the tyrosine kinase activity that is supposed to be the cause of the disease and its complications.JAK2V617F mutation primarily affects the proliferation of the red cell lineage but its impact on the phenotype of circulating RBCs is poorly documented. Our group showed increased adhesion of PV RBCs to endothelial cells mediated by erythroid Lu/BCAM and endothelial laminin a5 chain [4]. Recently, we showed that this abnormal adhesion stems from JAK2V617F-mediated Lu/BCAM activation through a Rap1/Akt signaling pathway [5]. Blocking this pathway by JAK2 specific inhibitors led to significant decrease of PV RBC adhesion [5]. Here, we extended our findings by a quantitative analysis to determine the relationship between the rate of RBC adhesion to laminin and the JAK2V617F allele burden.We first asked whether variation between patients in RBC adhesion was related to Lu/BCAM expression on the surface of PV RBCs. Indeed, Lu/BCAM erythroid expression is heterogeneous within the same individual, including a variable surface expression level and a proportion of Lu/BCAM-negative RBCs. We performed adhesion assays with blood samples from 17 JAK2V617F-positive PV patients, with no cytotoxic antiproliferative treatment, and determined the number of RBCs adhering to laminin at 3 dyn/cm 2 .The adhesion values were variable, ranging from 14 to 1021 RBCs/mm 2 (Supporting Information Table S1). The percentage of Lu/BCAM-positive RBCs as assessed by flow cytometry ranged from 38% to 90%, and the expression level, estimated by the mean fluorescence intensity (MFI), from 1249 to 3153 (Supporting Information Table S1). Because Lu/BCAM phosphorylation is driven by a JAK2V617F-dependent pathway in PV RBCs [5], we asked whether RBC adhesion was influenced by JAK2V617F expression level. To date the most commonly used tool for evaluating the mutational load in one individual is by measuring the mutant allele burden in blood cell DNA. The JAK2V617F allele burden was determined for the 17 PV patients using an allele specific quantitative PCR (MutaquantV R , Qiagen) and ranged from 20 to 68% (Supporting Information Table S1). RBC adhesion values were plotted against the percentage of JAK2V617F. Statistical analysis showed a significant correlation between RBC adhesion and JAK2V617F percentage (r 5 0.571 and P 5 0.016, Fig. 1C) that was improved when the interval of Lu/ BCAM-positive RBCs was restricted to 65-90% (r 5 0.849 and P 5 0.0009, n 5 11 patients), indicating that for equivalent Lu/BCAM expression, higher JAK2V617F levels would drive higher RBC adhesion to laminin (Fig. 1D).As RBCs are enucleated, it is not possible to estimate the level of JAK2V617F using DNA or RNA based PCR assays. However, clonogenic assays showed that patients with higher JAK2V617F mutant allele burden in blood DNA carried more mutated erythroid progenitor cells [6]. Our study revealed RBC adhesion as a biological marker to estimate the V617F mutational load at the protein level in PV RBCs; although indirect, this marker has the potential of informing about the ...

Although not regularly transfused, patients with non-transfusion-dependent thalassemia (NTDT) are prone to iron overload and its complications. Their molecular, phenotypical and laboratory characteristics vary in different populations and there is a need to document local prevailing patterns. We have reviewed the records of our patients with NTDT in Kuwait and documented their clinical and molecular characteristics in addition to iron status [serum ferritin and liver magnetic resonance imaging (MRI) T2*], management and complications. There were 41 patients, made up of 20 with β-thalassemia intermedia (β-TI), 18 with Hb H (β4) disease and three with Hb E (HBB: c.79G > A)-β-thalassemia (Hb E-β-thal); their ages ranged from 3 to 36 years (mean 12.5 ± 7.7). While 18 (43.9%) had been transfused at least once, only three (7.3%) had been transfused on multiple occasions. Three patients had serum ferritin >500 ng/mL; while four of 38 had mild or moderate liver iron overload. Seven (35.0%) of the β-TI patients were managed with hydroxyurea (HU) with good response. Other complications included five patients with gallstones and one each of hypothyroidism and moyamoya. The most common mutations among the β-TI patients were IVS-II-1 (G > A) and IVS-I-6 (T > C), while among the Hb H patients, the Saudi α2-globin gene polyadenylation (polyA) (AATAAA > AATAAG) mutation was responsible for all cases either as homozygotes (61.1%) or compound heterozygotes with the α-thal-2 (-α(3.7)) allele (33.3%). Although the pattern of NTDT in Kuwaiti patients is generally mild, there is a need to follow them to adulthood as the complications are cumulative and more prevalent in this group.

To describe outcomes of human leukocyte antigen-matched sibling bone marrow transplantation (BMT) in 12patients: 9 with β-thalassemia major (TM) and 1 each with severe aplastic anemia, myelodysplastic syndrome and congenital dyserythropoietic anemia. The median age at transplantation was 9years and the median follow-up was 24months. Among the TM patients, 6 were in Pesaro risk class II and 3 in class III. The median neutrophil recovery and platelet recovery were 19 and 25days, respectively. None of the patients had primary graft failure. At the last follow-up, 11 out of 12 patients were alive, free of disease and living normal quality of life. Most of the patients (75%) had acute GVHD grade I-II, in the skin. None of the patients had grade IV regimen-related toxicity. Six patients developed Gram-negative while two had Gram-positive bacteremia, one had probable lung aspergillosis and four developed febrile neutropenia without identifiable microbiological growth during the aplastic phase. Cytomegalovirus reactivation occurred in five patients. We lost one patient who developed severe acute graft-versus-host disease (GVHD) affecting the skin, gut and liver, who later progressed to severe chronic GVHD and severe interstitial pneumonitis. In conclusion, allogenic BMT is feasible and safe in pediatric patients with hematological disorders in Kuwait.