S. Chandrakala

Down-regulation of miR-199b associated with imatinib drug resistance in 9q34.1 deleted BCR/ABL positive CML patients.

Chronic myeloid leukemia (CML) occurs due to t(9,22) (q34;q11) and molecularly BCR/ABL gene fusion. About 15-18% Philadelphia positive CML patients have gene deletions around the translocation breakpoints on 9q34.1. The microRNAs (miRNAs), namely miR-219-2 and miR-199b, centromeric to the ABL1 gene are frequently lost in CML patients. We have designed a study to determine miR-219-2 and miR-199b expression levels which would help to understand the prognosis of imatinib therapy. A total of 150 CML patients were analyzed to identify 9q deletion. Fluorescent in-situ hybridization (FISH) was performed using BCR/ABL dual color, dual fusion probe to study the signal pattern and BAC probes for miR-199b and miR-219-2 (RP11-339B21 and RP11-395P17) to study the miRNA deletions. The expression level of miRNA was analyzed by real-time polymerase chain reaction (RT-PCR). FISH analysis revealed 9q34.1 deletion in 34 (23%) CML patients. The deletions were not detected using BAC probes for miRNAs in 9q deleted patients. The expression analysis showed down-regulation of miR-199b and miR-219-2 in the 9q deleted patients (34 CML) as compared to a pool of patients without deletion. However, miR-199b (9q34.11) was significantly (p=0.001) down-regulated compared to miR-219-2. The follow-up study showed that the miR-199b was found to be strongly associated with imatinib resistance, as 44.11% patients showed resistance to imatinib therapy. Hence, the deletion in 9q34.1 region (ABL) plays an important role in disease pathogenesis. Eventually, miRNAs can provide new therapeutic strategies and can be used as a prognostic indicator.

Does HbF induction by hydroxycarbamide work through MIR210 in sickle cell anaemia patients

Sickle cell disease (SCD) is characterized by chronic haemolytic anaemia as a result of a single point mutation in the HBB (b-globin) gene. Hydroxycarbamide (HC, also termed hydroxyurea) is efficacious for sickle cell anaemia (SCA) patients. We previously reported significant improvement in the clinical condition of 98% of SCA patients after HC therapy (Italia et al, 2009). The efficacy of HC is due to its ability to increase fetal haemoglobin (HbF). However, HbF induction by HC is highly variable among patients, and the mechanism of HbF induction is not yet clear, although direct transcriptional effects and altered cell cycle kinetics have been proposed (Lou et al, 2009). Understanding the developmental progression of globin gene expression, reactivation of HBG1/HBG2 (c-globin genes) expression and HbF production in the adult is an important therapeutic strategy for SCA (Rogers et al, 2008). MicroRNAs (miRNAs) are potent molecular regulators that modulate the expression of protein-coding genes by inhibiting mRNA translation. They have been described to have a possible role in globin switching and can modulate transcriptional erythroid-specific regulators (Fornari et al, 2013). Thus, we hypothesized that miRNA regulation may be involved in HC-mediated HbF induction. Kosaka et al (2008) analysed 324 human miRNAs to investigate their role in erythroid maturation; interestingly MIR210 was one of the miRNAs that was significantly elevated when UT-7 cells were stimulated with erythropoietin and MIR210 expression was elevated during erythroid maturation in vitro. The authors suggested that MIR210 might play a regulatory role in erythroid maturation. Hence, we studied MIR210 expression patterns in SCA patients treated with HC to determine whether MIR210 is involved in the induction of HbF by HC. We collected peripheral blood samples from 10 SCA patients after obtaining informed consent, at baseline and then 3 months after HC therapy (10 mg/kg/d). The control group comprised of 10 normal individuals. HbF levels were measured by high performance liquid chromatography (VARIANT haemoglobin Testing System; Bio-rad Laboratory, Hercules, CA, USA). The mean HbF levels increased from 16 87 2 98% at baseline to 30 19 2 37% in SCA patients after 3 months of HC therapy (P < 0 01) (Fig 1A). CD71 (also termed TFRC) is the receptor for the ironbinding protein transferrin and is present on reticulocytes and erythroid progenitors. Hence, CD71 erythroid cells were enriched after CD45 leucocyte depletion using magneticactivated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany). This ‘double-MACS’ procedure yielded approximate recovery rates of 28–45% for each step. The average recovery of CD71 cells was 2 3 9 10 cells per ml from normal healthy controls, 5 32 9 10 and 3 527 9 10 cells per ml from SCA patients at baseline and after 3 months of HC therapy, respectively. The purity of CD71 populations was >87% and contained mainly erythroid progenitors, primarily reticulocytes and nucleated erythrocytes. We further extracted total RNA and miRNA (Ambion mirVana kit; Life Technologies, Carlsbad, CA, USA). MIR210 (HGNC: 31587) expression was determined by quantitative polymerase chain reaction using MIR451A (HGNC: 32053) as the reference gene for normalization (Walker et al, 2011) and Taqman probes (part 4427975; Applied Biosystems, Carlsbad, CA, USA). The baseline study identified a 4 01 1 41-fold increase in MIR210 expression in the untreated SCA patients compared to healthy normal controls. MIR210 expression was significantly up-regulated in the patients after HC therapy with a fold increase of 7 49 1 031 (P < 0 05) when compared to baseline, suggesting a role of MIR210 in HbF induction. Figure 1B shows the relative quantification of MIR210 in the SCA patients after HC therapy. Analysis of HbF levels and MIR210 expression in paired samples (baseline and 3 months after HC therapy) for each patient by Spearman’s correlation coefficient indicated a significant association between HbF and MIR210 expression in response to HC therapy (P < 0 0001) (Fig 2A). Thus, the increase in MIR210 expression was associated with HC-mediated induction of HbF. We also analysed HBG2 (HGNC: 4832; c-globin gene) expression in all 3 groups by TaqMan hybridization probe (HBG2; HB HS00361131_g1; Applied Biosystems, Carlsbad, CA, USA) using human 18S rRNA (HS99999901_s1; Applied

Can hydroxyurea serve as a free radical scavenger and reduce iron overload in β-thalassemia patients?

Abstract In this study, we hypothesize that hydroxyurea could provide an additional benefit as a free radical scavenger and/or iron chelator in β-thalassemia patients with iron overload. Twenty-one β-thalassemia intermedia patients who presented between 3 and 17 years but later required regular blood transfusions were enrolled for hydroxyurea therapy for a year. Fourteen patients responded to the therapy with hemoglobin levels maintained above 7.5 g/dl without transfusions. Hydroxyurea was discontinued after 6 months in seven patients who did not respond to the therapy and had to be continued on regular blood transfusions. We observed a statistically significant decrease in serum ferritin levels from 4194 ± 4850 ng/ml to 2129 ± 2380 ng/ml among the responders and from 2955 ± 2909 ng/ml to 2040 ± 2432 ng/ml among the non-responders and statistically significant decrease in labile iron pool from 18678.7 ± 10067.4 mean fluorescence intensity (MFI) to 14888.5 ± 5284.0 MFI among responders and from 17986.3 ± 9079.8 MFI to 15634.8 ± 8976.9 MFI among the non-responders after therapy. Phosphatidylserine externalization also showed a statistically significant decrease from 44.2 ± 22.2 MFI to 16.6 ± 6.7 MFI among the responders and from 46.9 ± 33.1 MFI to 39.8 ± 7.4 MFI among the non-responders along with a statistically significant decrease in the levels of reactive oxygen species from 72.8 ± 35.5 MFI to 29.0 ± 8.3 MFI among the responders and from 80.9 ± 41.4 MFI to 40.5 ± 15.8 MFI among the non-responders after therapy. A statistically significant increase in reduced glutathione levels was also observed from 430.8 ± 201.1 MFI to 715.5 ± 292.4 MFI among the responders and from 359.6 ± 165.6 MFI to 450.3 ± 279.5 MFI among the non-responders after therapy. This suggests the possible additional role of hydroxyurea as a free radical scavenger and/or iron chelator but requires a larger study for substantiation.

Mutations in Intron 1 and Intron 22 Inversion Negative Haemophilia A Patients from Western India

Despite increased awareness and diagnostic facilities, 70–80% of the haemophilia A (HA) patients still remain undiagnosed in India. Very little data is available on prevalent mutations in HA from this country. We report fifty mutations in seventy one Indian HA patients, of which twenty were novel. Ten novel missense mutations [p.Leu11Pro (p.Leu-8Pro), p.Tyr155Ser (p.Tyr136Ser), p.Ile405Thr (p.Ile386Thr), p.Gly582Val (p.Gly563Val) p.Thr696Ile (p.Thr677Ile), p.Tyr737Cys (p.Tyr718Cys), p.Pro1999Arg (p.Pro1980Arg), p.Ser2082Thr (p.Ser2063Thr), p.Leu2197Trp (p.Leu2178Trp), p.Asp2317Glu (p.Asp2298Glu)] two nonsense [p.Lys396* (p.Lys377*), p.Ser2205* (p.Ser2186*)], one insertion [p.Glu1268_Asp1269ins (p.Glu1249_Asp1250)] and seven deletions [p.Leu882del (p.Leu863del), p.Met701del (p.Met682del), p.Leu1223del (p.Leu1204del), p.Trp1961_Tyr1962del (p.Trp1942_Tyr1943del) p.Glu1988del (p.Glu1969del), p.His1841del (p.His1822del), p.Ser2205del (p.Ser2186del)] were identified. Double mutations (p.Asp2317Glu; p.Thr696Ile) were observed in a moderate HA case. Mutations [p. Arg612Cys (p.Arg593Cys), p.Arg2326Gln (p.Arg2307Gln)] known to be predisposing to inhibitors to factor VIII (FVIII) were identified in two patients. 4.6% of the cases were found to be cross reacting material positive (CRM+ve). A wide heterogeneity in the nature of mutations was seen in the present study which has been successfully used for carrier detection and antenatal diagnosis in 10 families affected with severe to moderate HA.

Paradoxical Bleeding and Thrombosis in a Patient With Afibrinogenemia and Fibrinogen Mumbai Mutation

OBJECTIVES Thrombosis is rarely reported in cases of afibrinogenemia and is generally associated with thrombophilia or replacement therapy. Often, it is difficult to predict whether the patients will bleed or whether they are exposed to the risk of thrombosis. METHODS We report a patient with afibrinogenemia who presented with complete thrombosis of right hepatic, portal, and splenic veins and who described a lifelong history of bleeding. Direct sequencing of the three fibrinogen genes was performed to identify the mutation. RESULTS DNA sequencing showed the presence of a homozygous for G8017A substitution in exon 8 of the fibrinogen β-chain gene, resulting in a G434D missense mutation (Fibrinogen Mumbai). CONCLUSIONS Presence of both bleeding and thrombotic manifestations in a patient with afibrinogenemia in the presence of other associated risk factors warrants a very careful individualized approach in the management of patients with afibrinogenemia.

Role of lupus anticoagulants in immediate acting inhibitor positivity in congenital haemophilia A patients

OBJECTIVES Presence of lupus anticoagulants (LA) in haemophilia and their interference in coagulation assays is well-known. Factor VIII (FVIII) inhibitors are generally time and temperature dependent whereas LAs are immediate acting inhibitors (IAIs). The present study reports the challenges in laboratory detection of both progressive and non-progressive, specific FVIII inhibitors in the presence of LA. METHODS From 2012 through 2015, 4900 HA patients were screened for inhibitors. APTT based inhibitor screening tests and Nijmegen-modified Bethesda assay (NBA) were done in all samples. LA test and FVIII inhibitors by ELISA were done in patients with IAIs. RESULTS Out of 451 patients positive for inhibitors in the initial screening tests, classical and progressive FVIII inhibitors were observed in 398 patients while 53 had IAIs showing no/partial correction in 1:1 mixtures of NPP and patient plasma. In 27 patients, both FVIII and FIX activity levels were <1%, resulting in difficulty in diagnosis. In 48 HA patients with IAIs, 42 were LA positive. 4 patients were found to have only LA with false positive results in NBA while 38 had a combination of LA and FVIII inhibitors. Six patients were LA negative and had only FVIII IAIs. Five (62.5%) of 8 HA patients initiated on immune tolerance induction (ITI) also were positive for IAIs. CONCLUSION The findings emphasizes the presence of specific FVIII inhibitors in congenital HA with absence of time dependent inactivation kinetics in a small proportion of cases. ELISA or chromogenic assays along with LA testing can offer accurate laboratory diagnosis in patients with coexisting LA.