Gene Expression Profiling of Reticulocytes in Patients with Hereditary Spherocytosis after Depleting Globin Gene Transcripts
Authors: Das Reena , Anu Aggarwal , Jamwal Manu , Sharma Prashant , Sachdeva Man Updesh Singh , Bansal Deepak , Malhotra Pankaj 
Published: November 2019

Abstract
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.