The Clinical Utility Of Next-Generation Sequencing In The Diagnosis Of Polycythemia

Polycythemia or erythrocytosis is a disorder characterized by expansion of the RBC mass, and can be primary or secondary. Primary polycythemia is caused by an acquired or inherited mutation, it includes polycythemia vera and familial/congenital variants. e.g. due to gain of function mutations in the erythropoietin receptor (EPOR), or mutations in the hypoxia sensing pathway. Secondary polycythemia is caused by a circulating factor stimulating erythropoiesis, usually erythropoietin (EPO). It is most often due to an EPO response to hypoxia, but can also result from an EPO-secreting tumor. Either primary or secondary polycythemias can be inherited, i.e. due to germline mutations. The hypoxic response, mediated by hypoxia inducible transcription factors (HIFs), is central to the control and development of many essential biological functions, including erythropoiesis. Mutations in this pathway, causing polycythemia, have been identified in negative regulators of HIFs, such as the von Hippel-Lindau (VHL) gene, the HIF-prolyl-hydroxylase 2 (PHD2) gene, and gain-of-function mutations of the HIF-2-alpha (HIF2A) gene. Routine diagnostic testing can be challenging with specialized testing often only available in specialized research laboratories. Comprehensive, coding region analysis of all candidate genes by selective amplification of DNA regions of interest by PCR followed by the sequencing and analysis of amplified DNA fragments involved can be daunting due to molecular heterogeneity of causative genes as well as the size of the genes involved. Targeted molecular analysis is now being developed for both acquired (somatic) mutations or inherited (germline mutations) causing acquired and inherited diseases. We developed a novel, high-throughput, sensitive sequencing assay for diagnosis of congenital causes of polycythemias and polycythemia vera. Our diagnostic panel includes 9 genes and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Custom 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 10 patients with clinically suspected polycythemia, and 30 normal controls were tested in our assay. Whole blood genomic DNA was isolated from the patients and targeted gene capture performed. Mutations in the target genes were identified in 3/10 patients, two of these being novel. All identified mutations were confirmed by Sanger sequencing. In one of these patients, a child with increased RBC mass, with EPO hypersensitive BFU-E colonies, a novel pathogenic, nonsense mutation was found in exon 8 of the EPOR gene (Q434X) resulting protein truncation and absence of the C-terminal negative regulatory domain of the receptor. In a different patient with suspected primary congenital/familial polycythemia due to EPO hypersensitive BFU-E colonies, we identified a novel pathogenic mutation in exon 3 of the MPL gene. This novel mutation, a single base deletion causes a frame-shift in codon 126 (F126L) and early termination at codon 130. To the best of our knowledge, this is the first report of a loss of function mutation in the MPL gene in a patient with polycythemia. Analysis of greater cohort of polycythemic patients is now in progress.