👤 Maria J Pereira

Higher tumor size correlates with poor prognosis and is an independent predictive survival factor in oral squamous cell carcinoma (OSCC) patients. However, the molecular events underlining OSCC tumor evolution are poorly understood. We aimed to investigate if large OSCC tumors show different cell cycle gene transcriptional signature compared to small tumors. Seventeen fresh OSCC tumor samples with different tumor sizes (T) were included in the study. Tumors were from the tongue or from the floor of the mouth, and only three patients were nonsmokers. Samples were categorized according to clinical tumor size in tumors ≤2 cm (T1, n = 5) or tumors >2 cm (T2, n = 9; T3, n = 2; T4, n = 1). The group of tumors ≤2 cm was considered the reference group, while the larger tumors were considered the test group. We assessed the expression of 84 cell cycle genes by qRT-PCR array and normalized it to the expression of two housekeeping genes. Results were analyzed according to the formula 2(⁻DeltaCt). A five-fold change cutoff was used, and p values <0.05 were considered statistically significant. Ki-67 immunohistochemistry was performed to estimate cell proliferation index. Twenty-nine genes were downregulated in the test group (larger tumors) compared to the reference group (smaller tumors). Among these genes, 13 reached statistical significance: ANAPC4, CUL1, SUMO1, KPNA2, MAD2L2, CCNG2, E2F4, NBN, CUL2, PCNA, TFDP1, KNTC1, and ATR. Ki-67 labeling index was similar in both tumor groups. Our findings suggest that the transcriptional activity of specific cell cycle genes varies according to the size of OSCC tumor, which probably reflects tumor molecular evolution and adaptation to the microenvironment. Show less

Atonal homolog1 (Atoh1) encodes a basic helix-loop-helix protein that is the first transcription factor to be expressed in differentiating hair cells. Previous work suggests that expression of Atoh1 in prosensory precursors is necessary for the differentiation and survival of hair cells, but it is not clear whether Atoh1 is required exclusively for these processes, or whether it regulates other functions later during hair cell maturation. We used EGFP-tagged Atoh1 knock-in mice to demonstrate for the first time that Atoh1 protein is expressed in hair cell precursors several days before the appearance of differentiated markers, but not in the broad pattern expected of a proneural gene. We conditionally deleted Atoh1 at different points in hair cell development and observe a rapid onset of hair cell defects, suggesting that the Atoh1 protein is unstable in differentiating hair cells and is necessary through an extended phase of their differentiation. Conditional deletion of Atoh1 reveals multiple functions in hair cell survival, maturation of stereociliary bundles, and auditory function. We show the presence of distinct critical periods for Atoh1 in each of these functions, suggesting that Atoh1 may be directly regulating many aspects of hair cell function. Finally, we show that the supporting cell death that accompanies loss of Atoh1 in hair cells is likely caused by the abortive trans-differentiation of supporting cells into hair cells. Together our data suggest that Atoh1 regulates multiple aspects of hair cell development and function. Show less

Hypertrophic cardiomyopathy (HC) is the most prevalent genetic cardiac disease caused by a mutation in sarcomeres, Z-disks, or calcium-handling genes and is characterized by unexplained left ventricular hypertrophy. The aim of this study was to determine the genetic profile of Brazilian patients with HC and correlate the genotype with the phenotype. We included 268 index patients from São Paulo city and 3 other cities in Brazil and extracted their DNA from whole blood. We amplified the coding sequencing of MYH7, MYBPC3, and TNNT2 genes and sequenced them with an automatic sequencer. We identified causal mutations in 131 patients (48.8%). Seventy-eight (59.5%) were in the MYH7 gene, 50 (38.2%) in the MYBPC3 gene, and 3 (2.3%) in the TNNT2 gene. We identified 69 mutations, 24 not previously described. Patients with an identified mutation were younger at diagnosis and at current age, had a higher mean heart rate and higher nonsustained ventricular tachycardia frequency compared with those without a mutation. Patients with MYH7 gene mutations had a larger left atrium and higher frequency of atrial fibrillation than did patients with MYBPC3 gene mutations. The presence of a mutation in one of the genes suggests a worse prognosis. Mutations in the MYH7 gene, rather than in the MYBPC3 gene, were also related to a worse prognosis. This is the first work characterizing HC molecular epidemiology in the Brazilian population for the 3 most important genes. Show less

The enzyme 17β-hydroxysteroid dehydrogenase type 3 (17-β-HSD3) catalyzes the conversion of androstenedione to testosterone in the testes, and its deficiency is a rare disorder of sex development in 46,XY individuals. It can lead to a wide range of phenotypic features, with variable hormonal profiles. We report four patients with the 46,XY karyotype and 17-β-HSD3 deficiency, showing different degrees of genital ambiguity, increased androstenedione and decreased testosterone levels, and testosterone to androstenedione ratio < 0.8. In three of the patients, diagnosis was only determined due to the presence of signs of virilization at puberty. All patients had been raised as females, and female gender identity was maintained in all of them. Compound heterozygosis for c.277+2T>G novel mutation, and c.277+4A>T mutation, both located within the intron 3 splice donor site of the HSD17B3 gene, were identified in case 3. In addition, homozygosis for the missense p.Ala203Val, p.Gly289Ser, p.Arg80Gln mutations were found upon HSD17B3 gene sequencing in cases 1, 2, and 4, respectively. Show less

Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant. Show less

Sarcomeric hypertrophic cardiomyopathy has heterogeneous phenotypic expressions, of which sudden cardiac death is the most feared. A genetic diagnosis is essential to identify subjects at risk in each family. The spectrum of disease-causing mutations in the Portuguese population is unknown. Seventy-seven unrelated probands with hypertrophic cardiomyopathy were systematically screened for mutations by PCR and sequencing of five sarcomeric genes: MYBPC3, MYH7, TNNT2, TNNI3 and MYL2. Familial cosegregation analysis was performed in most patients. Thirty-four different mutations were identified in 41 (53%) index patients, 71% with familial hypertrophic cardiomyopathy. The most frequently involved gene was MYBPC3 (66%) with 22 different mutations (8 novel) in 27 patients, followed by MYH7 (22%), TNNT2 (12%) and TNNI3 (2.6%). In three patients (7%), two mutations were found in MYBPC3 and/or MYH7. Additionally, 276 relatives were screened, leading to the identification of a mean of three other affected relatives for each pedigree with the familial form of the disease. Disease-associated mutations were identified mostly in familial hypertrophic cardiomyopathy, corroborating the idea that rarely studied genes may be implicated in sporadic forms. Private mutations are the rule, MYBPC3 being the most commonly involved gene. Mutations in MYBPC3 and MYH7 accounted for most cases of sarcomere-related disease. Multiple mutations in these genes may occur, which highlights the importance of screening both. The detection of novel mutations strongly suggests that all coding regions should be systematically screened. Genotyping in hypertrophic cardiomyopathy enables a more precise diagnosis of the disease, with implications for risk stratification and genetic counseling. Show less

Markers at the pericentriolar material 1 gene (PCM1) have shown genetic association with schizophrenia in both a University College London (UCL) and a USA-based case-control sample. In this paper we report a statistically significant replication of the PCM1 association in a large Scottish case-control sample from Aberdeen. Resequencing of the genomic DNA from research volunteers who had inherited haplotypes associated with schizophrenia showed a threonine to isoleucine missense mutation in exon 24 which was likely to change the structure and function of PCM1 (rs370429). This mutation was found only as a heterozygote in 98 schizophrenic research subjects and controls out of 2246 case and control research subjects. Among the 98 carriers of rs370429, 67 were affected with schizophrenia. The same alleles and haplotypes were associated with schizophrenia in both the London and Aberdeen samples. Another potential aetiological base pair change in PCM1 was rs445422, which altered a splice site signal. A further mutation, rs208747, was shown by electrophoretic mobility shift assays to create or destroy a promoter transcription factor site. Five further non-synonymous changes in exons were also found. Genotyping of the new variants discovered in the UCL case-control sample strengthened the evidence for allelic and haplotypic association (P=0.02-0.0002). Given the number and identity of the haplotypes associated with schizophrenia, further aetiological base pair changes must exist within and around the PCM1 gene. PCM1 protein has been shown to interact directly with the disrupted-in-schizophrenia 1 (DISC1) protein, Bardet-Biedl syndrome 4, and Huntingtin-associated protein 1, and is important in neuronal cell growth. In a separate study we found that clozapine but not haloperidol downregulated PCM1 expression in the mouse brain. We hypothesize that mutant PCM1 may be responsible for causing a subtype of schizophrenia through abnormal cell division and abnormal regeneration in dividing cells in the central nervous system. This is supported by our previous finding of orbitofrontal volumetric deficits in PCM1-associated schizophrenia patients as opposed to temporal pole deficits in non-PCM1-associated schizophrenia patients. Caution needs to be exercised in interpreting the actual biological effects of the mutations we have found without further cell biology. However, the DNA changes we have found deserve widespread genotyping in multiple case-control populations. Show less

Recent studies have reported large common regions of homozygosity (ROHs) that are the result of autozygosity, that is, the cooccurrence within individuals of long haplotypes that have a high frequency in the population. A recent study reports that such regions are found more commonly in individuals with schizophrenia compared with controls, and identified nine 'risk ROHs' that were individually more common in cases. Of these, four contained or neighboured genes associated with schizophrenia (NOS1AP/UHMK1, ATF2, NSF and PIK3C3). We have applied the same methodology to a UK sample of 506 cases with bipolar disorder and 510 controls. There was no overall excess of common ROHs among bipolar individuals. With one exception, the haplotypes accounting for the ROHs appeared to be distributed according to the Hardy-Weinberg equilibrium. One ROH was individually more common among cases (uncorrected P = 0.0003). This ROH spanned the chromosome 2p23.3 gene ITSN2 (the gene for intersectin 2 isoform 2). However, inspection of the homozygous haplotypes and haplotype-based tests for association failed to provide a clearer understanding of why this ROH was occurring more commonly. Overall, we conclude that, in contrast with schizophrenia, common ROHs are rarely associated with susceptibility to bipolar disorder. This supports the idea that predominantly different genes are increasing susceptibility to schizophrenia and bipolar affective disorders. Show less

In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. Here, we show that the latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process. Show less

Little is known about the degree to which behavioural, biological, and genetic traits contribute to within-person variation in serum cholesterol. Materials and Methods The authors studied within-person variation in serum total and high density lipoprotein (HDL) cholesterol in 458 participants of 27 dietary intervention studies in Wageningen, The Netherlands, from 1976 to 1995. For a median of 4 days between blood draws, the geometric mean of the within-person standard deviation was 0.13 mmol/l ( approximately 5 mg/dl, coefficient of variation = 3.0%) for total cholesterol and 0.04 mmol/l ( approximately 1.5 mg/dl, coefficient of variation = 3.0%) for HDL cholesterol. In mixed-model linear regressions using within-person variance as the dependent variable and including lipid concentration and covariates listed below, within-person variance of both total cholesterol and HDL cholesterol was higher for greater number of days between blood draws and for self-selected diet rather than investigator-controlled diet. Within-person variance of total cholesterol only was higher for non-standardized versus standardized phlebotomy protocol and for female sex. The authors found evidence that the APOA4 -347 (12/22 genotype) and MTP -493 (11 genotype) polymorphisms may increase the within-person variation in total cholesterol. Under certain study design (self-selected diet, use of non-standardized phlebotomy protocol) or participant characteristics (female, certain polymorphisms) within-person lipid variance is increased and required sample size will be greater. These findings may have important implications for the time and cost of such interventions. Show less