Reliable genetic diagnosis of NCF1 (p47phox)-deficient chronic granulomatous disease using high-throughput sequencing
Amy P Hsu1, Eric Karlins2, Justin Lack3,4
1Immunopathogenesis Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD, United States.
Insights
A new bioinformatic method enables genetic diagnosis for NCF1-CGD patients, including those with non-deletion mutations. This approach uses existing sequencing data to identify NCF1 gene mutations, improving diagnostic accuracy for chronic granulomatous disease.
Area of Science:
- Immunology and Genetics
- Bioinformatics and Computational Biology
Background:
- Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the NADPH oxidase complex.
- NCF1 (p47phox) gene mutations account for a significant portion of CGD cases, but genetic diagnosis is challenging due to pseudogenes.
- Current diagnostic methods for NCF1-CGD, such as DHR and immunoblotting, can be insufficient for identifying all mutation types.
Purpose of the Study:
- To develop and validate a bioinformatic method for the genetic diagnosis of NCF1-CGD.
- To identify both deletion (ΔGT) and non-deletion mutations in the NCF1 gene using sequencing data.
- To investigate the role of pseudogene NCF1B and NCF1C in NCF1-CGD pathogenesis.
Main Methods:
- Development of a bioinformatic pipeline utilizing existing short or long-read sequencing data.
- Analysis of sequencing data from 48 NCF1-CGD patients and carriers.
- Comparison of NCF1 sequences from NCF1-CGD patients with healthy controls (1000Genomes cohort).
Main Results:
- Successfully identified both ΔGT and non-ΔGT NCF1 gene mutations in NCF1-CGD patients.
- Confirmed that ΔGT mutations result from pseudogene (NCF1B/NCF1C) sequence replacement in the NCF1 locus.
- Observed reciprocal pseudogene replacement by NCF1 in some healthy individuals, highlighting complex genetic interactions.
Conclusions:
- The developed bioinformatic method provides a means for genetic diagnosis in NCF1-CGD patients, including those with previously undiagnosed mutations.
- Reanalysis of existing sequencing data can yield definitive genetic diagnoses, improving patient management.
- The methodology holds potential for application in diagnosing other genetic disorders involving pseudogenes.
Introduction:
Chronic granulomatous disease is caused by mutations in any of the 6 components of the phagocytic NADPH oxidase complex including gp91phox, p47phox, p22phox, p40phox, p67phox, 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 NCF1 (p47phox) due to two, nearly identical, pseudogenes (NCF1B, NCF1C). The majority of NCF1-CGD patients carry a 2-base deletion caused by crossover between NCF1 and NCF1B or NCF1C. Currently, NCF1 deficiency is diagnosed functionally: a characteristic DHR with low levels of residual ROS, loss of p47phox 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 NCF1 gene mutations. Additionally, we confirm that the presence of ΔGT in NCF1 is due to pseudogene copy into the NCF1 locus. We compare NCF1 sequence from NCF1-CGD patients to cohorts of non-NCF1-CGD and healthy controls (1000Genomes), demonstrating pseudogene replacement of NCF1 in NCF1-CGD as well as the reciprocal replacement of NCF1B or NCF1C by NCF1 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.
