Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

BACKGROUND: The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. OBJECTIVE: The aim of this study was to detect the following mutations: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. METHODS: Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. RESULTS: Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L-positive patient with primary myelofibrosis had more severe anemia than other patients with primary myelofibrosis. CONCLUSIONS: This study demonstrates that karyotyping for JAK2 and MPL mutations is useful in the diagnosis of myeloproliferative neoplasms. The precise pathogenetic contribution of these alterations is still unclear. However, this study adds more information about the pathophysiology of polycythemia vera, essential thrombocythemia and primary myelofibrosis.Universidade Federal de São Paulo (UNIFESP) Hematology DepartmentUniversidade Federal de São Paulo (UNIFESP) Rheumatology DepartmentUniversidade Federal de São Paulo (UNIFESP) Genetics DepartmentUNIFESP, Hematology DepartmentUNIFESP, Rheumatology DepartmentUNIFESP, Genetics DepartmentSciEL

Epigenetic regulation of metalloproteinases and their inhibitors in rotator cuff tears

Rotator cuff tear is a common orthopedic condition. Metalloproteinases (MMP) and their inhibitors (TIMP) seem to play a role in the development of joint injuries and in the failure of tissue healing. However, the mechanisms of regulation of gene expression in tendons are still unknown. Epigenetic mechanisms, such as DNA methylation and microRNAs regulation, are involved in the dynamic control of gene expression. Here, the mRNA expression and DNA methylation status of MMPs (MMP1, MMP2, MMP3, MMP9, MMP13, and MMP14) and TIMPs (TIMP1-3) and the expression of miR-29 family members in ruptured supraspinatus tendons were compared with non-injured tendons of individuals without this lesion. Additionally, the gene expression and methylation status at the edge of the ruptured tendon were compared with macroscopically non-injured rotator cuff tendon samples from the anterior and posterior regions of patients with tendon tears. Moreover, the possible associations between the molecular alterations and the clinical and histologic characteristics were investigated. Dysregulated expression and DNA methylation of MMP and TIMP genes were found across the rotator cuff tendon samples of patients with supraspinatus tears. These alterations were influenced at least in part by age at surgery, sex, smoking habit, tear size, and duration of symptoms. Alterations in the studied MMP and TIMP genes may contribute to the presence of microcysts, fissures, necrosis, and neovascularization in tendons and may thus be involved in the tendon healing process. In conclusion, MMPs and their inhibitors are regulated by epigenetic modifications and may play a role in rotator cuff tears.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Sao Paulo, Dept Ortopedia & Traumatol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Disciplina Genet, Dept Morfol & Genet, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Patol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Ortopedia & Traumatol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Disciplina Genet, Dept Morfol & Genet, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Patol, Sao Paulo, SP, BrazilFAPESP: 2011/22548-2FAPESP: 2012/14768-5FAPESP: 2016/01392-8FAPESP: 2016/15785-1FAPESP: 2013/10691-0FAPESP: 2013/10791-5Web of Scienc

Cytogenetic study and search for mutations in JAK2 and MPL genes in Polycythemia vera, Primary myelofibrosis and Essential thrombocythemia

Introcuction: Polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are clonal disorders of hematopoietic stem cell and clinical and biological aspects have in common that hinder their diagnosis, however, cytogenetic and molecular studies represent important tools to assist in this procedure. Therefore, the investigation of the presence of JAK2 V617F, mutations in exon 12 of JAK2, MPL W515K of MPLW515L and cytogenetic alterations per karyotype and FISH can provide a more detailed view for the diagnosis and prognosis of these diseases. In this study such cytogenetic and molecular changes were correlated with degree of fibrosis in cases of MF, the number of leukocytes, platelets, hemoglobin and age at diagnosis in PV, ET and MF Method: The karyotype by G-band was performed on samples of bone marrow, grown in culture for short duration (24h) without mitogens and processed as usual (Chauffaille, 2006). The sample (1 ml) was intended to FISH with probes for the regions: 20q12, 20q13.12, 13q14.3, 13qter, 8p11.1-q11.1 and 9q12. The investigation of JAK2 V617F and MPL W515K/L mutations was performed on DNA from peripheral blood by real time PCR, using the kit MutaScreenTM JAK2 (IPSOGEN). The search for mutations in exon 12 of JAK2 was performed by direct sequencing. Results: Chromosomal abnormalities were observed in 11.8% of PV, 17.6% of MF and none of the ET, no relation between clinical data and assessed chromosomal alterations. Chromosomal abnormalities were not amplified by FISH. JAK2 V617F was observed in 90% of PV, 42.8% of MF and 47% of ET. Patients with JAK2 V617F negative PV showed lower levels of platelets in relation to V617F positive PV (p <0.0001). MF V617F negative and MPL W515L positive showed higher degrees of fibrosis than V617F negative (p =0.003). Was not detected the presence of mutations in exon 12 of JAK2 in PV patients. MPL W515L was observed in a case of MF and a TE. No MPL W515K mutation was found in patients with ET and MF. The ET patient MPL W515L positive showed no clinical different from other patients with ET, whereas the patient with MF MPL W515L showed positive clinical more aggressive when compared to other patients with MF. The number of clonal abnormalities showed no difference in the clinical data evaluated. Conclusions: Different types of clonal abnormalities in myeloproliferative neoplasms exalt their different pathophysiological mechanism, aiding in the diagnosis and understanding of the biology of these diseases. This study allowed the cytogenetics and molecular characterization of PV, MF and ET.Objetivos: Descrever as alteracoes cromossomicas em policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primaria (MF). Verificar se a taxa de anormalidades cromossomicas e ampliada pela FISH nos casos com cariotipo normal e ausencia de metafases. Detectar a incidencia da mutacao JAK2 V617F em PV, TE e MF; de mutacoes no exon 12 do JAK2 em PV e de mutacoes MPL W515K/L em TE e MF. Correlacionar as alteracoes citogeneticas e moleculares encontradas com o grau de fibrose medular nos casos de MF; numero de leucocitos, plaquetas, hemoglobinas; e idade, ao diagnostico, em todos pacientes. Metodo: O estudo foi realizado em 20 casos de PV, 17 de TE e 21 de MF. O cariotipo por banda G foi realizado em amostras de medula ossea, semeadas em cultura de curta duracao (24h), sem mitogenos e processadas de forma habitual (Chauffaille, 2006). Parte da amostra (1mL) foi destinada a FISH, com sondas para as regioes: 20q12, 20q13.12, 13q14.3, 13qter (subtelomerica), 8p11.1-q11.1 (ƒ¿-satellite) e 9q12 (satellite III). A pesquisa das mutacoes JAK2V617F e MPL W515K/L foi realizada em DNA de sangue periferico, por PCR em tempo real, utilizando-se o kit JAK2 MutaScreenTM (Ipsogen). A pesquisa de mutacoes no exon 12 do JAK2 foi realizada por sequenciamento direto. Resultados: As alteracoes cromossomicas foram observadas em 11,8% das PV, 17,6% das MF e nenhuma das TE, nao havendo relacao entre dados clinicos avaliados e alteracoes cromossomicas. As anormalidades cromossomicas nao foram ampliadas pela FISH. JAK2 V617F foi observada em 90% das PV, 42,8% das MF e 47% das TE. Os pacientes com PV JAK2 V617F negativos apresentaram menores niveis de plaquetas em relacao aos PV V617F positivos (p<0,0001). MF V617F positivos apresentaram maiores graus de fibrose do que os V617F negativos (p=0,003). Nao foi detectada a presenca de mutacoes no exon 12 do JAK2 em pacientes com PV. MPL W515L foi observada em um caso de MF e em um de TE. Nao foi encontrada a mutacao MPL W515K nos pacientes com TE e MF. A paciente com TE MPL W515L positivo nao apresentou quadro clinico diferente dos demais pacientes com TE, enquanto que a paciente com MF MPL W515L positivo apresentou quadro clinico mais agressivo quando comparada aos demais pacientes com MF. O numero de alteracoes clonais nao mostrou diferenca quanto aos dados clinicos avaliados. Conclusoes: Os diferentes tipos de alteracoes clonais em neoplasias mieloproliferativas exaltam seus diferentes mecanismos fisiopatogenicos, auxiliando no diagnostico e compreensao da biologia destas doencas. Este estudo permitiu a caracterizacao citogenetica e molecular de PV, MF e TE.TEDEBV UNIFESP: Teses e dissertaçõe

Rare single‐nucleotide variants in oculo‐auriculo‐vertebral spectrum (OAVS)

Abstract Background Oculo‐auriculo‐vertebral spectrum (OAVS) is a craniofacial developmental disorder that affects structures derived from the first and second pharyngeal arches. The clinically heterogeneous phenotype involves mandibular, oral, and ear development anomalies. Etiology is complex and poorly understood. Genetic factors have been associated, evidenced by chromosomal abnormalities affecting different genomic regions and genes. However, known pathogenic single‐nucleotide variants (SNVs) have only been identified in MYT1 in a restricted number of patients. Therefore, investigations of SNVs on candidate genes may reveal other pathogenic mechanisms. Methods In a cohort of 73 patients, coding and untranslated regions (UTR) of 10 candidate genes (CRKL, YPEL1, MAPK1, NKX3‐2, HMX1, MYT1, OTX2, GSC, PUF60, HOXA2) were sequenced. Rare SNVs were selected and in silico predictions were performed to ascertain pathogenicity. Likely pathogenic variants were validated by Sanger sequencing and heritability was assessed when possible. Results Four likely pathogenic variants in heterozygous state were identified in different patients. Two SNVs were located in the 5’UTR of YPEL1; one in the 3’UTR of CRKL and one in the 3’UTR of OTX2. Conclusion Our work described variants in candidate genes for OAVS and supported the genetic heterogeneity of the spectrum

The Complex Network between MYC Oncogene and microRNAs in Gastric Cancer: An Overview

Despite the advancements in cancer treatments, gastric cancer is still one of the leading causes of death worldwide. In this context, it is of great interest to discover new and more effective ways of treating this disease. Accumulated evidences have demonstrated the amplification of 8q24.21 region in gastric tumors. Furthermore, this is the region where the widely known MYC oncogene and different microRNAs are located. MYC deregulation is key in tumorigenesis in various types of tissues, once it is associated with cell proliferation, survival, and drug resistance. microRNAs are a class of noncoding RNAs that negatively regulate the protein translation, and which deregulation is related with gastric cancer development. However, little is understood about the interactions between microRNAs and MYC. Here, we overview the MYC role and its relationship with the microRNAs network in gastric cancer aiming to identify potential targets useful to be used in clinic, not only as biomarkers, but also as molecules for development of promising therapies

Distribution of the clinical and histological variables of rotator cuff tear patients and controls.

<p>Distribution of the clinical and histological variables of rotator cuff tear patients and controls.</p

Clinical variations and DNA methylation in rotator cuff tears.

<p>A) Increased DNA methylation frequency at <i>MMP1</i> promoter in females; B) Reduced DNA methylation frequency at CpG site +49 of <i>TIMP2</i> in females; C) Increased DNA methylation frequency at CpG site -400 of <i>MMP1</i> in smokers; D) Reduced DNA methylation frequency at CpG site -19 of <i>TIMP2</i> in smokers; E) Increased DNA methylation frequency in CpG sites of <i>MMP13</i> in samples of patients with tears greater than 2 cm. *Significant difference between groups by Mann-Whitney test (p<0.05).</p

Correlation between DNA methylation and age at surgery in rotator cuff tears.

<p>A) CpG site -536 of <i>MMP1</i>; B) CpG site -454 of <i>MMP1</i>; C) CpG site -400 of <i>MMP1</i>; D) CpG site -234 of <i>TIMP3</i>; E) CpG site +27 of <i>TIMP3</i>; F) CpG site +117 of <i>TIMP3</i>.</p