Manoela M. Ortega

Early mammalian erythropoiesis requires the Dot1L methyltransferase

Histone methylation is an important regulator of gene expression; its coordinated activity is critical in complex developmental processes such as hematopoiesis. Disruptor of telomere silencing 1-like (DOT1L) is a unique histone methyltransferase that specifically methylates histone H3 at lysine 79. We analyzed Dot1L-mutant mice to determine influence of this enzyme on embryonic hematopoiesis. Mutant mice developed more slowly than wild-type embryos and died between embryonic days 10.5 and 13.5, displaying a striking anemia, especially apparent in small vessels of the yolk sac. Further, a severe, selective defect in erythroid, but not myeloid, differentiation was observed. Erythroid progenitors failed to develop normally, showing retarded progression through the cell cycle, accumulation during G₀/G₁ stage, and marked increase in apoptosis in response to erythroid growth factors. GATA2, a factor essential for early erythropoiesis, was significantly reduced in Dot1L-deficient cells, whereas expression of PU.1, a transcription factor that inhibits erythropoiesis and promotes myelopoiesis, was increased. These data suggest a model whereby DOT1L-dependent lysine 79 of histone H3 methylation serves as a critical regulator of a differentiation switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.1 transcription, thus controlling numbers of circulating erythroid and myeloid cells.

Fractal Characteristics of May-Grünwald-Giemsa Stained Chromatin Are Independent Prognostic Factors for Survival in Multiple Myeloma

Background The use of computerized image analysis for the study of nuclear texture features has provided important prognostic information for several neoplasias. Recently fractal characteristics of the chromatin structure in routinely stained smears have shown to be independent prognostic factors in acute leukemia. In the present study we investigated the influence of the fractal dimension (FD) of chromatin on survival of patients with multiple myeloma. Methodology We analyzed 67 newly diagnosed patients from our Institution treated in the Brazilian Multiple Myeloma Study Group. Diagnostic work-up consisted of peripheral blood counts, bone marrow cytology, bone radiograms, serum biochemistry and cytogenetics. The International Staging System (ISS) was used. In every patient, at least 40 digital nuclear images from diagnostic May-Grünwald-Giemsa stained bone marrow smears were acquired and transformed into pseudo-3D images. FD was determined by the Minkowski-Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R2 values in the log-log plots. The influence of diagnostic features on overall survival was analyzed in Cox regressions. Patients that underwent autologous bone marrow transplantation were censored at the day of transplantation. Principal Findings Median age was 56 years. According to ISS, 14% of the patients were stage I, 39% were stage II and 47% were stage III. Additional features of a bad prognosis were observed in 46% of the cases. When stratifying for ISS, both FD and its goodness-of-fit were significant prognostic factors in univariate analyses. Patients with higher FD values or lower goodness-of-fit showed a worse outcome. In the multivariate Cox-regression, FD, R2, and ISS stage entered the final model, which showed to be stable in a bootstrap resampling study. Conclusions Fractal characteristics of the chromatin texture in routine cytological preparations revealed relevant prognostic information in patients with multiple myeloma.

Mutações do gene cystic fibrosis transmembrane conductance regulator e deleções dos genes glutationa S-transferase em pacientes com fibrose cística no Brasil

OBJECTIVE To determine the effects that mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and deletion of the glutathione S-transferase (GST) genes mu-1 (GSTM1) and theta-1 (GSTT1) have on the clinical course of cystic fibrosis (CF) in patients residing in the southeastern region of Brazil. METHODS The study sample consisted of all consecutive CF patients treated at the Hospital de Clínicas School of Medical Sciences of the State University at Campinas between March of 2002 and March of 2005. We included 66 CF patients. Genomic DNA was analyzed by polymerase chain reaction and restriction endonuclease digestion for the identification of the genotypes. RESULTS The DF508 mutation of the CFTR gene was found in 44 patients (66.7%). The null genotypes GSTM1, GSTT1 and GSTM1/GSTT1 were found in 40.9%, 15.2%, and 3.0% of the patients, respectively. The DF508 CFTR mutation was more common in patients diagnosed with CF before 2.5 years of age than in those diagnosed later (75.5% vs. 41.2%; p = 0.008). The frequency of the DF508 CFTR mutation, as well as of the GSTM1 and GSTT1 genotypes, was not found to be associated with gender, ethnicity, pulmonary disease status, or pancreatic disease status. CONCLUSIONS When the patients were stratified by clinical and epidemiological features, the frequencies of the GSTM1 and GSTT1 null genotypes were similar, suggesting that the inherited absence of these enzymatic pathways does not alter the course of CF. However, the high frequency of the DF508 CFTR mutation found in younger children suggests that it influences the age at diagnosis of CF in this region of Brazil.

Polymorphisms of glutathione S‐transferase mu1 (GSTM1) and theta 1 (GSTT1) genes in chronic myeloid leukaemia

To the Editor: The environmental exposure to cytotoxic and genotoxic agents, particularly those derived from benzene, may be associated with an increased risk of chronic myeloid leukaemia (CML) (1). The enzymes of the glutathione S-transferase (GST) system reduce molecules of chemical agents to less toxic levels. Gene codings for the GST mu1 (GSTM1) and theta 1 (GSTT1) proteins are polymorphic in humans and are homozygous null in approximately 50% and 20% of normal individuals, respectively, which results in a lack of the active proteins (2). An increase in homozygous GSTT1 null genotype was observed in CML patients from India (3). As GSTT1 is involved in the metabolism of ethylene oxide (EO), a genotoxic agent capable of producing heritable translocations and the frequency of spontaneous chromosome abnormalities, presumably because of EO, is higher in GSTT1 null individuals (4), it was hypothesised that the loss of the GSTT1 genes might influence the production of Philadelphia (Ph) chromosome, leading to CML. Cancer is the second most common cause of death in south-eastern Brazil and chemical agentsrelated diseases are a serious health problem in this area of the country (5, 6). For this reason, the identification of the GSTM1 and GSTT1 gene polymorphisms in CML patients, from an area in which there is a potential exposure to cytotoxic and genotoxic agents, was considered necessary in order to test whether homozygous null genotypes influenced the risk for disease. Genomic DNA obtained from bone marrow samples of 125 CML patients [73 male, 52 female; age (mean ± SD) 39.0 ± 16.4 yr; 108 White people, 17 African–Americans] and peripheral blood of 341 racially matched controls [198 male, 143 female; age (mean ± SD) 53.0 ± 4.3 yr; 296 White people, 45 African–Americans] was analysed using the multiplex-polymerase chain reaction (7, 8). The diagnosis of CML was based on haematological, cytogenetic and molecular analyses. The statistical significance of the differences between groups was calculated by chi-square or Fischer’s exact test. Crude odds ratio (OR) was given within 95% confidence intervals (CI) and was ageand gender-adjusted. We observed similar frequencies of GSTM1, GSTT1 and combined null genotypes in patients and controls (43.2% vs. 43.7%, P 1⁄4 1.00; 18.4% vs. 17.6%, P 1⁄4 0.95; and 7.2% vs. 7.9%, P 1⁄4 0.75 respectively). Individuals with GSTM1 (OR 1⁄4 0.98; 95% CI: 0.65–1.50), GSTT1 (OR 1⁄4 1.06; 95% CI: 0.62–1.80) and combined null genotypes (OR 1⁄4 0.84; 95% CI: 0.36–1.96) were at similar risk of CML compared with those without the null genotypes. We also examined the frequency of the polymorphisms in patients in relation to clinical variables (Table 1). Similar frequencies of the GSTM1, GSTT1 and combined null genotypes in patients under 50 yr or older, gender and ethnic origin were seen. The frequency of the GSTM1 null genotype was lower in patients in the accelerated phase or with blast crisis than those seen in patients with chronic phase (20.0% vs. 49.0%, P 1⁄4 0.01). Similar frequency of the GSTT1 and combined null genotypes was found in patients with distinct phases of the disease. In conclusion, our results indicate that the inherited absence of this carcinogen detoxification pathway could be unimportant for the Ph chromosome production and aetiology of CML. However, the role of the GSTM1 null genotype in different phases of this disease requires confirmation by large-scale molecular epidemiological studies.

GSTM1 and codon 72 P53 polymorphism in multiple myeloma

The GSTM1 and GSTT1 genes reduce the effects of exposure to cytotoxic agents. Both genes have a null variant allele in which the entire gene is absent. On the other hand, a common polymorphism of the tumour suppressor P53 gene results in either arginine (A) or proline (P) at amino-acid position 72. The A and P alleles code proteins with distinct functions in apoptosis and DNA repair and have been associated with variable risks for several cancers. However, their roles in multiple myeloma (MM) are still unknown. We tested in study whether the GSTM1, GSTT1 and P53 genotypes altered the risk for MM in Brazilian patients. Genomic DNA from 106 patients and 230 controls were analysed by polymerase chain reaction-based methods for identification of the genotypes. Similar frequencies of the GSTM1, GSTT1 and P53 genotypes were seen in patients and controls. Individuals with the distinct genotypes had similar risks for disease. In contrast, an excess of the GSTM1 null (45.1 vs 17.2%, P = 0.009), the P53 PP+AP (70.4 vs 44.8%, P = 0.041) and the GSTM1 null plus P53 PP+AP (29.6 vs 10.3%, P = 0.004) genotypes were seen in MM patients at stage III compared with those at stages I + II. Our data suggest that the GSTM1, GSTT1 and P53 genotypes do not influence the risk for MM. However, the inherited presence of the variant codon 72 P53 allele, described here for the first time, and the absence of the GSTM1 detoxification pathway, seem to act in disease progression in our country.

N-RAS and K-RAS gene mutations in Brazilian patients with multiple myeloma.

Point mutations affecting codons 12, 13 (exon 1) and 61 (exon 2) of the N-RAS gene and codons 12 and 13 (exon 1) of the K-RAS gene are identified in approximately 30.0% and 10.0%, respectively, of multiple myeloma (MM) patients living in the northern hemisphere. To date, there are no reports about the prevalence of RAS gene mutations in MM Brazilian patients, and this comprised the aim of the present study. DNA from bone marrow aspirates of 252 patients with MM (139 males and 113 females; aged 59.33 ± 11.95 years) were investigated for whole exons 1 and 2 of the N-RAS gene and whole exon 1 of the K-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Fifty-three out of 252 (21.03%) MM patients presented RAS mutations. Heterozygous mutations at codons 4, 10 (exon 1), 61 and 65 (exon 2) of the N-RAS gene were identified in seven out of 252 (2.78%) patients. K-RAS heterozygous mutations at codons 7, 12, 13 (exon 1) were seen in 46 out of 252 (18.25%) patients. To the best of our knowledge, the mutation at codon 7 of K-RAS gene is reported for the first time in MM. Taken together, these results suggest that Brazilian MM patients are characterized by: (i) a low prevalence of RAS mutation and (ii) RAS mutations located at distinct regions of the critical codons of the N-RAS and K-RAS genes.

ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells

ANKHD1 is a multiple ankyrin repeat containing protein, highly expressed in cancers, such as acute leukemia. The present study was undertaken to determine the expression and functional significance of ANKHD1 in human Multiple Myeloma (MM). We found that ANKHD1 is highly expressed in MM patient cells and cell lines. In vitro, lentiviral mediated ANKHD1‐shRNA inhibited proliferation and delayed S to G2M cell cycle progression in glucocorticoid resistant (U266) and sensitive (MM1S) MM cells. Further ANKHD1 silencing resulted in upregulation of cyclin dependent kinase inhibitor p21 irrespective of the p53 status of the MM cell lines. These data suggest that ANKHD1 might have a role in MM cell proliferation and cell cycle progression by regulating expression of p21.

The rare t(6;8) (q27;p11) translocation in a case of chronic myeloid neoplasm mimicking polycythemia vera

A distinct entity associated with 8p11 rearrangement, characterised by chronic myeloid hyperplasia and fast progression to high grade lymphoma (B or T) or acute myeloid leukaemia (AML), was described by MacDonald et al. [1] and Inhorn et al. [2]. The 8p11 region was the site of a recurrent breakpoint in disease, which was combined with nine different partners, 6q27, 7q34, 9q33, 12p11, 12q15, 13q12, 17q23, 19q13, 22q22 [1–15]. The FGFR1 gene, located on 8p11 region, was described as fused to FGFR1OP, TIF1, CEP1, FGFROP2, CPSF6, ZNF198, MYO18A, HERV-K or BCR genes in each distinct translocation [3–9,12– 15]. All resulting fusion proteins had constitutive tyrosine kinase activity and activated multiple signal transduction pathways [3,9]. The entity was recently recognised as 8p11 myeloproliferative syndrome (MPS) in the 2008 WHO classification system for chronic myeloid neoplasm (CMN) [16]. Among the specific rearrangements of the 8p11 MPS, the t(6;8) was a recurrent translocation in only eight cases [10–15], including three cases previously diagnosed as polycythemia vera (PV) based on clinical features, blood count and analysis of bone marrow aspirate [12,14,15]. We present, herein, a case of CMN with a t(6;8)(q27;p11), which was initially diagnosed as having PV in July 2003. The patient was a 12-yearold child. She was referred to our service because of polycythemia and leukocytosis. Plethora was the unique abnormality found at physical examination. Hematological data were as follows: hemoglobin, 18.7 g/dL; hematocrit, 58.6%; VCM, 70 fL; HCM, 22.3 pg; CHCM, 31.9 g/dL; reticulocyte, 1.4%; WBC, 14.5 6 10/L (metamyelocyte: 2.0%, band: 10.0%, segmented: 52.0%, eosinophil: 2.0%, basophil: 7.0%, lymphocyte: 23.0%; monocyte: 4.0%); and platelets, 240.0 6 10/L. Serum measurements were as follows: iron: 43.0 mg/dL (range: 49.0–151.0), total iron binding capacity: 443.0 mg/dL (range: 242.0–450.0), ferritin: 22.4 ng/mL (range: 10.0–200.0), vitamin B12: 1,521.0 pg/mL (range: 271.0–976.0), and creatinine: 0.7 mg/dL (range: 0.3–1.0). The Cr-EDTA-glomerular filtration rate was 114.0 mL/min/1.73 m (range: 100.0+20.0) and arterial oxygen saturation was 97.0%. Normal hemoglobin electrophoresis and negative tests for unstable hemoglobin were also obtained. Computed tomography scan revealed mild splenomegaly. Bone marrow (BM) smear and biopsy revealed hyperplasia of the erythroid, megakaryocytic and granulocytic lineages without fibrosis or eosinophilia; myeloid/erythroid ratio was 1.9 (range: 2.0– 4.0). No abnormalities of the BCR and ABL genes were seen by reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) technique performed on interphase nuclei, using the LSI BCR/ABL ES 32-191022 probe (Vysis, Downers Grove, IL). The diagnosis of PV was

Clinical features related to xeroderma pigmentosum in a Brazilian patient diagnosed at advanced age

Xeroderma pigmentosum is a rare autosomal recessive genetic disease characterized by extreme sensitivity due to solar radiation and deficiency in excision repair DNA. Those factors promote a set of skin abnormalities such as keratosis, hyperpigmentation, tumors in areas exposed to sunlight, and ocular and, eventually, neurological disorders. In the present review, we summarize the main clinical features related to a case of xeroderma pigmentosum in a man who was not diagnosed until he was 45 years old.

Polymorphisms of VEGF, GSTM1 and GSTT1 genes in multiple myeloma risk

To the Editor Angiogenesis (AG) has been implicated in both the origin and the progression of multiple myeloma (MM). The vascular endothelial growth factor (VEGF) has been described as an essential stimulator of AG [1,2]. The VEGF C936T polymorphism has been associated with a variable inherited ability to form blood vessels: the wild C allele produces higher VEGF levels than the variant T allele [3,4]. The wild-type 936CC genotype was identified in 70–80% of individuals of distinct ethnic populations, with an important role for the risk of solid tumours [4] but with an unknown influence for MM development. Multiple myeloma has also been seen as an environmental occupational disease [5]. The glutathione S-transferase (GST) enzymes serve to inactivate chemical agents [6]. The genes encoding these enzymes, GSTM1 and the GSTT1, are polymorphic and may be homozygous null in 10–60% of individuals from different populations, resulting in a lack of expression of the proteins [7]. The GSTM1 deletion was previously associated with MM progression [8,9] and MM risk [10], possibly due to a lack of carcinogen inactivation, but no influence of other GST genotypes for risk of the disease was seen [11]. The GSTs can also induce AG via regulation of the hypoxia-inducible factor-1a (HIF-1a), a transcription factor that regulates VEGF transcription in response to changes in the availability of oxygen [12,13]. The GSTM1 [14] and the GSTT1 [15] genes were linked to a high angiogenic phenotype in breast tumours compared with the respective null genotypes, but their roles on AG in MM are unknown. Cancer is the second-leading cause of death in southeastern Brazil, and MM induces death in elderly individuals [16]. Environment-related diseases have also been described as a serious health problem in this region [17,18]. Thus, we analysed herein the genotypes of the VEGF C936T, the GSTM1 and the GSTT1 genes in MM patients and controls from south-eastern Brazil, with the purpose of verifying whether they correlate with an increased risk of the disease. The case group comprised 150 MM patients at diagnosis (median age: 54years, range: 29–86; 69 women, 81 men; 119 White people, 31 African-Brazilians; 50 with tumours at stages I+II, 95 with tumours at stage III, five with unstaged tumour) seen at the University Hospital from December 2003 to August 2009. The control group was composed of 150 blood donors (median age: 55years, range: 25–60; 73 women, 77 men; 125 White people, 25 African-Brazilians) from the same time period and the same University Hospital in order to provide a representative group of the general population that seeks medical