Reliable genetic diagnosis of (p47)-deficient chronic granulomatous disease using high-throughput sequencing.

INTRODUCTION

Chronic granulomatous disease is caused by mutations in any of the 6 components of the phagocytic NADPH oxidase complex including gp91, p47, p22, p40, p67, or EROS. Functional assays include reactive oxygen species (ROS) production, flow cytometry, and immunoblotting for NADPH proteins. The advent of high-throughput sequencing allows genetic diagnosis for all components except (p47) due to two, nearly identical, pseudogenes (, ). The majority of NCF1-CGD patients carry a 2-base deletion caused by crossover between and or . Currently, NCF1 deficiency is diagnosed functionally: a characteristic DHR with low levels of residual ROS, loss of p47 on immunoblot, or digital droplet PCR or Gene-scan to enumerate intact (GTGT) or deleted (ΔGT). While this provides patients a clinical CGD diagnosis, for the 20% of NCF1-CGD patients with a non-ΔGT mutation a definitive genetic diagnosis is still lacking.

METHODS

We developed a bioinformatic method using existing short or long-read sequencing data from 48 NCF1-CGD patients or carriers.

RESULTS

We identified both ΔGT and non-ΔGT gene mutations. Additionally, we confirm that the presence of ΔGT in is due to pseudogene copy into the locus. We compare sequence from NCF1-CGD patients to cohorts of non-NCF1-CGD and healthy controls (1000Genomes), demonstrating pseudogene replacement of in NCF1-CGD as well as the reciprocal replacement of or by in some healthy controls.

DISCUSSION

With this method, reanalysis of existing sequence data may provide genetic diagnosis to NCF1-CGD patients. This technique may be modified for other diagnostically relevant pseudogenes.