Clinical Application of Massively Parallel Sequencing in the Diagnosis of Hereditary Hemolytic and Dyserythropoietic Anemias
Authors: Lesmana Harry , Christakopoulos Georgios E , Seu Katie Giger , Risinger Mary , Duzkale Hatice , Dagaonkar Neha , Zhang Kejian , Kalfa Theodosia A 
Published: October 2019

Abstract
The Hereditary Hemolytic Anemias (HHAs) are a genetically heterogeneous group of anemias characterized by decreased red blood cell (RBC) survival because of defects in hemoglobin, RBC membrane proteins or enzymes. The diagnosis of this group of disorders is complex and challenging requiring analysis of the morphology of RBCs, hemoglobin electrophoresis, and a battery of phenotypic assays. The phenotypic analysis is often problematic in transfusion dependent patients or at times of presentation with a hemolytic crisis as transfused blood or reticulocytosis confounds diagnostic testing. Molecular genetic testing has grown in popularity in the diagnosis of hereditary hemolytic anemias as it is not affected by transfusions or other clinical variables and provides additional insight into the mechanism of the disease. We have developed a Next Generation Sequencing (NGS) panel for HHA due to RBC membrane disorders and enzymopathies and congenital dyserythropoietic anemias (CDA). CDAs, although collectively rare, are included in the panel as they are occasionally misdiagnosed as hereditary spherocytosis (HS) due to their clinical characteristics of hemolysis, increased osmotic fragility, and splenomegaly albeit with inadequate reticulocytosis
We reviewed the results of 282 sequential HHA/CDA panels testing for patients with suspected HHA or CDA diagnosis, performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013-5/2016. Forty-three samples were omitted from the final analysis due to diagnosis of other disorders, indicating that negative results were true-negatives. For the analysis of the remaining 239 panels, all results were reviewed and categorized based on the type of testing ordered: comprehensive HHA/CDA (32 genes), RBC membrane disorders (13 genes), RBC enzyme disorders (14 genes), or CDA (6 genes). The protein-coding exons plus 25 bases of exon-intron junction as well as promoter sequences were included in the design. Genomic DNA was isolated from blood and target regions were enriched using the Haloplex technology. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software. All positive findings were confirmed by Sanger sequencing.
These 239 panels included 159 (66.5%) comprehensive HHA/CDA panels, 41 (17.2%) RBC membrane disorder panels, 10 (4.2%) RBC enzyme disorder panels, and 29 (12.1%) CDA panels. Overall, a diagnosis was confirmed or identified in 135 (56.5%) patients with specific genotype of hereditary spherocytosis in 52 patients; hereditary elliptocytosis in 15 patients; hereditary pyropoikilocytosis in 7 patients; hereditary stomatocytosis/xerocytosis in 12 patients; South East Asian Ovalocytosis in 1 patient; G6PD deficiency in 15 patients; pyruvate kinase deficiency in 17 patients; other rare RBC enzymopathies in 6 patients; and CDA in 10 patients. The clinical performance of RBC membrane disorder and RBC enzyme disorder panels were comparable between 68-70% in reaching a final diagnosis, while CDA panel confirmed final diagnosis in only 20% of suspected cases. The overall low prevalence, complexity of diagnosis with findings of dyserythropoiesis in bone marrow studies in patients with severe HHA, and evidence of locus heterogeneity in CDA might explain this result. Among patients with suspected RBC membrane disorders, approximately 14% were eventually diagnosed with hereditary xerocytosis (HX). HX diagnosis is critical to make in such patients since splenectomy is contraindicated due to the high risk of life-threatening thrombophilia complications.
In more than half (56.5%) of all cases with suspected hereditary hemolytic anemia, genetic testing provided or confirmed the diagnosis and optimized patients' clinical management. Further genetic counseling and testing for other at-risk family members was made possible by achieving molecular diagnosis. Genetic testing substantially altered management in approximately 14% of cases with suspected RBC membrane disorders due to the diagnosis of HX. In conclusion, genetic testing has a significant clinical utility and may facilitate and improve diagnosis, prognosis and management considerations in patients with hereditary hemolytic or dyserythropoietic anemia.
Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.