Phenotype-Genotype Spectrum of Stomatocytic Disorders Encountered in India Using Next Generation Sequencing

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.2326-2326
• Main Authors: Das, Reena, Jamwal, Manu, Aggarwal, Anu, Sharma, Prashant, Maitra, Arindam, Bansal, Deepak, Malhotra, Pankaj
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-114554

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 (