased autophagic flux, suggesting that FAM129A inhibits autophagy in these models. We next co-transfected PCCL3 cells with FAM129A and RET/PTC1 and tested autophagy in this context. Co-expression of FAM129A and RET/PTC1 oncogene in PCCL3 cells, inhibited RET/PTC1-induced autophagy. Together, our data suggest that, in normal cells FAM129A induces autophagy in order to maintain cell homeostasis and provide substrates under starvation conditions. Instead, in cancer cells, decreased autophagy may help the cells to overcome cell death. FAM129A regulates autophagy in a cell- and/or context-dependent manner. Our data reinforce the concept that autophagy can be used as a strategy for cancer treatment.

Inactivation of AMMECR1 is associated with growth, bone, and heart alterations.

We report five individuals with loss-of-function of the X-linked AMMECR1: a girl with a balanced X-autosome translocation and inactivation of the normal X-chromosome; two boys with maternally inherited and de novo nonsense variants; and two half-brothers with maternally inherited microdeletion variants. They present with short stature, cardiac and skeletal abnormalities, and hearing loss. Variants of unknown significance in AMMECR1 in four male patients from two families with partially overlapping phenotypes were previously reported. AMMECR1 is coexpressed with genes implicated in cell cycle regulation, five of which were previously associated with growth and bone alterations. Our knockdown of the zebrafish orthologous gene resulted in phenotypes reminiscent of patients' features. The increased transcript and encoded protein levels of AMMECR1L, an AMMECR1 paralog, in the t(X;9) patient's cells indicate a possible partial compensatory mechanism. AMMECR1 and AMMECR1L proteins dimerize and localize to the nucleus as suggested by their nucleic acid-binding RAGNYA folds. Our results suggest that AMMECR1 is potentially involved in cell cycle control and linked to a new syndrome with growth, bone, heart, and kidney alterations with or without elliptocytosis.

Novel lincRNA Susceptibility Gene and Its Role in Etiopathogenesis of Thyrotoxic Periodic Paralysis.

Thyrotoxic periodic paralysis (TPP) is a life-threatening neuromuscular complication of thyrotoxicosis characterized by muscle weakness and hypokalemia and with an unclear etiopathogenesis. However, the 17q24.3 locus had been genetically linked to TPP, in which the genetic variant rs312691 (TC genotype) in long intergenic noncoding RNA (lincRNA) CTD-2378E21.1 is located downstream of inward-rectifier potassium (Kir) channel genes [KCNJ2 and its antisense KCNJ2 (AS-KCNJ2)]. A TPP patient with a suppressed thyroid-stimulating hormone level, a high free thyroxine level of (5.8 ng/dL), and low serum potassium level of (2 mEq/L) was evaluated for Kir channel expression during and after recovery from thyrotoxicosis. We observed that circulating lincRNA and Kir expression varied in accordance with thyroid status and TC genotype. To endorse this association of a lincRNA-rs312691 variant with a genetic risk of TPP, an additional series of 37 patients with TPP and 32 patients with thyrotoxic without paralysis (TWP) were assessed. We verified that the risk of minor allele C was greater in TPP than in TWP (odds ratio, 5.289; P = 0.0062), and protective major allele T was more frequent than observed in the 1000 genome controls (odds ratio, 11.90; P < 0.0001). AS-KCNJ2 was downregulated during thyrotoxicosis in the TWP controls carrying allele T and were upregulated in those with TPP with risk allele C. Moreover, KCNJ2 (Kir2.1) expression was reduced during thyrotoxicosis and restored in euthyroid status. We further excluded any other coding variant by performing targeted exome sequencing mutational screening in 17q24.3. Our data suggest that high lincRNA AS-KCNJ2 and CDT-2378E21.1 expression, possibly driven by the triiodothyronine regulatory mechanism, reduces the Kir2.1 expression observed during thyrotoxicosis. This finding could contribute to the understanding of the reduced inward-rectifying current observed during muscle weakness in genetically susceptible TPP patients.

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.

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.

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. © 2015 Wiley Periodicals, Inc.

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.

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.

Deletion of 17p13 and LIS1 gene mutation in isolated lissencephaly sequence.

Classical lissencephaly is a neuroblast migration disorder that occurs either as isolated lissencephaly sequence or in association with malformation syndromes, such as the Miller-Dieker syndrome. In this work, alterations of the LIS1 gene in patients diagnosed as having isolated lissencephaly sequence were investigated. Ten patients were evaluated for the following aspects: classical cytogenetics by karyotyping using solid staining and G-banding; molecular cytogenetics using fluorescent in situ hybridization with a specific probe for the critical region of isolated lissencephaly sequence; and molecular analysis using deoxyribonucleic acid sequencing. Classical cytogenetic analysis indicated apparently normal karyotypes in all patients, but fluorescent in situ hybridization revealed a 17p13.3 microdeletion in one. In another patient, deoxyribonucleic acid sequencing disclosed a 1 base pair insertion in exon 4 within a sequence of eight consecutive adenine residues (162-163insA), a mutation that predicts a truncated protein. Two different polymorphisms were also detected: a T>C substitution in intron 6 (c.568 + 27bp T>C) and a C>T substitution in the nontranslated region of exon 11 (1250 C>T). These results indicate that cytogenetic analysis and molecular investigation of the LIS1 gene are not always sufficient to determine the disease etiology. These findings are consistent with previous studies and suggest the involvement of other genes in cortical malformation.

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.

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

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 66-97 years, 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 CVDxdepression 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.

Genética do autismo / Genetics of autism

O autismo é uma doença neuropsiquiátrica com profundas conseqüências sociofamilares. Inúmeros trabalhos investigaram pacientes e famílias com metodologia genético-clínica, citogenética e biologia molecular. Os resultados destes trabalhos apontam para um modelo multiloci com interação epistática associado à etiologia do autismo.