Diagnostic approaches for inherited hemolytic anemia in the genetic era

Abstract

Inherited hemolytic anemias (IHAs) are genetic diseases that present with anemia due to the increased destruction of circulating abnormal RBCs. The RBC abnormalities are classified into the three major disorders of membranopathies, hemoglobinopathies, and enzymopathies. Traditional diagnosis of IHA has been performed via a step-wise process combining clinical and laboratory findings. Nowadays, the etiology of IHA accounts for germline mutations of the responsible genes coding for the structural components of RBCs. Recent advances in molecular technologies, including next-generation sequencing, inspire us to apply these technologies as a first-line approach for the identification of potential mutations and to determine the novel causative genes in patients with IHAs. We herein review the concept and strategy for the genetic diagnosis of IHAs and provide an overview of the preparations for clinical applications of the new molecular technologies.

Keywords: Genetic testing; Inherited hemolytic anemia; Next-generation sequencing.

Fig. 1. A schematic representation of red blood cell (RBC) membrane structure with major functional components. The RBC membrane consists of three basic components: a lipid bilayer, transmembrane proteins, and a cytoskeletal network. The major transmembrane proteins are glycoproteins, band 3, and glycophorin. The most abundant protein in the membrane skeleton is spectrin, which is tethered to the phospholipid membrane.

Abbreviations: 4.1, protein band 4.1; 4.2, protein band 4.2; GLUT1, glucose transporter 1; GPA, glycophorin A; GPC, glycophorin C; Rh, rhesus polypeptide; RhAG, Rh-associated glycoprotein.

Fig. 2. Peripheral blood smear of inherited hemolytic anemia. (A) Hereditary spherocytosis, (B) hereditary elliptocytosis, (C) hereditary stomatocytosis, (D) β-thalassemia, (E) sickle cell anemia.

Fig. 3. Stepwise process for genetic-based diagnosis of hereditary spherocytosis.

Abbreviations: CBC, complete blood cell counting; HS, hereditary spherocytosis; LDH, lactate dehydrogenase; NGS, next-generation sequencing; RBC, red blood cell.

Fig. 4. Anaerobic glycolysis and antioxidant metabolic pathways of red blood cells.

Abbreviations: BPG, bisphosphoglyceric acid; DHAP, dihydroxyacetone phosphate; F6P, fructose 6-phosphate; FDP, fructose-1,6-diphosphate; G3P, glycerol 3-phosphate; G3PD, glyceraldehyde 3-phosphate dehydrogenase; G6P, glucose 6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; GCS, glutamylcysteine synthetase; GPI, glucose-phosphate isomerase; GS, glutathione synthetase; GSH, glutathione; GSSG, glutathione disulfide; HK, hexokinase; LD, lactate dehydrogenase; NADP, nicotinamide adenine dinucleotide phosphate; PEP, phosphoenolpyruvic acid; PFK, phosphofructokinase; PG, phosphoglyceric acid; PGK, phosphoglycerate kinase; PK, pyruvate kinase; Ru5P, ribose-5-phosphate isomerase.

Fig. 5. Overview of steps in the generation of NGS data and analysis.

Abbreviations: NGS, next-generation sequencing; dbSNP, NCBI dbSNP Build 141, http://www.ncbi.nlm.nih.gov/projects/SNP/; 1000Genomes, 1000 Genomes Project, http://www.1000genomes.org/; EVS, Exome Variant Server, http://evs.gs.washington.edu/EVS/; ExAC, Exome Aggregation Consortium database, http://exac.broadinstitute.org/; KRDGB, Korean Reference Genome Database, http://152.99.75.168/KRGDB/menuPages/intro.jsp; SIFT, http://sift.jcvi.org/; PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/; MutationTaster, http://www.mutationtaster.org/; Human splicing findinger, http://www.umd.be/HSF/; MaxEntScan, http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html.