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Inherited Factor VII (FVII) deficiency is commonest among the rare bleeding disorders. A small number of patients present in infancy with severe bleeding, and many may remain asymptomatic but detected before surgery/invasive procedures. Genetic testing may be helpful in predictive testing/prenatal diagnosis in severe cases.Characterisation of clinical and genotypic spectrum of patients with inherited FVII deficiency.Retro-prospectively, 35 cases with prolonged prothrombin time and FVII activity (FVII:C)50 IU/dl were subjected to targeted resequencing. After in-silico analysis, variant/s were validated by Sanger sequencing in index cases and family members. Haplotype analysis was done for F7 polymorphisms.Severe FVII deficiency was found in 50% of patients (FVII:C ≤1 IU/dl), and 42.9% were asymptomatic. Clinical severity assessment revealed 17% severe, 17% moderate and 22.9% patients with mild bleeds. FVII levels ranged from .3 to 38 IU/dl. Molecular analysis revealed variants in 30/35 cases, of which 17 were homozygous, 10 were compound heterozygous and 3 were heterozygous. Twelve genetic variants were identified, one promoter variant c.-30AC; seven missense (c.215CG, c.244TC, c.253GC, c.904GA, c.961CT, c.1109GT, c.1211GA), two deletions (c.21delG, c.868_870delATC), and one each of nonsense c.634CT and splice-site variant c.316+1GA. Recurrent variants c.1109GT and c.215CG were found in 17 and 8 cases, 12 of the former cases were homozygous. They had the same haplotype, indicating the founder effect in North Indians.This is the largest cohort of FVII genotyping from India, confirming heterogeneity in terms of clinical manifestations, FVII activity and zygosity of the variants with a limited genotypic phenotypic correlation.

Glanzmann thrombasthenia (GT) is an autosomal recessive platelet function disorder characterized by mucocutaneous bleeding as the most common clinical phenotype. Patients with GT have normal platelet counts, platelet morphology but reduced platelet aggregation in response to various agonists. Homozygosity or compound heterozygosity for variants in the ITGA2B/ITGB3 genes is the genetic basis for GT. Establishing a molecular diagnosis is definitive and is important for predictive testing. Using multi-gene panels is an accurate, faster, and cost-effective mode as compared to Sanger sequencing in large genes. We used a targeted resequencing based approach to identify pathogenic variants in eight cases in seven families. These variants were validated using Sanger sequencing in patients as well as family members and were predicted probably pathogenic using in-silico prediction tools. The variants include three missense (3/7 = 43%) (ITGA2B:c.1028 T > C, ITGA2B:c.1186G > A, ITGB3:c.1388G > C), two deletions (ITGA2B:c.559delG, ITGA2B:c.3092delT), one duplication (ITGA2B:c.1424_1427dupAGGT) and nonsense variant (ITGA2B:c.2578C > T, p.Gln860Ter). Except for one case which was compound heterozygous, the rest of the cases were homozygous. We found two novel variants that are reported for the first time in GT. The targeted resequencing based approach revealed varied genetic variants in North Indian patients, including two novels ones. The high yield of our panel indicates its suitability for usage in larger cohorts for the genetic diagnosis of GT patients. This approach is cost-effective and less cumbersome as compared to Sanger sequencing for these large size genes with multiple exons. The information so obtained is helpful in prenatal testing, carrier analysis, and genetic counseling.

Hemophilia A is an X-linked recessive disorder caused by genetic abnormalities in the F8 . Klinefelter syndrome is sex chromosome aneuploidy caused by nondisjunction during meiosis in the germ cells or mitotic cell divisions in the early embryonic cells. We here report an intriguing case of a prenatal diagnosis where a rare association of hemophilia A and Klinefelter syndrome was found in a fetus. This case highlights the diagnostic difficulty where the inverse-PCR for intron 22 inversion defect leading to hemophilia A did not amplify. Indirect molecular testing was done using multiallelic extragenic variable number tandem repeat (VNTR) DXS52 (St14) and polymorphic markers. The interpretation was further complicated by the presence of Klinefelter syndrome. This case highlights the challenges faced when such rare combinations are found during prenatal diagnosis.

Myeloid neoplasm with germline predisposition is a distinct entity in the WHO 2017 classification of tumors of hematopoietic and lymphoid tissues [1]. Among the three sub-categories in this group, ...

Introduction Hemolytic anemias are a group of disorders caused by the premature destruction of red blood cells with reticulocytosis. Common causes of inherited/congenital hemolysis are hemoglobinopathies and thalassemia syndromes, red blood cell membrane, and enzyme disorders. Most of the common causes (thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary spherocytosis, etc.) are diagnosed based on laboratory testing; however, for remaining causes laboratory tests are either inaccessible or cumbersome. We follow a stepwise diagnostic pipeline and red cell morphology is helpful with membrane disorders. Phenotypes vary from severe hemolysis (transfusion-dependent) to mild/asymptomatic patients. Undiagnosed haemolytic anemias are taken up for multi-gene panel-based targeted resequencing which is rapid, accurate, and cost-effective. The use of these panels expedites the diagnoses of inherited hemolytic anemias and is eventually helpful for evidence-based genetic counseling. Objectives This study aimed to determine the genetic defects in inherited/congenital hemolytic anemias which remained unexplained after routine laboratory tests. Methods Seventy-five families were enrolled based on the clinical and laboratory features of inherited/congenital hemolytic anemias. Common causes of inherited hemolysis are G6PD deficiency, hemoglobinopathies and thalassemia syndromes, autoimmune hemolytic anemias, hereditary spherocytosis, and pyruvate kinase (PK) deficiency were excluded on the basis of biochemical and molecular tests. DNA extraction was done QIAamp DNA Blood Mini Kit. Quantity and quality of DNA were verified using NanoDrop and Qubit Fluorometer respectively. DNA libraries were prepared using Amplicon custom panels for genes implicated in hemolytic anemias and sequenced on Illumina MiSeq Sequencer. Alignment and variant calling were done in Illumina Local run Manager and Variant annotation was done in Basespace VariantInterpretor. Sanger sequencing was done as orthogonal validation in the index case. Predictive testing was performed for the family members. Results After targeted resequencing of the total 75 index cases, 19 patients were found to have red blood cell enzymopathies, 15 patients had stomatocytosis, 13 had membranopathies and three patients had unstable hemoglobins. In 8 patients cause was not established either only heterozygous variant was found for autosomal recessive or due to the lack of samples of family members for screening. Seventeen cases remained unexplained even after next-generation sequencing. Out of 19 patients, unexpected PK deficiency was found in 12 patients and G6PD deficiency was found in 3 patients; despite the enzyme assay being normal in these cases. We also found 2 patients with glucose-6-phosphate isomerase deficiency. One case each with hexokinase deficiency and glutathione synthetase deficiency was found. Among 15 patients with stomatocytosis, 8 had Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5/ABCG8). These 8 patients showed the presence of stomatocytosis along with giant platelets on peripheral smear evaluation. Of the remaining 7 cases , 2 were found to have overhydrated hereditary stomatocytosis (RHAG) and dehydrated Stomatocytosis/xerocytosis was found in 5 (PIEZO1/KCNN4). We also found 13 cases of hemolytic anemia to have a genetic defect in red blood cell membrane protein-coding genes. Of these 5 had probably pathogenic variants in the ANK1 gene, 5 had a pathogenic variant in SPTA1, 2 had SPTB 2, and 1 patient SLC4A1. We also encountered 3 cases of unstable hemoglobins where no abnormality was noted in Hb-HPLC patterns. A total of seven patients underwent splenectomy and are transfusion free. Conclusions Our cohort of 75 families of hemolytic anemia of unexplained etiology showed a highly heterogeneous genetic spectrum. Of the total cases, the confirmed diagnosis was achieved in 67% of the patients. This approach of using a multi-gene panel is cost-effective and can provide a rapid and accurate diagnosis. Unexpected PK deficiency, G6PD deficiency, and unstable hemoglobins suggest that such cases can be missed. Providing accurate diagnosis in such cases provides evidence-based counseling and saves the families from inappropriate treatments. Disclosures No relevant conflicts of interest to declare.

Defects in various erythrocyte membrane proteins genes (ankyrin, band-3, β- and α-spectrin and protein 4·2) can cause hereditary spherocytosis (HS). This molecular heterogeneity of HS, together with co-inherited genetic modifiers, results in marked phenotypic variability among patients. We studied the molecular spectrum and genotype-phenotype correlations in 73 families (with 113 patients) with HS. Deleterious variants including nonsense (42%), deletions (18%), splice site (20%), missense (10%) and duplication/insertion (10%) were found in 47 patients. The variants detected included sporadic and dominantly-inherited defects in ANK1 (53·2%), SPTB (36·2%) and SLC4A1 (4·2%). Compound heterozygous variants in SPTA1 (6·4%) showed autosomal recessive inheritance. Alpha-spectrin variants were associated with severe anaemia and splenectomy alleviated symptoms. Co-inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in 15%. G6PD variants (n = 5) led to greater transfusion requirements (1-8 times) in males with HS. Homozygosity (41%) for the promoter variant of UGT1A1 (Gilbert syndrome) led to a significantly higher mean bilirubin level (126·54 µmol/l) with a higher frequency of cholelithiasis (30%) (P < 0·001). This first-ever south Asian study on the molecular spectrum of HS found ANK1 and SPTB genes variants to be the commonest with inheritance being sporadic/dominant. Next-generation sequencing provided a relatively sensitive and rapid tool for molecular diagnosis with a diagnostic yield of 64·4%.

Summary Introduction Laboratory diagnosis of hereditary spherocytosis (HS) relies on increased incubated red cell osmotic fragility test for screening. We evaluated the diagnostic role of eosin-5′-maleimide (EMA) binding test by flow cytometry in spherocytic and microcytic hypochromic hematological disorders in North Indians. Methods EMA binding test using flow cytometry was performed on 55 HS (40 families), 26 iron deficiency anemia (IDA), 32 β-thalassemia trait (βTT), and 10 autoimmune hemolytic anemia (AIHA) cases and 121 normals. Mean channel fluorescence (MCF) and coefficient of variation (CV) were studied. Different MCF parameters (MCF, MCF ratio, percent decrease MCF) and percent increase in CV were analyzed. Receiver operating characteristics analysis was performed to determine best cutoff values, sensitivity, and specificity for discriminating HS from other red cell disorders. Results MCF ratio of HS group was significantly lower than normals (0.67 ± 0.07 vs. 1.01 ± 0.05, P < 0.001) and other cases. All patients with HS showed MCF ratio to be ≤0.79. Four postsplenectomy cases with near-normal hemograms also revealed low MCF ratio, showing the specificity of the test. Conclusions EMA assay was efficient to diagnose cases of HS including postsplenectomy cases and shows no overlap with IDA, βTT, and AIHA.

Hereditary hemochromatosis (HH) is a rare disorder in Indians and is not associated with the common mutation Cys282Tyr in HFE gene found in Caucasians. Non-HFE HH can be associated with mutations in HJV, HAMP, TFR2 and SLC40A1 genes. Nineteen unrelated north Indian HH patients were detected after screening 258 chronic liver disease patients on the basis of increased transferrin saturation, ferritin levels >1000 ng/L and siderosis by Perl's stain on liver biopsy wherever available. Automated DNA sequencing was performed for the promoters and entire coding exons for HFE, HJV, HAMP, TFR2 and SLC40A1. A novel homozygous mutation at position p.Gly336Ter (c.1006 G>T) in exon 4 in HJV was identified in four adult unrelated patients. We encountered compound heterozygosity for p.Thr217Ile (c.650C>T) and p.His63Asp (c.187C>G) mutation of HFE gene in one patient. Two patients were compound heterozygous for two novel polymorphisms at c.−358 (G>A) and c.−36 (G>A) in 5′UTR of HJV gene. Our study shows a novel HJV gene mutation p.Gly336Ter as a recurrent mutation associated with HH in north Indians. Low index of suspicion, underlying nutritional iron deficiency and protective effect of menstrual blood loss may account for the late clinical presentation of juvenile HH.

Introduction Congenital dyserythropoietic anemias (CDA) are rare inherited red cell disorders characterized by ineffective erythropoiesis and inappropriate reticulocytopenia. CDAs are usually difficult to diagnose due to variable phenotypes and overlapping bone marrow (BM) morphology with other disorders. Numerous implicated causal genes make Sanger sequencing a less likely approach and hence, the use of targeted resequencing can expedite molecular diagnosis. This study aimed at determining the genetic spectrum of CDAs and translating the results into patient care. Methods Twenty nine patients with clinical and laboratory evidence suggestive of CDA and 1 patient suggestive of CDA with thrombocytopenia by BM morphology were studied. Various biochemical and molecular tests were done to exclude common hemolytic anemias. Common SEC23B: p.Tyr462Cys variant in our patients with CDA was screened by Sanger sequencing. DNA libraries were prepared using TruSight One Sequencing Panel and TruSeq Custom Amplicon Panel and sequenced on Illumina platform. After data analysis variants were classified and the most likely disease-causing variants were validated by Sanger sequencing followed by pedigree analysis. Results Out of 27 patients of suspected CDA, SEC23B: p.Tyr462Cys variant was found in 10 patients. Rest of the remaining 17 patients were subjected to targeted resequencing. Data analysis revealed novel potentially pathogenic variants in compound heterozygosity in SEC23B in 4 patients and 1 patient had a heterozygous variant in SEC23B. There could be the possibility of intronic or large indel in her. The variants were distributed throughout the SEC23B gene. Notably, in 7 patients with suspected CDA, the final molecular diagnosis were hemolytic anemias. Of them, 4 showed likely pathogenic variants in PKLR gene and 1 each had probably causal variant in MTRR, SPTB and PIEZO1 genes. In the patient's with pyruvate kinase deficiency, screening by enzyme assays were normal. Except for the patient with MTRR gene defect all 6 had transfusion dependent anemia and BM showed dyserythropoiesis. One patient each of GATA1 gene variant (novel) and a known pathogenic variant p.Glu325Lys in KLF1 gene (CDA type IV) was detected. Of 17 cases subjected to targeted resequencing the diagnosis was achieved in ~76% (13/17) of cases. The phenotypes correlated with the genetic defects found in the SEC23B gene. The homozygous and compound heterozygous defects in this gene cause CDA type II. As anticipated GATA1 gene defect (p.Val205Leu) was found in a patient of X-linked thrombocytopenia with dyserythropoietic anemia. Patient with KLF1 had high levels of fetal hemoglobin along with features of dyserythropoiesis in BM compatible with the phenotype of variant p.Glu325Lys causing CDA type IV. Phenotype-genotype correlation was discrepant in 7 cases of CDA. In 4 cases pyruvate kinase deficiency (PKLR) was found and each case of hereditary xerocytosis (PIEZO1), membrane defect (SPTB) and MTRR defect was found. Conclusion(s) CDA showed a highly varied etiology. Our experience demonstrates a high diagnostic yield (~76%) of targeted resequencing for molecular diagnosis of suspected CDAs. Discrepancy was noted in 41% (7/17) cases with suspected CDA which were diagnosed as hemolytic anemia after molecular analysis. Establishing the correct diagnosis of pyruvate kinase deficiency led to an evidence-based decision of splenectomy that eliminated transfusion dependence. In the patient with MTRR defect change in therapy was suggested. Prenatal diagnosis was done for 2 families, where in 1 of the family both the SEC23B variants were novel and in compound heterozygosity. This study highlights the importance of genetic testing in patients under frequent blood transfusions and suspected CDAs, to provide accurate diagnosis and therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Introduction Stomatocytes in peripheral blood are pathognomonic findings in multiple conditions along with hemolysis and reticulocytosis, often suggestive of erythrocyte membrane transport defects. These uncommon disorders are usually difficult to diagnose due to a wide range of overlapping phenotypes and a perception of stomatocytes being artefacts. Genes involved in these disorders are multiple (RHAG,SLC4A1, ABCG5, ABCG8, PIEZO1,KCNN4, ABCB6, SLC2A1 etc) rendering Sanger sequencing costly and labor intensive. Phenotypes vary from transfusion-dependent anemia to compensated hemolysis. Methods Seventeen patients were encountered in 12 families and enrolled in this study. Majority of the cases showed the presence of significant numbers of red blood cells showing stomatocytes with or without thrombocytopenia. Various hematological, biochemical and molecular tests were used to exclude thalassemia syndromes and hemoglobinopathies, glucose-6-phosphate dehydrogenase (G6PD) deficiency, autoimmune hemolytic anemia, hereditary spherocytosis (HS) and pyruvate kinase deficiency. Genomic DNA was extracted by the QIAamp DNA Blood Midi Kit and quantified on NanoDrop 2000 spectrophotometer and QubitFluorometer. DNA libraries were prepared using Illumina's custom panels (TruSight One Sequencing Panel and TruSeq Custom Amplicon v1.5) and sequenced on a MiSeq Sequencing System. MiSeq Reporter and VariantStudio were used for analysis, classification, and reporting of variants. Variants which were predicted pathogenic by in silico analysis using PolyPhen-2, SIFT, PROVEAN (http://provean.jcvi.org/), Mutpred (http://mutpred.mutdb.org/) and Human Splicing Finder as indicated, were subjected to Sanger sequencing in the patient and family members (where available). Results Of these 17 patients, 10 patients in 6 families were diagnosed to have Mediterranean stomatocytosis/ macrothrombocytopenia. All had the presence of stomatocytes along with macrothrombocytopenia, short stature, continuous abdominal discomfort and marked pleiotropic effects in different cases. This is a syndromic form of stomatocytosis and defects in ABCG5/ABCG8 genes were found and showed an autosomal recessive inheritance pattern. One case did not show any mutation. This number of cases suggests that this disorder is not rare in India and is probably underdiagnosed as patients have mild or moderate anemia and are often misdiagnosed as cases with HS. One of the patients also had coinheritance of G6PD deficiency (G6PD Kerela Kalyan). Patients with Mediterranean stomatocytosis/macrothrombocytopenia are advised to take sterol-absorption inhibitor 'ezetimibe' to reduce sterol accumulation. We also found 2 unrelated patients with stomatocytosis, reticulocytosis and splenomegaly with overhydrated hereditary stomatocytosis (OHSt) and pathogenic mutation in RHAG gene was found in both of them. The pattern of inheritance is sporadic or autosomal dominant. Splenectomy was deferred in a patient with OHSt as postsplenectomy thrombotic complications are known to occur and is contraindicated. Four patients in 3 families were found to have mutations in PIEZO1 gene which translates to red cell membrane mechanosensitive cation channel protein, causing xerocytosis/dehydrated hereditary stomatocytosis and 3 patients had severe anemia and were transfusion dependent. One patient showed the presence of stomatocytes and macrothrombocytopenia was found to have probably disease causing variant in SLC2A1 gene. She was incidentally undergoing treatment for infertility when the stomatocytosis was noted. Conclusions Stomatocytic disorders appear to be underdiagnosed in India which is compounded by the protean clinical manifestations, milder phenotypes, low index of suspicion and non-availability of molecular confirmation. Astute phenotype characterization is critical as it will help in establishing the causality of the variants identified and appropriate genetic counseling. Recently NGS for hemolytic anemias has led to rapid molecular characterization and accurate phenotypic correlation. Disclosures No relevant conflicts of interest to declare.

Introduction Diagnosis of inherited anemias is based on automated red cell indices, morphology and reticulocyte count in an appropriate clinical presentation and a stepwise diagnostic algorithm needs to be followed. Major inherited causes of hemolysis include the hemoglobinopathies, membranopathies and enzymopathies. In contrast, ineffective erythropoiesis, a term that refers to a disturbance in proliferation, differentiation and maturation of erythroblasts, includes megaloblastic anemia, myelodysplastic syndromes, thalassemias, and congenital dyserythropoietic anemias (CDA). Phenotypes vary from severe transfusion-dependent hemolytic anemia (HA) to fully compensated hemolysis. Patients are encountered where the entire spectrum of diagnostic tests cannot identify the etiology. Causal genes implicated are numerous, making a gene-by-gene approach time consuming, expensive and laborious. Hence, the use of targeted resequencing can expedite molecular diagnosis, genetic counseling and prenatal diagnosis if indicated. Objectives This study aimed at determining the spectrum of mutations in uncommon HA and CDA. Methods Sixty-one patients (HA=44, CDA=17) with clinical and laboratory evidence suggestive of HA/CDA were enrolled after excluding common causes of anemias. Various biochemical and molecular tests were used to exclude glucose-6-phosphate dehydrogenase (G6PD) deficiency, hemoglobinopathies, autoimmune HA, hereditary spherocytosis and pyruvate kinase (PK) deficiency. Peripheral blood genomic DNA was extracted using QIAamp DNA Blood Midi Kit, quantified on NanoDrop 2000 spectrophotometer and Qubit® 2.0 Fluorometer. Common G6PD, PKLR variants, SEC23B mutation were excluded by molecular tests. Since the genetic defects in Indian patients with unexplained HA/CDA are not available, we first tested 16 patients with a comprehensive commercially available sequencing panel that covers 4,813 clinically-relevant genes. We next sought to streamline testing and designed a customized panel of 55 genes (Illumina). Variants were annotated and classified and the most likely disease-causing variants were validated by Sanger sequencing in the patient and available affected and unaffected family members. Results Out of 61 patient's sequenced, unexpected pyruvate kinase (PK) deficiency were found in 11 patients. PK enzyme activity assay was within normal limits in all these cases. Three patients with G6PD deficiency, 2 patients with glucose-6-phosphate isomerase deficiency and 1 case with hexokinase deficiency were found. Of 5 patients with stomatocytes on peripheral blood film, 3 had Mediterranean stomatocytosis/ macrothrombocytopenia (ABCG5/ABCG8) and 2 were found to have overhydrated hereditary stomatocytosis (RHAG). We also found 6 cases of HA to have a mutation in erythrocyte membrane protein-coding genes. Heterozygous nonsense mutation in the ANK1 gene was found in 2 cases and compound heterozygous missense mutations in ANK1 were seen in 1. Missense mutations were also found in SPTB gene in other 3 cases. Xerocytosis or dehydrated hereditary stomatocytosis (PEIZO1) was found in 3 patients. Seventeen cases for suspected CDA were studied and SEC23B mutations were present in 7. In one patient only heterozygous mutation was found. There could be the possibility of intronic mutation or long deletion/insertion in her. Notably, 6 patients with CDA showed mutations in PKLR (n=3), 1 each had mutations in MTRR, SPTB and PIEZO1 genes. We also found a mutation in GATA1 gene in a patient of CDA with thrombocytopenia. Conclusion(s) HA/CDA showed highly varied etiology. Our experience demonstrates the high diagnostic yield (>75%) of targeted resequencing for molecular diagnosis of unexplained anemias. It is cost effective and can expedite the diagnosis where conventional testing is not helpful. Timely detection of the etiology was helpful in genetic counseling. It not only offered therapeutic benefits to our patients but may help us in future antenatal diagnosis if required. Therapeutic implications include performing splenectomy which can ameliorate the anemia in selected subgroups of patients (HS, PK deficiency, CDA type II). Disclosures No relevant conflicts of interest to declare.

Introduction Global transcriptome analysis of circulating reticulocytes using microarrays is challenging due to the high abundance of globin transcripts. In order to accurately measure the transcriptome in reticulocytes, we planned to study the reticulocytes transcriptome profile before and after globin depletion. Patients with Hereditary Spherocytosis (HS) were recruited because of high reticulocytosis and also there was no global trancriptome profile available for the disease to the best of our knowledge. Methods Reticulocytes were purified by passing EDTA anticoagulated red cell suspensions through a column of microcrystalline cellulose and α-cellulose mixture. The extraction of total RNA was done fresh prior to microarray analysis using TRIzol reagent (Invitrogen) and RNeasy columns as per manufacturer's instruction. The globin transcripts were depleted with starting quantity of 3 µg of total RNA.using GLOBINclearTMHuman kit (Ambion, Austin, TX). RNA quality were assessed before and after depletion using Agilent Technologies 2100 Bioanalyzer in each sample used for the study. The Agilent Low Input Quick Amp Labelling kit was used to generate cRNA with a sample input of 200 ng total RNA in single-color microarray analysis (SurePrint G3 Human Gene Expression v3 8x60K Microarray, G4858A- 072363). cRNA was hybridized for 17 hours at 65˚C. After thorough washing, the results were extracted with Feature Extraction Project software and analysed using GeneSpring v13.0. For this study, differentially expressed genes were identified according to the criteria: t-test unpaired p (Corr) cut-off = 0.05 and fold-change cut-off of 2.0. These differentially expressed genes were imported in PANTHER (Protein Analysis Through Evolutionary Relationships) classification system to classify the genes. Results Transcriptome profiling of reticulocyte RNA from HS patients revealed 5318 annotated transcripts and 2744 lnc-RNA/uncharacterized transcripts to be differentially regulated before globin depletion. After globin depletion increase (8196 annotated transcripts and 4292 lnc-RNA) in the number of differentially regulated genes were observed. An increase of 54% and 56% was seen in annotated transcripts and noncoding transcripts respectively after globin depletion. This increased coverage may be attributed to the removal of the globin transcript. Gene Ontology analysis for molecular function, biological process, cellular component and protein class did not reveal any difference in the percentage of the category, though the number of transcripts was more after depletion. There were approximately 7% more pathways in the globin transcript depleted samples. Namely, 2-arachidonoylglycerol biosynthesis, biotin biosynthesis, carnitine metabolism, cobalamin biosynthesis, coenzyme a linked carnitine metabolism, cysteine biosynthesis, flavin biosynthesis, phenylalanine biosynthesis, sulfate assimilation, tyrosine biosynthesis pathways, etc. were elucidated only after globin depletion. Conclusions The globin transcript reduction followed by microarray analysis represents a robust methodology for studying pathophysiology of hematologic diseases which are not related to globin chain disorders. Furthermore we describe the global transcriptome profile of HS for the first time. Globin transcript depletion elucidates the better number of the transcripts and eventually the pathways which may have been earlier masked under the abundance of globin mRNAs. Pathway analysis and validation experiments are required to explain the role of these differentially expressed genes in the pathophysiology of HS. Disclosures No relevant conflicts of interest to declare.