Next Generation Sequencing for Diagnostic Testing of Erythrocyte Cytoskeleton Disorders

Erythrocyte cytoskeleton disorders, a common cause of hereditary hemolytic anemia, consist of a genetically and phenotypically variable group of diseases that include hereditary spherocytosis (HS), elliptocytosis (HE), pyropoikilocytosis (HPP), and stomatocytosis (HSt) syndromes. The diagnosis is most commonly based on the morphology of the red blood cells (RBCs) on the blood smear and on ektacytometry and osmotic fragility studies. However, in infants and children with transfusion-dependent hemolytic anemia these studies are challenging to obtain since the patient has mostly transfused RBCs. Molecular diagnosis has not been easily attainable so far due to the number and large size of the genes involved in pathogenesis and due to the fact that each kindred has frequently a private mutation in the responsible gene(s) (Gallagher, Hematology, 2005).

We have developed a high-throughput assay for the diagnosis of known and discovery of new genetic mutations causing erythrocyte cytoskeleton disorders. The complete exon sequences of 24 genes encoding cytoskeleton proteins (spectrin a-chain (SPTA1), spectrin b-chain (SPTB), ankyrin 1 (ANK1), band 3 (SLC4A1), protein 4.1 (EPB41), protein 4.2 (EPB42), adducins (ADD), dematin (EPB49), tropomyosins (TPM), tropomodulins (TMOD), Rh-associated glycoprotein (RhAG), erythrocyte protein p55 (MPP1), stomatin (EPB72), Glut1-glucose transporter (SLC2A1), and K-Cl-cotransporter (SLC12A4, SLC12A6, SLC12A7) were determined using a Next Generation Sequencing technology. Analysis was performed on eleven patients diagnosed with erythrocyte cytoskeleton disorder along with a negative control. Informed consent was obtained from all subjects under an Institutional Review Board-approved protocol. The RainDance Technologies RDT1000 instrument was used for target enrichment covering the exons, 20 bases of exon/intron junctions, and 500 bases up and downstream of the 24 genes of interest. The products were then sequenced on an Illumina HiSeq2000 system. Bioinformatic analysis was performed in a blinded fashion as to the disease characteristics and inheritance mode of the patient using the GATK software package from the Broad Institute (DePristo et al, Nature Genetics, 2011).

Mutations predicted to have significant impact to the corresponding protein structure and therefore likely to cause disease were identified in 9 out of the 11 patients (Table 1). Shared pathogenic mutation(s) were identified blindly in family members, e.g. siblings HS-S1 and HS-S2, or parents sHS-M, sHS-F with child sHS. Two EPB49 mutations predicted to impair gene function were identified in an infant with clinical picture of HPP, implicating dematin in HPP pathogenesis. Immunoblotting of RBC membranes from this patient demonstrated decreased dematin.

Next generation sequencing for the genetically variable erythrocyte cytoskeleton disorders can provide a cost-effective and faster patient diagnosis compared with a gene-by-gene approach and it is a feasible diagnostic method in a transfusion-dependent child. Moreover, a precise genetic diagnosis can facilitate natural history studies to understand genotype-phenotype correlations in the erythrocyte cytoskeleton disorders and offer valuable insights into the structure-function relationship of the erythrocyte cytoskeleton proteins.

No relevant conflicts of interest to declare.