Milena Radmilovic

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.

Functional analysis of the role of the TPMT gene promoter VNTR polymorphism in TPMT gene transcription

AIMS Thiopurine S-methyltransferase (TPMT) activity is polymorphic, and a trimodal distribution has been demonstrated in Caucasians (low, intermediate and high methylator groups). The TPMT gene promoter contains a variable number of three GC-rich tandem repeats, namely A, B and C, ranging from three to nine in length in a A(n)B(m)C architecture. MATERIALS & METHODS Here, we investigated the influence of number and type of TPMT gene promoter tandem repeats on human TPMT gene transcription in K562 cells transiently transfected with reporter constructs bearing various variable number of tandem repeats (VNTR) and addressed the interaction of transcription factor binding to the VNTRs by electrophoretic mobility shift assays. RESULTS We found that the distribution patterns of VNTR alleles do not significantly differ among acute lymphoblastic leukemia patients, acute myeloid leukemia patients and normal individuals. We also demonstrated that the A repeat has a negative effect in TPMT gene transcription and that a positive regulatory element, identified immediately upstream to the VNTR region of the TPMT gene promoter, is indispensable for TPMT gene transcription. Our electrophoretic mobility shift assay analysis indicated that the Sp1 and Sp3 transcription factors bind to the VNTR repeats. CONCLUSION Overall, our data underline that both the number and type of VNTRs, as well as the upstream regulatory region of the TPMT gene promoter, determine the overall level of TPMT gene transcription. It remains to be seen whether these VNTRs can be employed as pharmacogenetic markers to individualize thiopurine therapy.

Genomic variation in the MAP3K5 gene is associated with β-thalassemia disease severity and hydroxyurea treatment efficacy

AIM In this study we explored the association between genetic variations in MAP3K5 and PDE7B genes, residing on chromosome 6q23, and disease severity in β-hemoglobinopathy patients, as well as the association between these variants with response to hydroxyurea (HU) treatment. Furthermore, we examined MAP3K5 expression in the context of high fetal hemoglobin (HbF) and upon HU treatment in erythroid progenitor cells from healthy and KLF1 haploinsufficient individuals. MATERIALS & METHODS For this purpose, we genotyped β-thalassemia intermedia and major patients and healthy controls, as well as a cohort of compound heterozygous sickle cell disease/β-thalassemia patients receiving HU as HbF augmentation treatment. Furthermore, we examined MAP3K5 expression in the context of high HbF and upon HU treatment in erythroid progenitor cells from healthy and KLF1 haploinsufficient individuals. RESULTS A short tandem repeat in the MAP3K5 promoter and two intronic MAP3K5 gene variants, as well as a PDE7B variant, are associated with low HbF levels and a severe disease phenotype. Moreover, MAP3K5 mRNA expression levels are altered in the context of high HbF and are affected by the presence of HU. Lastly, the abovementioned MAP3K5 variants are associated with HU treatment efficacy. CONCLUSION Our data suggest that these MAP3K5 variants are indicative of β-thalassemia disease severity and response to HU treatment.

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner

AIM TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. CONCLUSION These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.

The influence of novel transcriptional regulatory element in intron 14 on the expression of Janus kinase 2 gene in myeloproliferative neoplasms

The expression of Janus kinase 2 (JAK2) gene is altered in myeloproliferative neoplasms (MPN) and the regulation of transcription could be a mechanism that modulates JAK2 gene expression. We analyzed the transcriptional potential of single-nucleotide polymorphism (SNP) rs12343867 T > C in JAK2 intron 14, tagging 46/1 haplotype, and its influence on JAK2 gene expression. Functional analysis of JAK2 intron 14 was performed using the pBLCAT5 reporter system in K562 cells. Identification of the proteins binding to the intron 14 regulatory element was accomplished by electrophoretic mobility shift assay (EMSA) and supershift assays. Quantification of the expression of JAK2 gene in a cohort of 51 MPN patients and 12 healthy controls was performed by real-time quantitative polymerase chain reaction (RQ-PCR). Functional analysis revealed that the intronic DNA element harboring SNP rs12343867 T > C acts as a transcriptional repressor in vitro. The repressor activity was significantly attenuated by the presence of nucleotide C. Supershift analysis showed the enrolment of transcriptional factor Meis1 in this process. RQ-PCR experiments showed increased JAK2 expression in patients with the JAK2V617F mutation, with a significant difference between essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) patients. SNP rs12343867 showed no statistically significant influence on the expression of JAK2 gene in MNP patients.

Functional analysis of a novel KLF1 gene promoter variation associated with hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) is a rare hereditary condition resulting in elevated levels of fetal hemoglobin (HbF) in adults. Typical HPFH is associated with promoter mutations or large deletions affecting the human fetal globin (HBG1 and HBG2) genes, while genetic defects in other genes involved in human erythropoiesis, e.g. KLF1, also result in atypical HPFH. Here, we report the first KLF1 gene promoter mutation (KLF1:g.-148G > A) that is associated with increased HbF level. This mutation was shown to result in drastically reduced CAT reporter gene expression in K562 cells, compared to the wild-type sequence (p = 0.009) and also in reduced KLF1 gene expression in vivo. Furthermore, consistent with in silico analysis, electrophoretic mobility shift analysis showed that the KLF1:g.-148G > A mutation resides in a Sp1 binding site and further that this mutation leads to the ablation of Sp1 binding in vitro. These data suggest that the KLF1:g-148G > A mutation could play a role in increasing HbF levels in adults and further underlines the role of KLF1 as one of the key transcription factors involved in human fetal globin gene switching.

Thalassemia Syndromes in Serbia: An update

Thalassemia syndromes constitute a group of genetic disorders, widespread throughout the world. The present study contains data on thalassemia syndromes and their chromosomal environment obtained in Serbia over a period of 10 years. Ten different β-thalassemia (β-thal) mutations and two hemoglobin (Hb) variants were detected in 127 members of 68 families. Hb Lepore-Boston-Washington (Lepore-BW) (δ87Gln-β-IVS-II-8), a thalassemic Hb variant, was shown to be the most common cause of thalassemia in Serbia. Haplotype analyses of the β-globin gene clusters of healthy individuals as well as of individuals affected with β-thal showed that haplotype I was the most frequent haplotype in the Serbian population, followed by haplotypes II and IX. Two novel haplotypes were detected. Haplotype analyses showed the association between certain haplotypes and the most common thalassemic mutations. Results presented in this paper will update the Serbian national mutation database and contribute to a better understanding of genographic history of South European and Balkan populations.

Prognostic Impact of NPM1 Mutations in Serbian Adult Patients with Acute Myeloid Leukemia

Based on current findings, the presence of NPM1 mutations in acute myeloid leukemia (AML) patients is associated with an increased probability of complete remission (CR) and better overall survival (OS). We determined the incidence and prognostic relevance of NPM1 mutations, their association with FLT3 and IDH mutations, and other clinical characteristics in Serbian adult AML patients. Samples from 111 adult de novo AML patients, including 73 AML cases with a normal karyotype (NK-AML), were studied. NPM1, FLT3, and IDH mutations were detected by PCR and direct sequencing. NPM1 mutations were detected in 22.5% of patients. The presence of NPM1 mutations predicted a low CR rate and shorter OS. NPM1 mutations showed an association with both FLT3 and IDH mutations. Survival analysis based on NPM1/FLT3 mutational status revealed a lower OS for NPM1+/FLT3– compared to the NPM1–/FLT3– group in NK-AML patients. The lack of impact or unfavorable prognostic effect of NPM1 mutations found in this study can be assigned to a small cohort of analyzed AML patients, as can the presence of FLT3 and IDH mutations or other genetic lesions that cooperate with NPM1 mutations influencing prognosis.

Acknowledgements to Referees

Gregory A. Abel, Boston, USA Athanasios Aessopos, Athens, Greece Xabier Agirre, Navarra, Spain Graciela S. Alarcon, Birmingham, USA J.P. Allain, Cambridge, UK S.D. Anker, Berlin, Germany Jane F. Apperley, London, UK A. Arai, Tokyo, Japan Aderson Araújo, Recife, Brazil Luca Arcaini, Pavia, Italy Paolo Arese, Torino, Italy Maurizio Aricò, Florence, Italy Scott A. Armstrong, Boston, USA Roopen Arya, London, UK Giuseppe Avvisati, Rome, Italy Yesim Aydinok, Izmir, Turkey Ulrike Bacher, Hamburg, Germany Andrea Bacigalupo, Genoa, Italy Catherine Bagot, Glasgow, UK Carmen Baldazzi, Bologna, Italy Bernadett Balla, Budapest, Hungary David Barnett, Sheffield, UK Giovanni Barosi, Pavia, Italy Sharon L. Barrans, Leeds, UK Eva Bartova, Brno, Czech Republic Christian Bastard, Rouen, France Heiko Becker, Freiburg, Germany John M. Bennett, Rochester, USA M. Bennett, Afula, Israel J.A. Bernstein, Cincinnati, USA Erik Berntorp, Malmo, Sweden Alain Berrebi, Rehovot, Israel Caroline Besson, Le Kremlin-Bicêtre, France Wolfgang A. Bethge, Tübingen, Germany Deepa Bhojwani, Memphis, USA Janet J. Bijl, Montréal, Canada Douglas P. Blackall, Little Rock, USA Olga Blau, Berlin, Germany Dominique Bonnefont-Rousselot, Paris, France X. Bosch, Barcelona, Spain Reda Bouabdallah, Marseille, France Vassiliki A. Boussiotis, Boston, USA From September 1, 2011 to August 31, 2012 a number of additional referees assisted the editors with reviews of the submitted papers. The editors would like to thank the following colleagues: