Screening for Wiskott-Aldrich syndrome by flow cytometry

Abstract

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disease commonly associated with symptoms of immune deficiency. A diagnosis of WAS is suspected on the basis of clinical parameters including thrombocytopenia, eczema, recurring infections, and autoimmunity.1Candotti F. Clinical manifestations and pathophysiological mechanisms of the Wiskott-Aldrich syndrome.J Clin Immunol. 2018; 38: 13-27Crossref PubMed Scopus (104) Google Scholar WAS protein (WASP) is exclusively expressed in hematopoietic lineages. Lowered levels are thought to be a feature of WAS.2Jin Y. Mazza C. Christie J.R. Giliani S. Fiorini M. Mella P. et al.Mutations of the Wiskott-Aldrich syndrome protein (WASP): hotspots, effect on transcription, and translation and phenotype/genotype correlation.Blood. 2004; 104: 4010-4019Crossref PubMed Scopus (260) Google Scholar However, the diagnostic accuracy of flow cytometric WASP measurement has not been determined, and optimal diagnostic cutoff values are lacking. We therefore reviewed results from 107 individuals with suspected WAS to calculate the diagnostic accuracy of a rapid whole-blood flow cytometry–based assay and found it reliable for predicting mutations in WAS. WASP expression levels were determined in lymphocytes gated by CD45 versus side light scatter. A range of WASP protein expression was seen, from normal in healthy controls to those with reduced or poor WASP expression (Fig 1, A; see Fig E1, A-C, in this article's Online Repository at www.jacionline.org). Detailed methods can be found in this article's Online Repository at www.jacionline.org. The age at WASP staining ranged from 1 month to 32 years with a median of 1 year. All 58 persons with normal WAS sequence displayed WASP expression at levels comparable to those in healthy controls (mean staining ratio, 0.94 ± 0.15) while 41 of 49 patients with a genetic abnormality were found with reduced staining ratios (mean staining ratio, 0.34 ± 0.28) (Mann-Whitney nonparametric test P < .0001). We further divided the patients with genetic abnormalities into 5 categories, that is, missense (n = 30), insertion (n = 4), deletion (n = 8), gross deletion (n = 4), and nonsense (n = 3) (see Fig E2, A, in this article's Online Repository at www.jacionline.org). Table E1 in this article's Online Repository at www.jacionline.org lists all patients in this study. Every patient with gross deletions had poor WASP expression. Eight patients from the other mutation categories had normal WASP staining despite having a variant in WAS. Three of these patients carried variants of unknown clinical significance (VUCS). Interestingly, the c.538C>A(p.H180N) variant (staining ratio, 0.98) has been implicated in patients with X-linked thrombocytopenia (SCV000053274).3Landrum M.J. Lee J.M. Benson M. Brown G. Chao C. Chitipiralla S. et al.ClinVar: public archive of interpretations of clinically relevant variants.Nucleic Acids Res. 2016; 44: D862-D868Crossref PubMed Scopus (1521) Google Scholar It was not possible to rule out mosaicism or the influence of maternal cells with the available data; however, no patient included here was observed to have bimodal WASP staining. In addition, of the 8 patients with normal WASP expression, 3 mutations were intronic and 3 located at the C-terminus. Because the WASP antibody recognizes an epitope in the region between aa.146-265, it may still bind proteins bearing missense or C-terminal–truncating mutations.4Kawai S. Minegishi M. Ohashi Y. Sasahara Y. Kumaki S. Konno T. et al.Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations.J Immunol Methods. 2002; 260: 195-205Crossref PubMed Scopus (36) Google Scholar Six patients with missense mutations displayed staining ratios between 0.3 and 0.5. This “intermediate” staining ratio was not observed in other mutation groups, possibly due to low sample numbers. All missense mutations were “probably damaging” on PolyPhen-2 except c.995T>C (p.V332A) (0.0, benign), a known VUCS with a 0.84 staining ratio. Fold stability change (ΔΔG) predictions reported a range of slightly stabilizing to highly destabilizing effects (Fig 1, B).5Quan L. Lv Q. Zhang Y.S.T.R.U.M. structure-based prediction of protein stability changes upon single-point mutation.Bioinformatics. 2016; 32: 2936-2946Crossref PubMed Scopus (200) Google Scholar, 6Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. et al.A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref PubMed Scopus (5307) Google Scholar Certain damaging mutations might not influence the antibody epitope, leading to only slightly reduced antibody staining. Mutations that destabilize the protein could lead to degradation and poor detection, whereas stabilizing mutations could hinder protein function yet display normal levels of WASP staining. Highly accurate and reproducible staining is crucial for establishing diagnostic cutoff estimation. To evaluate staining consistency, we compared patients bearing similar WAS mutations. Here, the c.167C>T (p.A56V) mutation was found in 4 patients, c.356G>A (p.G119E) in 3, and c.559(+5) in 5. Results exhibited good reproducibility (see Fig E2, B). WAS scores were available for 28 of the 49 confirmed WAS cases (Table E1). Scores did not strongly correlate to WASP staining index or age of presentation (Fig 1, C; Fig E2, C and D).7Zhu Q. Zhang M. Blaese R.M. Derry J.M. Junker A. Francke U. et al.The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene.Blood. 1995; 86: 3797-3804PubMed Google Scholar, 8Buchbinder D. Nugent D.J. Fillipovich A.H. Wiskott-Aldrich syndrome: diagnosis, current management, and emerging treatments.Appl Clin Genet. 2014; 7: 55-66Crossref PubMed Scopus (79) Google Scholar Interestingly, patients with autoimmunity (WAS score 5) were all low WASP expressers. No malignancies were reported in any of the 28 patients with available scores. From the receiver-operating characteristic curve, when all entries were considered, a cutoff staining ratio of 0.60 returned a sensitivity and specificity of 82.6% and 100%, respectively. Removing 3 VUCS entries improved the sensitivity and specificity to 89.1% and 100%, respectively (Fig 1, D). By removing VUCS entries, the overall accuracy of the test improved from 92.3% to 95.2%, validating the assay ability in predicting WAS genetic abnormalities. All patients with reduced WASP expression carried a WAS variant but patients suspicious of WAS expressing normal WASP should not be excluded from WAS sequencing because 8 of 49 patients (16.3%) with WAS variants expressed normal WASP. Normal WASP level is thus insufficient to rule out WAS. WASP quantification is also useful for lineage-specific donor chimerism monitoring following allogeneic hematopoietic cell transplantation. Percent total blood genetic chimerism correlated with specific leukocyte subset WASP chimerism in 2 patients studied longitudinally (Fig 1, E; Fig E2, E; see Table E2 in this article's Online Repository at www.jacionline.org). For each patient, sorting lymphocyte subsets before genotyping yielded very comparable specific subset genotype-phenotype results (Fig E2, F). Future efforts to improve the accuracy of WAS screening diagnostics could include the development of a functional assay demonstrating defective actin remodeling during lymphocyte polarization.9Houmadi R. Guipouy D. Rey-Barroso J. Vasconcelos Z. Cornet J. Manghi M. et al.The Wiskott-Aldrich syndrome protein contributes to the assembly of the LFA-1 nanocluster belt at the lytic synapse.Cell Rep. 2018; 22: 979-991Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Such an assay might potentially also be useful in diagnosing X-linked thrombocytopenia and X-linked neutropenia, and would be especially useful for the evaluation of potential patients with WAS with novel mutations of unknown significance with unperturbed WASP staining. Patients with residual WASP function may be associated with milder clinical phenotypes, and a functional assay that could demonstrate lowered but not defective function in this population may allow disease risk stratification as well as diagnosis via one assay. In summary, we demonstrated the efficacy of a whole-blood flow cytometry–based staining protocol as a tool to quickly diagnose patients with WAS with up to 89% sensitivity and 100% specificity. However, WAS diagnosis cannot be excluded in clinically suspicious cases when WASP is present. Inclusion of clinical markers and other possible functional assays in a combined algorithm could improve the overall accuracy for diagnosing WAS. Institutional review board approval was obtained for this study. All reported results of WASP testing performed at the Diagnostic Immunology Laboratory at Cincinnati Children's Hospital between June 2010 and October 2016 were retrieved. After removing incomplete or repeated entries, entries with a bimodal staining pattern in which mean channel fluorescence (MCF) cannot be correctly determined, and selecting only for entries with completed Sanger sequencing of WAS, 49 samples with genetically confirmed WAS mutations and 58 samples with no apparent mutation in WAS were collected. From these 107 samples, analyses of diagnostic accuracy were performed. All but 1 sample with a mutation in WAS were male. The only female sample was a heterozygous carrier with normal levels of WASP staining and therefore included in the control group for analysis. Whole-blood samples from patients and healthy controls were surface stained for CD45 (BD Biosciences, La Jolla, Calif), fixed, and then permeabilized before being incubated with anti-WASP antibody (BD Biosciences, clone 5A5).E1Kawai S. Minegishi M. Ohashi Y. Sasahara Y. Kumaki S. Konno T. et al.Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations.J Immunol Methods. 2002; 260: 195-205Crossref PubMed Scopus (42) Google Scholar Cells were acquired on a FACS Canto II (BD Biosciences) and analyzed with FCS Express (v4, De Novo Software, Glendale, Calif). Results were expressed as the gated lymphocyte arithmetic MCF ratio of WASP from the patient against the lymphocyte MCF of WASP from a freshly drawn healthy control. In samples analyzed for posttransplant engraftment screening, additional antibodies against CD3 (clone SK7), CD14 (MφP9), CD19 (SJ25C1), and CD56 (MY31) were added into the surface staining step. For this assay, results were expressed as the percentage WASP-positive cells for each leukocyte subpopulation. Genomic DNA from peripheral whole blood was isolated followed by PCR-based amplification and bidirectional Sanger sequencing (Applied Biosystems, Foster City, Calif). Exons including intron boundaries of the WAS transcript NM_000377.1 were captured and sequenced. Mutations in untranslated exons, regulatory, and further into introns were not sequenced. Large deletions involving 1 or more whole exons were detected using comparative genomic hybridization array using custom 180K array (Agilent Technologies, Inc, Santa Clara, Calif) and CytoSure (Oxford Gene Technology, Oxfordshire, United Kingdom). Single-nucleotide substitutions, small deletions, and insertions were analyzed using Sequencher software (GeneCodes, Ann Arbor, Mich). Variants were mapped to human genome reference sequence (hg19). Where variants fell below 1% occurrence in frequency, they were analyzed with gnomAD database (gnomad.broadinstitute.org), SIFT, MutationTaster, and PolyPhen-2 in silico predictions.E2Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nat Protoc. 2009; 4: 1073-1081Crossref PubMed Scopus (5006) Google Scholar, E3Schwarz J.M. Cooper D.N. Schuelke M. Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age.Nat Methods. 2014; 11: 361-362Crossref PubMed Scopus (2419) Google Scholar, E4Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. et al.A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref PubMed Scopus (9288) Google Scholar Finally, all variants were classified into pathogenic, benign, or uncertain clinical significance (VUCS) on the basis of published evidence of function in literature of the specific variants and American College of Medical Genetics guidelines.E5Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. et al.Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (14616) Google Scholar In silico prediction were not concordant, suggesting the need for improvements. Fig E2Further characterization of the WAS cohort. A, WASP staining index of 107 patients divided by genetic findings. B, Reproducible staining results when comparing patients with similar mutations. C and D, Poor association seen between WASP expression, WAS clinical score, and age of presentation. E, Posttransplant patient studied longitudinally showing correlated blood genetic chimerism and WASP levels. F, Isolated lymphocyte subsets show similar levels of genetic chimerism and WASP expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Graphs, receiver operating characteristic curves, and accuracy calculations were created using Prism (v7, GraphPad, La Jolla, Calif). Optimal cutoff value was determined by the point on the receiver operating characteristic curve with the largest sensitivity + specificity combined value. Table E1WASP staining, WAS genetic evaluation, and WAS scores of patients included in this studyNo.WASP stain indexPresentationageVariantreportedPMIDdbSNPfrequency (%)genomADSIFTMutationTaster scorePolyPhen-2WAS scoreLowest platelet count ± 6 mo of WASP samplingEczemaInfectionsAutoimmunediseaseMissense/intronic 10.2810 moc.134C>Tp.(Thr45Met)7753869,19817875,2316046925525159rs132630273NA0.01Disease causing128 K/mcLMildMinorNone 20.28NAc.167C>Tp.(Ala56Val)7795648,19817875,25091438rs132630269NA0.24Disease causing1NA 30.389 yc.167C>Tp.(Ala56Val)286 K/mcLMildNoneNone 40.24NAc.167C>Tp.(Ala56Val)NA 50.386 yc.167C>Tp.(Ala56Val)330 K/mcLPersistent but manageableMinorNone 60.16NAc.193A>Cp.(Thr65Pro)NANANA0.14Polymorphism0.990NA 70.33NAc.229G>Ap.(Asp77Asn)NANANA0Disease causing0.994NA 80.40NAc.229G>Ap.(Asp77Asn)NA 90.32NAc.231T>Ap.(Asp77Glu)NANANA0Disease causing0.999NA 100.131 wkc.245C>Ap.(Ser82Tyr)NANANA0Disease causing1137 K/mcLNoneNoneNone 110.192 yc.257G>Ap.(Arg86His)8069912,19817875,23160469,rs132630268NA0.01Disease causing119 K/mcLPersistent but manageableNoneHSP 120.13NAc.319T>Ap.(Tyr107Asn)NANANA0.1Polymorphism1NA 130.183 moc.356G>Ap.(Gly119Glu)8931701,19817875NANA0.34Disease causing1533 K/mcLMildRecurringITP 140.191 moc.356G>Ap.(Gly119Glu)525 K/mcLMildRecurringITP 150.171 moc.356G>Ap.(Gly119Glu)532 K/mcLMildRecurringITP 160.181 wkc.383T>Cp.(Phe128Ser)8931701NANA0.01Disease causing1351 K/mcLPersistent but manageableRecurringNone 170.19NAc.397G>Ap.(Glu133Lys)7753869,19817875NANA0.04Disease causing1NA 180.83NAc.463+3G>Cp.?NANA0.0019Predicted loss at donor site 3bp upstream (34.8%)NA 190.176 moc.506-1G>Cp.?Skip of exon 6 is likely, MaxEnt, NNSPLICE, and HSF algorithms predict loss of acceptor site (100%)280 K/mcLMildRecurringNone 200.98NAc.538C>Ap.(His180Asn)20173115,24728327NA0.095NANANA2180 K/mcLNoneMinorNone 210.175 moc.559+5G>Cp.?NAClinVar -Likely pathogenicNANANA221 K/mcLMildNoneNone 220.204 moc.559+5G>Cp.?212 K/mcLPersistent but manageableRecurringNone 230.261 moc.559+5G>Ap.?9326235,25525159ClinVar -Likely pathogenicNANANA545 K/mcLMildMinorITP 240.18NAc.559+5G>Ap.?NA 250.1410 moc.559+5G>Ap.?534 K/mcLMildMinorITP 260.20NAc.778-2A>Gp.?8595430NANANANANANA 270.84NAc.995T>Cp.(Val332Ala)17400488,22995991,24728327,24369837,rs27377990.470.4Polymorphism0NA 281.18NAc.1315C>Tp.(Arg439Trp)NANANA0.01Disease causing1NA 290.50NAc.1339-2A>Gp.?Skip of exon 11 is likely. MaxEnt, NNSPLICE, and HSF algorithms predict loss of acceptor site (100%)314 K/mcLPersistent but manageableNoneNone 300.25NAc.1453G>Ap.(Asp485Asn)11298372,19817875,RCV000482823.10Disease causing1NAGross deletion 310.133 moPartial gene hemizygous deletion Exon 1-Intron 1 by array CGH arr[hg19] Xp11.23(48,533,049-48,542,539)x0520 K/mcLNoneRecurringAIHA 320.15NAFull gene hemizygous deletion Exon 1-Exon 12. Confirmed by array CGH arr[hg19] Xp11.23(48,533,302-48,613,923)x0515 K/mcLDifficult to controlRecurringITP 330.19NAFull gene hemizygous deletion Exon 1-Exon 12. Confirmed by array CGH arr[hg19] Xp11.23(48,533,302-48,613,923)x0NA 340.202 moPresumed whole gene deletion.22 K/mcLNoneMinorNoneInsertion 350.93NAc. 273(+11_+12) ins Cp.?rs7827937220.46NANANANA 360.24NAc.827_828 ins22p.?NANANANANANA240 K/mcLMildMinorNone 370.18NAc.1271dupp.(Leu425Profs*70)8528198NANANANANANA 380.84NAc.1434_1436dupp.(Ser478dup)NANANANANANADeletion 390.144 moc.101delGp.(Arg34Hisfs*11)NANANANANANA323 K/mcLPersistent but manageableNoneNone 400.164 moc.107_108delTTp.(Phe36X)NANANANANANA51 K/mcLPersistent but manageableMinorNone 410.201 wkc.336 delCp.(Phe113Serfs*14)15284122NANANANANA216 K/mcLMildNoneNone 420.143 moc.391_407del17p.(Glu131Leufs*32)NANANANANANA517 K/cmLPersistent but manageableMinorITP, colitis 431.15 moc.777+3_777+6delGAGT, p.?10737997NANANANANA25 K/mcLMildMinorNone 440.16NAc.852delCp.(Glu285Argfs*23)NANANANANANANA 450.244 moc.1114_1115delp.(Pro372Serfs*122)NANANANANANA56 K/mcLNoneLife threateningITP, IAHA 460.20NAc.1271delGp.(Gly424Alafs*21)7753869NANANANANANANonsense 470.171 wkc.607C>Tp.(Q203*)8682510NANANANANA545 K/mcLMildMinorAutoimmune neutropenia 480.14NAc.862A>Tp.(Lys288*)21488158,25525159NANANANANANA 490.805 moc.1468C>Tp.(Gln490*)NANANANANANA318 K/mcLPersistent but manageableMinorNoneSIFT score [0-1]: Deleterious score ≤0.05, Tolerated >0.05.PolyPhen-2 score [0-1]: Benign, 0-0.15; Probably damaging, 0.85-1.dbSNP, Single Nucleotide Polymorphism database; genomAD, Genome Aggregation Database; NA, not available; SIFT, Sorting Intolerant from Tolerant. Open table in a new tab Table E2Genetic chimerism and WASP-positive leukocyte subset staining in transplanted patientsTransplant patient no.Sample no.(chronological)Overall donor genetic chimerismPercentage of WASP-positive cells per subsetT cellNK cellsB cellsMonocytes11869487456226096762131341767313274479474192259999100999961009910010010079898100991002140664654262546847572736073516332465785868325617949683866680587547769816072458717959733697284577538107082617437115982627231126486637819 Open table in a new tab SIFT score [0-1]: Deleterious score ≤0.05, Tolerated >0.05. PolyPhen-2 score [0-1]: Benign, 0-0.15; Probably damaging, 0.85-1. dbSNP, Single Nucleotide Polymorphism database; genomAD, Genome Aggregation Database; NA, not available; SIFT, Sorting Intolerant from Tolerant.