The Clinical Utility Of Next-Generation Sequencing In The Diagnosis Of Hereditary Hemolytic Anemias

Hereditary hemolytic anemia encompasses a diverse group of genetically and phenotypically heterogeneous disorders that are characterized by increased red cell destruction, with consequences ranging from relatively harmless to severe life-threatening anemia. Moreover, red cell hemolysis leads to increased production of bilirubin, a breakdown product of hemoglobin, which in neonates places them at risk for extreme jaundice and its consequences. Two of the more common genetic causes of hereditary hemolytic anemia, excluding hemoglobinopathies, can be attributed to defects in either the red cell cytoskeleton or enzyme deficiency (e.g. G6PD, PKLR). Morphological and biochemical diagnosis of hereditary hemolytic anemia due to defects in RBC cytoskeleton or enzyme deficiency is routinely performed in many laboratories. However, routine studies can be challenging, particularly in transfusion-dependent infants and children since these patients have mostly transfused RBCs. Molecular diagnosis has also been challenging not only due to molecular heterogeneity but also due to the number and size of the genes involved. We developed a novel, high-throughput, sensitive sequencing assay for diagnosis of the molecular causes of the two major types of hereditary hemolytic anemia described above. Our diagnostic panel includes 25 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using HaloPlex as described by the manufacturer (Agilent Technologies, Santa Clara, CA). One hundred base-pair paired-end sequencing was done on a HiSeq 2000 system (Illumina, San Diego, CA). Bioinformatic analysis was based on an “in house” pipeline using standard open-source software. A total of 19 patients with unexplained hemolytic anemia, and 30 normal controls were tested in our assay. Mutations in the appropriate genes were identified in 17/19 patients, many of these being novel. All identified mutations were confirmed by Sanger sequencing. In silico prediction of the impact resulting from the novel mutations was performed using two web-based software packages, Sift and Polyphen. Where possible, inheritance of pathogenic mutations was determined in immediate relatives. One of the cases we investigated involved a neonate with unexplained jaundice and subsequent, significantly compensated, anemia without family history of a hemolytic disorder. Routine studies were suggestive for hereditary spherocytosis due to the presence of microspherocytes on the proband’s blood film, increased osmotic fragility, and decreased eosin-5-maleimide stained red cells. Two pathogenic mutations, in compound heterozygosity, were identified in the SPTA1 gene (α-spectrin). A previously reported mutation α^LEPRA, known to be associated with recessive spectrin-deficient HS, and a novel mutation in intron 45 +1 (c.6530+1G>A) disrupting the consensus splice site. Screening of other relevant genes failed to reveal additional mutations. Studies of his parents revealed both to be heterozygous carriers with the asymptomatic mother harboring the α^LEPRA mutation and the asymptomatic father harboring the novel mutation. Our results demonstrate the clinical utility of this assay for molecular diagnosis and genetic counseling for parents at risk of having affected children. Next-generation sequencing provides a cost-effective and rapid approach to molecular diagnosis, especially in cases where traditional testing has failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in several cases with no family history. Furthermore, we have validated its clinical utility in neonates risk for hyperbillirubinemia, as well as, in patients with transfusion dependent hemolytic anemia.