Diagnostic paradigm for evaluation of male patients with chronic granulomatous disease, based on the dihydrorhodamine 123 assay.
BACKGROUND
Chronic granulomatous disease (CGD) is a phagocyte disorder caused by mutations in nicotinamide dinucleotide phosphate (NADPH) oxidase subunits. The dihydrorhodamine 123 (DHR) assay is an effective test for CGD that for most patients also might help to differentiate between the 2 most common forms, X-linked (X) gp91(phox) defect CGD and autosomal recessive (AR) p47(phox) defect CGD. However, some male patients with X-CGD have DHR patterns that overlap the AR-CGD pattern.


OBJECTIVE
The purpose of this investigation was to develop a diagnostic paradigm to delineate male patients with X-CGD expressing a DHR pattern suggestive of p47(phox) deficiency.


METHODS
The DHR assay measured change in fluorescence of DHR-loaded granulocytes after phorbol myristate acetate (PMA) stimulation. Western blot analysis measured the presence of NADPH oxidase subunits gp91(phox), p47(phox), p67(phox), and p22(phox). CYBB exonic sequencing was performed on PCR-amplified genomic DNA through use of intronic flanking primers. Ferricytochrome-c assay evaluated specific superoxide production by PMA-stimulated granulocytes.


RESULTS
Although 83% of patients with X-CGD have virtually no neutrophil DHR activity, we found that 17% of patients, proven to have X-CGD by other criteria, have modest DHR activity that is most consistent with p47(phox) deficiency. We describe a diagnostic paradigm to deal with such patients, and we present 2 cases, along with results of additional studies, including carrier evaluation, protein assessment, and mutation analysis, that are useful in establishing the genotype under these circumstances. DHR assays from the 2 patients described showed a fluorescence shift most characteristic of p47(phox)-AR-CGD; however, each of the patients' mothers showed mosaicism with a bimodal DHR pattern. Patient 1 had some gp91(phox) protein with a Y41D mutation and modest superoxide activity. Patient 2 had a normal level of gp91(phox) protein with a C537R mutation without detectable superoxide activity, as determined by ferricytochrome-c assay, despite the modest DHR activity.


CONCLUSIONS
Evaluation of male patients with CGD with modest DHR activity should initially include evaluation of potential female carriers for mosaicism with the use of the DHR assay. In circumstances in which this is uninformative, patients should be referred to centers capable of additional testing, including Western blot analysis and CYBB mutation analysis, to clarify the disease genotype.
Jirapongsananuruk
Orathai
O
Department of Laboratory Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Building 10, Room 2C-306, 10 Center Drive, MSC 1508, Bethesda, MD 20892, USA.
Malech
Harry L
HL
Kuhns
Douglas B
DB
Niemela
Julie E
JE
Brown
Margaret R
MR
Anderson-Cohen
Mindy
M
Fleisher
Thomas A
TA
D000328:Adult; D001483:Base Sequence; D002648:Child; D041321:Chromosomes, Human, X / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D005456:Fluorescent Dyes; D005808:Genes, Recessive; D008040:Genetic Linkage; D006105:Granulomatous Disease, Chronic / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D009030:Mosaicism; D009154:Mutation; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000235:genetics; D012235:Rhodamines; D013481:Superoxides / Q000378:metabolism
D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D012235:Rhodamines; C058319:dihydrorhodamine 123; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1