DHR CGD.pdf

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Background: Chronic granulomatous disease (CGD) is aphagocyte disorder caused by mutations in nicotinamide dinu-cleotide phosphate (NADPH) oxidase subunits. The dihy-drorhodamine 123 (DHR) assay is an effective test for CGDthat for most patients also might help to differentiate betweenthe 2 most common forms, X-linked (X) gp91phox defect CGDand autosomal recessive (AR) p47phox defect CGD. However,some male patients with X-CGD have DHR patterns that over-lap the AR-CGD pattern.Objective: The purpose of this investigation was to develop adiagnostic paradigm to delineate male patients with X-CGDexpressing a DHR pattern suggestive of p47phox deficiency.Methods: The DHR assay measured change in fluorescence ofDHR-loaded granulocytes after phorbol myristate acetate(PMA) stimulation. Western blot analysis measured the pres-ence of NADPH oxidase subunits gp91phox, p47phox, p67phox,and p22phox. CYBB exonic sequencing was performed on PCR-amplified genomic DNA through use of intronic flankingprimers. Ferricytochrome-c assay evaluated specific superox-ide production by PMA-stimulated granulocytes.Results: Although 83% of patients with X-CGD have virtuallyno neutrophil DHR activity, we found that 17% of patients,proven to have X-CGD by other criteria, have modest DHRactivity that is most consistent with p47phox deficiency. Wedescribe a diagnostic paradigm to deal with such patients, andwe present 2 cases, along with results of additional studies,including carrier evaluation, protein assessment, and mutationanalysis, that are useful in establishing the genotype underthese circumstances. DHR assays from the 2 patients describedshowed a fluorescence shift most characteristic of p47phox-AR-CGD; however, each of the patients mothers showedmosaicism with a bimodal DHR pattern. Patient 1 had somegp91phox protein with a Y41D mutation and modest superoxideactivity. Patient 2 had a normal level of gp91phox protein with aC537R mutation without detectable superoxide activity, asdetermined by ferricytochrome-c assay, despite the modestDHR activity.

Conclusions: Evaluation of male patients with CGD with mod-est DHR activity should initially include evaluation of potentialfemale carriers for mosaicism with the use of the DHR assay.In circumstances in which this is uninformative, patientsshould be referred to centers capable of additional testing,including Western blot analysis and CYBB mutation analysis,to clarify the disease genotype. (J Allergy Clin Immunol2003;111:374-9.)Key words: Chronic granulomatous disease, CGD, CYBB, dihy-drorhodamine assay, DHR, gp91phox, immune deficiency, mutationanalysis, phagocyte defect

Chronic granulomatous disease (CGD) is an inheritedphagocyte disorder of the reduced nicotinamide dinu-cleotide phosphate (NADPH) oxidase complex resultingin defective superoxide generation and intracellularkilling.1 Patients with CGD have recurrent life-threaten-ing bacterial and fungal infections, formation of chronicgranulomas, and poor wound healing.1 Genetic defects ofthis disease have been identified, and the most commonpresentation (70%2) is caused by mutations in the CYBBgene (GenBank AF469757-69) on the X chromosome(Xp21.1) coding for the gp91phox protein. This proteinand p22phox are the 2 membrane subunits of flavocy-tochrome b558; the protein is an essential component ofthe phagocyte NADPH oxidase system. The remainder ofpatients with CGD have an autosomal recessive (AR)form; of these patients, the majority have a deficiency ofp47phox protein, whereas deficiencies of p67phox orp22phox protein are far less frequent (

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firm the disease genotype. In this study, 2 such patientsare presented as examples for the laboratory evaluationof CGD, and a diagnostic paradigm is developed with theDHR used as the first step, followed by additional testingas needed to define the CGD genotype.

METHODSPatients

Studies were conducted with patients, their family members,and normal control subjects after informed consent was obtained(IRB approved research protocol 93-I-0119, National Institutes ofHealth [NIH]).

Patient 1An 8-year-old boy followed at the NIH for 1 year had first been

seen at a referral medical center at the age of 6 years for fever ofunknown origin that had persisted for 9 months. Chest radiographyrevealed a dense pulmonary infiltrate in the left upper lobe. Open lungbiopsy showed granuloma formation with positive culture forAspergillus fumigatus. NBT and myeloperoxidase test results werereported as normal. The child was treated with amphotericin for 6weeks; this was followed by treatment with oral itraconazole. Themedical history was positive for BCG infection at the vaccination site,multiple sinus infections, and chronic cough but no specific infectionssuggestive of CGD until he was 6 years old. After antifungal therapyat the age of 6 years, blood was sent to the NIH for a DHR assay; theresult was abnormal, the stimulation index (SI) being 35.9 (control,199). The patient was then referred to the NIH at 7 years of agebecause of the recurrent fever and persistent infiltrate.

Patient 2A 34-year-old man followed at the NIH for 5 years had a history

of multiple infections throughout life and inflammatory bowel dis-ease that had necessitated a diverting colostomy. Infections includedcervical abscess at 6 years of age, pneumonia at 7 years, Staphylo-coccal liver abscess at 11 and 15 years, and perirectal fistulae at 19years. He was identified as having X-CGD at 7 years, as evidencedby an abnormal NBT test result in the patient and his X-CGD broth-er. His mother had mosaicism confirmed by the NBT assay.

DHR assayDHR assay was performed according to the previously described

methods of Vowells et al.7 In brief, after red blood cell lysis, leuko-cytes were loaded with DHR (Molecular Probe, Eugene, Ore) at37C for 5 minutes in the presence of catalase (Sigma Chemical, StLouis, Mo). After DHR loading, cells were stimulated with phorbolmyristate acetate (PMA; Sigma Chemical) for 14 minutes andimmediately analyzed by flow cytometry.

Flow cytometryDHR samples were analyzed by using Cell Quest on a FACScan

flow cytometer (Becton Dickinson Immunocytometry Systems, SanJose, Calif). Twenty thousand viable granulocytes determined by

forward and side scatterplot were collected, and FL-2 (585 nm) wasanalyzed. A sample from a healthy subject was used as a positivecontrol for each assay. The SI was calculated as the ratio of geo-metric mean channel fluorescence intensity of PMA-stimulated andunstimulated granulocytes.4

Western blot analysisSDS-PAGE immunoblot detection of cytosolic and membrane

NADPH oxidase components was performed as described.8 Diiso-propyl fluorophosphatetreated granulocyte pellets were solubilizedin sample buffer (5 106 cells/100 L sample buffer) by sonication.The lysates (1 106 cell equivalents/lane) were then separated oneither NuPage Novex 10% gels (p47phox and p67phox) or 4% to 12%gradient gels (p22phox and gp91phox; Invitrogen, Carlsbad, Calif) andtransferred to Immobilon-P (Millipore Corp, Bedford, Mass). Themembranes were probed with goat anti-p22phox, anti-p47phox, anti-p67phox, and anti-gp91phox heteroantisera. Immunoblots were thenincubated with peroxidase-conjugated rabbit antigoat IgG (SigmaChemical Co) and developed with 3,3,5,5 tetramethylbenzidine(BioFX Laboratories, Owings Mill, Md).

CYBB mutation analysisGenomic DNA from patients PBMCs was isolated with the use of

the Puregene kit (Gentra systems, Minneapolis, Minn), according tothe manufacturers instructions. The 13 CYBB exons with their adja-cent intronic sequences were amplified with flanking primer combi-nations.9,10 After 35 cycles of thermal cycling, the PCR products werepurified by gel filtration (Edge Biosystems, Gaithersburg, Md) anddirectly sequenced by ABI Prism BigDye terminators (AppliedBiosystems, Foster City, Calif) and nested primers specific for eachexon. After 25 cycles of thermal cycling, sequencing extension prod-ucts were purified by paramagnetic particle separation (RapXtractDye Terminator Removal Kit, Prolinx, Bothell, Wash) and analyzedwith a 3100 Genetic Analyzer (Applied Biosystems). To detect poten-tial PCR artifacts, all mutations were confirmed by sequencing a sec-ond PCR product amplified from DNA from the original subject or acarrier relative of which no discrepancies were encountered.

Ferricytochrome-c reduction assayThe specific detection of O2 production was measured by the

superoxide dismutaseinhibitable reduction of ferricytochrome-c.Briefly, granulocytes (1 106/mL) suspended in HBSS containing 150L ferricytochrome-c were incubated at 37C for 10 or 60 minutes inthe absence or presence of PMA (100 ng/mL). The reaction wasstopped at 4C and the supernatant fluid was harvested after cen-trifuging. An identically treated tube containing superoxide dismutase(100 g/mL) served as the blank for each set of conditions. The reduc-tion of ferricytochrome-c was assayed with an analytic wavelength of549.5 nm and background wavelengths at the isobestic points, 541 and556 nm. The data were converted to nanomoles of O2/106 cells per 10or 60 minutes by using a micromolar extinction coefficient of 0.0211.

RESULTS

The DHR flow cytometry assay has proved to be arapid and sensitive screening test for CGD. This assaycan differentiate between X-CGD and p47phox-AR-CGDin the majority of patients, on the basis of distinctiveDHR histogram SI and pattern. Vowells et al7 demon-strated that the geometric mean SI of granulocytes fromnormal subjects was 127.9 (range, 85.2 to 264.6), where-as the SIs from patients with CGD with defectivegp91phox and p47phox were 1.3 (range, 0.9 to 2.2) and13.2 (range, 3.5 to 52.1), respectively.4 Fig 1 (left panel,

Abbreviations usedAR: Autosomal recessive

CGD: Chronic granulomatous diseaseDHR: Dihydrorhodamine 123

NADPH: Nicotinamide dinucleotide phosphateNBT: Nitroblue tetrazoliumPMA: Phorbol myristate acetate

SI: Stimulation index

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FIG 1. In the left panel, typical DHR histograms and SIs in a normal subject and in p47phox-AR-CGD, XL-CGDand XL-CGD carriers are shown (top to bottom, respectively). In the right panel, DHR histograms and SIs inpatient 1, patient 1s mother, patient 2, and patient 2s mother are shown (top to bottom, respectively).




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top to bottom) shows typical DHR histograms and SIs innormal, p47phox-AR-CGD, X-CGD, and X-CGD carri-ers. Fig 1 (right panel, top to bottom) shows patient 1, themother of patient 1, patient 2, and the mother of patient2. The 2 patients described in this report had DHR pat-terns resembling p47phox-AR-CGD with SIs of 44.2 and17.1, respectively, plus broad histograms. The DHR pat-tern from the mothers showed bimodal distribution con-sistent with their obligate X-CGD carrier status. Thismosaic pattern in the mothers is characterized by abimodal histogram demonstrating one peak (M1) similarto their sons DHR results and a second peak (M2) simi-lar to results seen with control cells. Although the resultsfrom the Vowell report are applicable for most patientswith CGD, there is a subset of X-CGD that shows anoverlapping SI and pattern with p47phox-AR-CGD. Ourexpanded DHR assay database at the NIH now includes75 patients with X-CGD, of whom 13 (17%) have an SIof >4.5. These patients with X-CGD with modest DHRactivity included both patients with and patients withoutgp91phox protein production. In contrast, of the 31patients with AR-CGD who were evaluated, only 2both malehad SIs of 4.5, addi-tional studies are warranted. The simplest means ofestablishing the genotype in this group of patients is todemonstrate mosaicism in a potential female carrier, aswas the case in the 2 cases presented. However, when thisapproach is not informative, protein analysis and/ormutation analysis should be performed. In addition, theferricytochrome-c assay provides additional functionaldata regarding the in vitro production of reactive oxygenspecies. However, it is important to point out that thisassay fails to distinguish between X-CGD and AR-CGD.

In the 2 cases presented, different results wereobserved with additional confirmatory testing. Westernblot analysis from patient 1 revealed a small amount of anormal-sized gp91phox protein (Fig 2); this is unlike whatis seen in most patients with X-CGD, who do not demon-strate any protein. The Western blot also demonstratesdecreased but identifiable p22phox protein. This findingfits with our observation of a large number of gp91phox-deficient patients, who consistently show that a smallamount of p22phox protein can be identified by overde-veloping the gel. The only circumstance in which we seetotal absence of p22phox protein is AR-CGD related todocumented mutations in p22phox. Superoxide produc-tion per 106 neutrophils at 10 and 60 minutes was signif-icantly decreased: 12.6 and 51.9 nmol compared withcontrol values of 35.8 and 151.7 nmol, respectively (rest-ing cells produced 1.1 nmol at both time points), andthese findings were confirmed on repeat evaluation.Western blot analysis from patient 2 revealed a normalamount of a normal-sized gp91phox protein (Fig 2).Superoxide production per 106 neutrophils at 10 and 60minutes was absent: 0.5 and 0.9 nmol compared withcontrol values of 51.2 and 263.1 nmol, respectively (rest-ing cell produced 0.8 nmol at both time points). CYBBmutation analysis for patient 1 showed a point mutationin exon 2, yielding an amino acid change 41 Tyr Aspthat was confirmed by the presence of the same mutationon one X chromosome from his mother (Fig 3). A pointmutation in exon 13 was identified for patient 2, result-ing in 537 Cys Arg (data not shown).

DISCUSSION

The most common form of X-CGD is caused by largedeletions, nonsense mutations, and splice-junction muta-tions in the CYBB gene, leading to an absence ofgp91phox protein (X910).1 Fewer than 10% of X-CGD

FIG 2. Western immunoblot analysis measuring gp 91phox, p67phox, p47phox, and p22phox protein in the granu-locyte fractions of patient 1 (left panel) and patient 2 (right panel) compared with control subjects and a patientwith typical XL-CGD.

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cases have a gp91phox protein level ranging from 1% to25% of normal (X91).1 X91 phagocytes typically gen-erate reduced but measurable amounts of superoxide. Insome X-CGD cases, normal amounts of nonfunctionalgp91phox protein are present (X91+). The X91 and X91+forms are both due to a missense mutations or smallinframe deletions.1,11 These patients often have variableclinical symptoms and might present at an older age. Thephenotype of patient 1 is most compatible with X91with decreased quantity of gp91phox protein expressionbut, interestingly, with approximately one third of controlsuperoxide production. The clinical history in this patientis puzzling, in that this relatively high level of in vitrosuperoxide activity appears to be insufficient for normalhost defense. CYBB mutation analysis demonstrated amutation in exon 2 on the N-terminal domain. Thisdomain serves multiple functions, including binding ofgp91phox protein in the membrane, interaction withp22phox protein, and binding of the heme groups.12 Thereare a number of potential explanations for diminishedgp91phox protein in this patient, including decreased sta-bility or impaired folding of the protein as well asimpaired membrane integration.11 However, we couldnot find decreased DHR activity or intracellular stainingfor gp91phox protein when the patients blood was heldovernight for additional testing. Thus, the functionalbasis of the defect for patient 1 remains to be defined.

The phenotype of patient 2 is most compatible withX91+, given the normal quantity of gp91phox proteinexpression and the absence of superoxide production.Interestingly, despite the lack of superoxide generation,there must be electron leakage that reduces DHR, yield-ing an increased SI compared with the typical X-CGDDHR histogram. This patients mutation is in exon 13within the NADPH-binding domain. Mutations in thisdomain previously have been reported to cause the X91+phenotype as a result of decreased NADPH binding or

lack of interaction with the cytosolic NADPH-oxidasecomponents.12,13 This probably is the explanation for thenormal quantity of gp91phox protein observed in theabsence of superoxide production. We noted previouslythat granulocytes from several other patients with X-CGDevaluated in our laboratory were found to have a DHRpattern typical for p47phox-AR-CGD, and their cells werealso observed to express some gp91phox protein. Howev-er, we have also seen patients with X91 CGD whosegranulocytes have modest DHR activity typical for AR-CGD. This suggests that some electron flow to DHRoccurs in the absence of NADPH oxidase activity, but thenature of the electron flow to DHR in the absence ofsuperoxide production is not known. Furthermore, thefact that the 2 patients had similar DHR patterns butshowed different results with the ferricytochrome-c assaypoints out that these 2 assays measure different end prod-ucts. In addition, these findings indicate that even patientswith CGD who have some level of in vitro superoxideproduction, as reflected by the ferricytochrome-c assay,might have recurrent severe infections.

We conclude that the majority (83%) of patients withX-CGD show a unique DHR assay pattern that is diag-nostic for this genotype. The remainder (17%) of patientswith X-CGD have a DHR assay histogram that overlapswith that of p47phox-AR-CGD; they require additionaltesting to identify the genotype. Evaluation of a malepatient with modest DHR activity (SI, >4.5) shouldinclude DHR assay of obligate and/or potential carriersto establish mosaicism. If this is unrevealing, it might benecessary to refer the patient to a specialized center capa-ble of specialized protein and CYBB mutation analysis.

We thank Pablo Patino for his excellent technical assistance.

Nucleotide sequenceThe nucleotide sequence data used in this report have been sub-

mitted to GenBank with the accession number AF 469757-69.

FIG 3. CYBB mutation analysis in patient 1 revealed a point mutation T G in exon 2 at nucleotide 133(nucleotide number in accordance with cDNA sequence described by Orkin,10 in which the start of transla-tion is +1 and the A of the ATG start codon is 13; see full genomic DNA sequence in GenBank files AF469757-69). This results in alteration of amino acid 41 Tyr Asp. Patients mothers sequence showed het-erozygosity in this mutation.




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