Sukesh C. Nair

Identification of factor VIII gene mutations in 101 patients with haemophilia A: mutation analysis by inversion screening and multiplex PCR and CSGE and molecular modelling of 10 novel missense substitutions

Summary.  Haemophilia A (HA) is an X‐linked bleeding disorder caused by diverse mutations in the human coagulation factor VIII (FVIII) gene. We have analysed DNA from 109 unrelated Indian patients with HA for their FVIII gene defects. Among these patients 89 (82%) had severe (FVIII:C <1%) HA, 11 (10%) had moderate (FVIII:C 1–5%) HA and nine (8%) had mild (FVIII:C 5–30%) HA. These patients were first screened for the common intron 22 and intron 1 inversions. Inversion negative samples were screened for point mutations by a multiplex PCR and conformation sensitive gel electrophoresis strategy. Mutations were identified in 101 of the 109 patients. These included two (2%) intron 1 and 51 (51%) intron 22 inversions, four (4%) gross deletions and 44 (43%) point mutations. Twenty‐nine novel causative mutations, including 11 missense, seven frameshift, five nonsense mutations, three splice site defects and three gross deletions were detected. Ten of the novel missense mutations were studied by molecular modelling. Two different (Thr2253Pro and Pro1392fs) mutations were seen in four unrelated families and FVIII gene haplotyping suggested a common founder effect. Seven of these 109 patients had inhibitors. Among them, four had intron 22 inversions, one had a novel gross deletion (delexon 2–9) and one a nonsense mutation (Trp1535Stop). In one of these patients, no mutation could be identified in the FVIII gene. A Thr2253Pro novel mutation and an intron 22 inversion were identified in two female haemophiliacs. The data from this study suggests that the spectrum of gene defects in Indian patients with HA is as heterogeneous as reported in other populations.

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

Comparison of Newly Diagnosed and Relapsed Patients with Acute Promyelocytic Leukemia Treated with Arsenic Trioxide: Insight into Mechanisms of Resistance

There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens.

Management of Hemophilia in Patients with Inhibitors: The Perspective from Developing Countries

Data are limited on inhibitors in people with hemophilia (PWH) in developing countries. There is a perception that the overall prevalence of inhibitors, ranging from 7 to 19% in different reports, may be lower in these countries as compared with that reported from developed countries. This is possible given the fact that most patients are treated after 2 years of age with plasma-derived clotting factor concentrates. Whether genetic or other environmental factors also contribute to this needs further evaluation. There is a need to develop laboratory infrastructure and establish quality control programs for laboratory tests for inhibitors in developing countries. Management options vary widely given the socioeconomic diversity among these countries. Significant individualization of approach to management is therefore required depending on the available resources, particularly with regard to the use of bypassing agents. The limited data on immune tolerance induction with some low-dose regimens deserve further evaluation. Even in resource-constrained environments, education and a policy of systematic screening of patients associated with judicious use of bypassing agents can significantly improve the care of PWH who develop inhibitors.

Fracture neck of femur in haemophilia A – experience from a cohort of 11 patients from a tertiary centre in India

Summary. We describe here the management of eleven patients with fracture neck of femur. Excepting one patient all had severe haemophilia A. Nine patients were less than 50 years of age. Eight out of eleven patients had fracture after trivial trauma. Nine patients had closed reduction and one patient open reduction. The patient with non union had a Valgus osteotomy. All fractures united. The average time to union was 11 weeks (range:8–16). We followed either a low dose intermittent or a low dose continuous infusion factor support protocol for the management of these patients. The median dose of factor support was 252 u/kg (range: 136–580). The average duration of factor support was 9 days (range: 7–10). Two patients had aggravation of pre existing knee stiffness following post operative immobilisation. No other major complication was observed in this cohort of patients. To conclude, management of fracture neck of femur in patients with haemophilia is no different from general population if an adequate haemostasis is achieved.

Clinical manifestations of combined factor V and VIII deficiency: A series of 37 cases from a single center in India†

We describe here the clinical manifestations of 37 patients with combined coagulation factor (F) V and FVIII deficiency, which is the commonest multiple coagulation factor deficiency state. Only a few cases are reported in the literature from India. Prolonged bleed post injury/ surgery (62%) is the commonest manifestation in our series, and epistaxis (19%) is rare in our patients in comparison to other series described in the literature. Although the frequency of bleeding manifestations differs among various reports, there is no evidence for increased bleeding manifestation due to combined deficiency of two coagulation factors as against a single coagulation factor. To the best of our knowledge, this is the largest series of this disorder described so far.

Mean reticulocyte volume enhances the utility of red cell mean sphered cell volume in differentiating peripheral blood spherocytes of hereditary spherocytosis from other causes

CONTEXT Mean sphered cell volume (MSCV) and mean reticulocyte volume (MRV) are additional reticulocyte parameters generated while processing the blood samples on Beckman coulter LH 755 in the reticulocyte mode using the volume, conductivity and scatter technology. It has been observed that the difference between mean corpuscular volume (MCV) and MSCV is higher in the cases of hereditary spherocytosis (HS) and this difference is increasingly being utilized as a screening tool for spherocytes. In addition now there have been new observations that reticulocyte volume in cases of HS is less as compared to normal reticulocyte. AIMS Our aim was to test the usefulness of reticulocyte parameters like MSCV and MRV in distinguishing cases of HS and autoimmune hemolytic anemia (AIHA). MATERIALS AND METHODS This is a retrospective and partly prospective study where peripheral blood ethylenediaminetetraacetic acid samples from cases of HS (n = 57) and AIHA (n = 29) were processed on LH 755 in both the differential and the reticulocyte mode. The data generated were analyzed and compared with data from normal healthy donors (n = 46). RESULTS Using an algorithm of MCV - MSCV >10 and MRV - MSCV <25, a sensitivity of 84.2% and specificity of 94.7% was observed in cases of HS. CONCLUSIONS With the reticulocyte analysis, we may now have a simple and cheap additional tool for screening of HS.

Lack of grading agreement among international hemostasis external quality assessment programs

&NA; Laboratory quality programs rely on internal quality control and external quality assessment (EQA). EQA programs provide unknown specimens for the laboratory to test. The laboratorys result is compared with other (peer) laboratories performing the same test. EQA programs assign target values using a variety of methods statistical tools and performance assessment of ‘pass’ or ‘fail’ is made. EQA provider members of the international organization, external quality assurance in thrombosis and hemostasis, took part in a study to compare outcome of performance analysis using the same data set of laboratory results. Eleven EQA organizations using eight different analytical approaches participated. Data for a normal and prolonged activated partial thromboplastin time (aPTT) and a normal and reduced factor VIII (FVIII) from 218 laboratories were sent to the EQA providers who analyzed the data set using their method of evaluation for aPTT and FVIII, determining the performance for each laboratory record in the data set. Providers also summarized their statistical approach to assignment of target values and laboratory performance. Each laboratory record in the data set was graded pass/fail by all EQA providers for each of the four analytes. There was a lack of agreement of pass/fail grading among EQA programs. Discordance in the grading was 17.9 and 11% of normal and prolonged aPTT results, respectively, and 20.2 and 17.4% of normal and reduced FVIII results, respectively. All EQA programs in this study employed statistical methods compliant with the International Standardization Organization (ISO), ISO 13528, yet the evaluation of laboratory results for all four analytes showed remarkable grading discordance.

Molecular basis of Wiskott–Aldrich syndrome in patients from India

To the Editor: Wiskott–Aldrich syndrome (WAS) [OMIM: 301000] is an X-linked immunodeficiency disease characterized by thrombocytopenia and small platelets, eczema, recurrent infections, with an increased risk for autoimmunity and malignancy (1, 2). The gene responsible for this syndrome, WAS, comprises 12 exons and approximately 1.8 kb in length (3). WAS encodes a 502-amino-acid protein (WASp) that is expressed selectively in hematopoietic stem cell– derived lineages and is involved in cell signaling and cytoskeleton reorganization (4). A milder allelic variant caused by WAS gene mutation leads to X-linked thrombocytopenia (XLT), a congenital disorder characterized by thrombocytopenia and small platelets but, in general, without the other complications of WAS (5). So far, ~369mutations have been reported in WAS gene (http://www.hgmd.cf.ac.uk/ ac/gene.php?gene=WAS). Detection of additional mutations in this gene is important for the precise genetic diagnosis in families affected by this disorder as well as for studying the molecular basis of this disease (6). We report here for the first time the WAS gene mutations identified in patients with WAS/XLT from India and their genotype–phenotype correlations. Ten patients from eight families were evaluated at the Department of Haematology, Christian Medical College, Vellore, with clinical features suggestive of WAS on written, informed consent. All patients underwent hematological and biochemical evaluation (Table 1). Blood was collected in citrated buffer and in ethylenediamine tetra-acetic acid (EDTA) from the probands and, whenever available, from family members. Similar samples were also collected from healthy controls. Blood smears were stained using modified Giemsa–Wright stain (Beckman, Duarte, CA, USA) and evaluated for morphology. Platelet count and the mean platelet volume (MPV) were estimated in a cell counter (Coulter LH 755; Beckman Coulter). A mononuclear cell suspension in phosphate-buffered saline (PBS) was isolated from heparinized blood by Ficoll gradient centrifugation. The cells were fixed (Fix and Perm Medium; Invitrogen, Carlsbad, CA, USA) and permeabilized (Fix and Perm medium B; Invitrogen) for intracellular staining and incubated with phycoerythrin (PE)-conjugated antibody against WASP DI (SantaCruz Biotechnology, SantaCruz, CA, USA) and WAS B9 (Santa Cruz Biotechnology) with appropriate IgG1 and IgG2a controls (BD Pharmingen, San Jose, CA, USA). After processing, cells were analyzed for intracellular bound florescence in a flow cytometer (BD FACS Calibur, Manifield, MA, USA). The patient WASP expression was compared to normal controls processed at the same time, and the data are expressed as percentage of normal WASP-positive cells (7). Genomic DNA from EDTA anti-coagulated blood was isolated by standard phenol–chloroform method. The human WAS gene exonic and flanking intronic regions were amplified by twelve pairs of primers as described previously (8). Nucleotide changes in the amplified fragments were screened by a conformation-sensitive gel electrophoresis (CSGE) and DNA sequencing strategy (9). Samples displaying abnormal CSGE patterns were sequenced by the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) on an ABI 3130 genetic analyzer (PE Applied Biosystems, Foster City, CA, USA). All the novel mutations identified were confirmed as unique to the patients by screening 100 normal control alleles. The normal controls were recruited based on a written informed consent. Mutations at or near the splice junction consensus sequences were analyzed by ‘Splice Site Prediction program’ (http://www.fruitfly.org/seq_tools/splice.html) to predict changes in RNA splicing. The clinical features, and hematological and molecular genetic data of all the 10 patients are detailed in Table 1. Diagnosis was based on low platelet count (9000–95 000/ mm), presence of small platelets (mean platelet volume – 6.2 ± 1.5) and reduced levels of WASp by flow cytometry. Of these, two were sibling pairs (WAS-9/WAS-10; WAS15/WAS-35). The median age at first clinical symptoms of these patients was 2.5 yr (range 1–12 yr). Six of them had a family history of bleeding. To differentiate between classical WAS and XLT in these patients, the disease was scored from 1 to 5 based on the presence of thrombocytopenia, small platelets, eczema, immunodeficiency, infections, autoimmunity or malignancy and congenital neutropenia as described previously (10). In patients who were lost to follow-up, we could not comprehensively analyze the biochemical or genetic data, and this was a limitation to our study. Genotypic analysis revealed mutations in eight of ten patients (Table 1). Among them, three had nonsense mutations, two splice site variations, two deletions, and one missense mutation. Two of these eight mutations were novel. These included a single ‘T-nucleotide’ deletion (c.108delT),

The t(8;14)(q24.1;q32) and its variant translocations: A study of 34 cases

BACKGROUND The t(8;14)(q24.1;q32) and its variants - the t(2;8)(p12;q24.1) and t(8;22)(q24.1;q11.2) are associated with B-cell neoplasia and result in MYC/immunoglobulin (IG) gene rearrangement. PATIENTS AND METHODS We correlated the cytogenetic, molecular and clinico-pathological findings of patients with 8q24 translocations seen in the Department of Haematology, Christian Medical College, Vellore, from January 2003 to December 2015. RESULTS There were 34 patients with 8q24 translocations (31, ALL and three myeloma). The t(8;14) was seen in 25 patients, t(8;22) in seven and t(2;8) in two. The salient findings were as follows: 85% males; 79% adults, median age 37 years; L3 morphology in 61%; mature B immunophenotype in 77%; extra-medullary disease in 41%; additional abnormalities in 28 (85%), notably, structural abnormalities of chromosome 1q (41%) and 13q (9%) and monosomy 13 (15%); complex karyotypes in 68%. There were two double-hit lymphoma/leukemia, one with a t(14;18)(q32;q21) and the other with a t(3;14)(q27;q11.2), associated with nodal high grade B cell lymphoma and dermal leukemic infiltrates respectively. Only 13 samples were processed for DNA PCR and all these samples were positive for MYC-IgH (c-gamma type) rearrangement. Only in one patient, in addition to c-gamma, c-alpha rearrangement was also detected. CONCLUSION The frequency (1.7%) and distribution of these translocations in our series and the association with 1q and 13q abnormalities is similar to the literature. Trisomies 7 and 12 were seen in less than 10% of our patients.