Greice A. Molfetta

Role of NFKB2 on the early myeloid differentiation of CD34+ hematopoietic stem/progenitor cells.

To better understand the early events regulating lineage-specific hematopoietic differentiation, we analyzed the transcriptional profiles of CD34+ human hematopoietic stem and progenitor cells (HSPCs) subjected to differentiation stimulus. CD34+ cells were cultured for 12 and 40h in liquid cultures with supplemented media favoring myeloid or erythroid commitment. Serial analysis of gene expression (SAGE) was employed to generate four independent libraries. By analyzing the differentially expressed regulated transcripts between the un-stimulated and the stimulated CD34+ cells, we observed a set of genes that was initially up-regulated at 12h but were then down-regulated at 40h, exclusively after myeloid stimulus. Among those we found transcripts for NFKB2, RELB, IL1B, LTB, LTBR, TNFRSF4, TGFB1, and IKBKA. Also, the inhibitor NFKBIA (IKBA) was more expressed at 12h. All those transcripts code for signaling proteins of the nuclear factor kappa B pathway. NFKB2 is a subunit of the NF-κB transcription factor that with RELB mediates the non-canonical NF-κB pathway. Interference RNA (RNAi) against NFKB1, NFKB2 and control RNAi were transfected into bone marrow CD34+HSPC. The percentage and the size of the myeloid colonies derived from the CD34+ cells decreased after inhibition of NFKB2. Altogether, our results indicate that NFKB2 gene has a role in the early commitment of CD34+HSPC towards the myeloid lineage.

Mitochondrial genome instability in colorectal adenoma and adenocarcinoma.

Mitochondrial dysfunction is regarded as a hallmark of cancer progression. In the current study, we evaluated mitochondrial genome instability and copy number in colorectal cancer using Next Generation Sequencing approach and qPCR, respectively. The results revealed higher levels of heteroplasmy and depletion of the relative mtDNA copy number in colorectal adenocarcinoma. Adenocarcinoma samples also presented an increased number of mutations in nuclear genes encoding proteins which functions are related with mitochondria fusion, fission and localization. Moreover, we found a set of mitochondrial and nuclear genes, which cooperate in the same mitochondrial function simultaneously mutated in adenocarcinoma. In summary, these results support an important role for mitochondrial function and genomic instability in colorectal tumorigenesis.

Placenta-Enriched LincRNAs MIR503HG and LINC00629 Decrease Migration and Invasion Potential of JEG-3 Cell Line.

LINC00629 and MIR503HG are long intergenic non-coding RNAs (lincRNAs) mapped on chromosome X (Xq26), a region enriched for genes associated with human reproduction. Genes highly expressed in normal reproductive tissues and cancers (CT genes) are well known as potential tumor biomarkers. This study aimed to characterize the structure, expression, function and regulation mechanism of MIR503HG and LINC00629 lincRNAs. According to our data, MIR503HG expression was almost exclusive to placenta and LINC00629 was highly expressed in placenta and other reproductive tissues. Further analysis, using a cancer cell lines panel, showed that MIR503HG and LINC00629 were expressed in 50% and 100% of the cancer cell lines, respectively. MIR503HG was expressed predominantly in the nucleus of JEG-3 choriocarcinoma cells. We observed a positively correlated expression between MIR503HG and LINC00629, and between the lincRNAs and neighboring miRNAs. Also, both LINC00629 and MIR503GH could be negatively regulated by DNA methylation in an indirect way. Additionally, we identified new transcripts for MIR503HG and LINC00629 that are relatively conserved when compared to other primates. Furthermore, we found that overexpression of MIR503HG2 and the three-exon LINC00629 new isoforms decreased invasion and migration potential of JEG-3 tumor cell line. In conclusion, our results suggest that lincRNAs MIR503HG and LINC00629 impaired migration and invasion capacities in a choriocarcinoma in vitro model, indicating a potential role in human reproduction and tumorigenesis. Moreover, the MIR503HG expression pattern found here could indicate a putative new tumor biomarker.

Biochemical and genetic analysis of butyrylcholinesterase (BChE) in a family, due to prolonged neuromuscular blockade after the use of succinylcholine

Butyrylcholinesterase (BChE) is a plasma enzyme that catalyzes the hydrolysis of choline esters, including the muscle-relaxant succinylcholine and mivacurium. Patients who present sustained neuromuscular blockade after using succinylcholine usually carry BChE variants with reduced enzyme activity or an acquired BChE deficiency. We report here the molecular basis of the BCHE gene underlying the slow catabolism of succinylcholine in a patient who underwent endoscopic nasal surgery. We measured the enzyme activity of BChE and extracted genomic DNA in order to study the promoter region and all exons of the BCHE gene of the patient, her parents and siblings. PCR products were sequenced and compared with reference sequences from GenBank. We detected that the patient and one of her brothers have two homozygous mutations: nt1615 GCA > ACA (Ala539Thr), responsible for the K variant, and nt209 GAT > GGT (Asp70Gly), which produces the atypical variant A. Her parents and two of her brothers were found to be heterozygous for the AK allele, and another brother is homozygous for the normal allele. Sequence analysis of exon 1 including 5′UTR showed that the proband and her brother are homozygous for –116GG. The AK/AK genotype is considered the most frequent in hereditary hypocholinesterasemia (44%). This work demonstrates the importance of defining the phenotype and genotype of the BCHE gene in patients who are subjected to neuromuscular block by succinylcholine, because of the risk of prolonged neuromuscular paralysis.

A further case of a Prader-Willi syndrome phenotype in a patient with Angelman syndrome molecular defect

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct human neurogenetic disorders; however, a clinical overlap between AS and PWS has been identified. We report on a further case of a patient showing the PWS phenotype with the AS molecular defect. Despite the PWS phenotype, the DNA methylation analysis of SNRPN revealed an AS pattern. Cytogenetic and FISH analysis showed normal chromosomes 15 and microsatellite analysis showed heterozygous loci inside and outside the 15q11-13 region. The presence of these atypical cases could be more frequent than previously expected and we reinforce that the DNA methylation analysis is important for the correct diagnosis of severe mental deficiency, congenital hypotonia and obesity.

Clinical-neurologic, cytogenetic and molecular aspects of the Prader-Willi and Angelman syndromes

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are human neurogenetic disorders involving the imprinting mechanism, at the 15q11-13 chromosome region. The predominant genetic defects in PW are 15q11-13 deletions of paternal origin and maternal chromosome 15 uniparental disomy. In contrast, maternal deletions and paternal chromosome 15 uniparental disomy are associated with a different neurogenetic disorder, the AS. In both disorders, these mutations are associated with parent-of-origin specific methylation at several 15q11-13 loci. We studied 5 patients suspect of PWS and 4 patients suspect of AS who were referred to the Medical Genetics Unit at the University Hospital of Medical School from Ribeirão Preto. Our objective was to establish the correct clinical and etiological diagnosis in these cases. We used conventional cytogenetics, methylation analysis with the probe KB17 (CpG island of the SNRPN gene) by Southern blotting after digestion with the Xba I and Not I restriction enzymes. We studied in patients and their parents the segregation of the (CA)*** repeats polymorphisms by PCR, using the primers 196 and IR4-3R. All the patients had normal conventional cytogenetical analysis. We confirmed 3 cases of PWS: one by de novo deletion, one by maternal chromosome 15 uniparental disomy and one case with no defined cause determined by the used primers. We confirmed 2 cases of AS, caused by de novo deletion at the 15q11-13 region, and one case with normal molecular analysis but with strong clinical characteristics.

Microarray profiles of ex vivo expanded hematopoietic stem cells show induction of genes involved in noncanonical Wnt signaling.

The low number of hematopoietic stem cells (HSC) in umbilical cord blood (UCB) is directly related to increased risk of transplant failure. Effective ex vivo expansion of HSC has been tried for many years, with conflicting results because of the inability to reproduce in vitro HSC proliferation in the same way it occurs in vivo. We compared freshly isolated HSC with their expanded counterparts by microarray analysis and detected activation of the noncanonical Wnt (wingless-type MMTV integration site family) pathway. Study of early alterations during ex vivo UCB-HSC expansion could contribute to improvement of ex vivo expansion systems.

1031-1034delTAAC (Leu125Stop): a novel familial UBE3A mutation causing Angelman syndrome in two siblings showing distinct phenotypes

BackgroundMore than 50 mutations in the UBE3A gene (E6-AP ubiquitin protein ligase gene) have been found in Angelman syndrome patients with no deletion, no uniparental disomy, and no imprinting defect.Case PresentationWe here describe a novel UBE3A frameshift mutation in two siblings who have inherited it from their asymptomatic mother. Despite carrying the same UBE3A mutation, the proband shows a more severe phenotype whereas his sister shows a milder phenotype presenting the typical AS features.ConclusionsWe hypothesized that the mutation Leu125Stop causes both severe and milder phenotypes. Potential mechanisms include: i) maybe the proband has an additional problem (genetic or environmental) besides the UBE3A mutation; ii) since the two siblings have different fathers, the UBE3A mutation is interacting with a different genetic variant in the proband that, by itself, does not cause problems but in combination with the UBE3A mutation causes the severe phenotype; iii) this UBE3A mutation alone can cause either typical AS or the severe clinical picture seen in the proband.

ProbFAST: Probabilistic functional analysis system tool.

BackgroundThe post-genomic era has brought new challenges regarding the understanding of the organization and function of the human genome. Many of these challenges are centered on the meaning of differential gene regulation under distinct biological conditions and can be performed by analyzing the Multiple Differential Expression (MDE) of genes associated with normal and abnormal biological processes. Currently MDE analyses are limited to usual methods of differential expression initially designed for paired analysis.ResultsWe proposed a web platform named ProbFAST for MDE analysis which uses Bayesian inference to identify key genes that are intuitively prioritized by means of probabilities. A simulated study revealed that our method gives a better performance when compared to other approaches and when applied to public expression data, we demonstrated its flexibility to obtain relevant genes biologically associated with normal and abnormal biological processes.ConclusionsProbFAST is a free accessible web-based application that enables MDE analysis on a global scale. It offers an efficient methodological approach for MDE analysis of a set of genes that are turned on and off related to functional information during the evolution of a tumor or tissue differentiation. ProbFAST server can be accessed at http://gdm.fmrp.usp.br/probfast.

A non-functional galanin receptor-2 in a multiple sclerosis patient

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.