Bali, P

Borte, S

Buchholz, B

Ehl, S.

Felgentreff, K

Fisch, P

Grimbacher, B

Hershfield, Ms

La Marca, Giancarlo

Neumann, C

Santisteban, I

Sass, Jo

Schlesier, M

Schwarz, K

Speckmann, C

Wiech, E

English

Florence Research

Letter to the EditorDelayed-onset adenosine deaminase defi-ciency: Strategies for an early diagnosisTo the Editor:Adenosine deaminase (ADA) deficiency is a well-known causefor severe combined immunodeficiency (SCID). However,hypomorphic mutations in the ADA gene can lead to a differentimmunodeficiency with variable phenotype including signs of im-mune dysregulation.1,2 Early diagnosis and therapeutic manage-ment often remain a challenge. We report on 2 ADA-deficientpatients with delayed-onset (P1) and late-onset1 phenotype (P2)illustrating these difficulties and describe how newborn screeningcould have facilitated early diagnosis in one of these patients.The clinical and laboratory findings of our patients are sum-marized in Table I. Chronic lymphopenia and elevated IgE levelswere key findings in routine laboratory investigations. Furtheranalysis of lymphocyte subsets identified a T(1)BlowNKlow phe-notype. The T-cell receptor repertoire was diverse.The presence of autoimmune phenomena (Table I) in the contextof panlymphopenia prompted us to determine ADA levels3 thatwere undetectable in dried blood spot punches. Correspondingly,total adenine deoxyribonucleotides levels were elevated. Subse-quent sequencing of the ADA gene revealed compound heterozy-gous mutations in P1 and P2. Both patients carried the sameintron 8 splice donor site mutation (c.78011G>A), while the sec-ondmutationwas c.637G>T (predicting a novelVal213Phe substi-tution in exon 7) in P1 and c.396dupA (p.Val133SerfsX38) in P2.Expression of a cDNA encoding the V213F mutation in an Esche-richia coli system yielded about 1% of wild-type ADA activity,classifying it as a ‘‘group III allele,’’ which is in line with missensemutations found in previous late-onset ADA-deficient patients.1While P1 was lost to follow-up after diagnosis, we initiatedenzyme replacement therapy (ERT) in P2. He achieved immunereconstitution within 4 weeks as determined by the normalizationof cell numbers, T-cell proliferation, and pneumococcal vaccineresponses. A marked drop in ADA activity 3 months later (Fig 1)was due to the presence of neutralizing antibodies. An increase inthe polyethylene glycol–modified ADA dose resulted in sustainednormalization ofADAactivity and continuous systemic detoxifica-tion. The chronic cough of P2 cleared within weeks after the initi-ation of ERT, and former radiological signs of interstitial alveolitisresolved completely as found in a recent computed tomographyscan. Improvement in pulmonary symptoms correlated with thelevel of detoxification rather than with the later occurring immunereconstitution, in line with previous reported observations.4The clinical course of our patients and other individualssummarized in Felgentreff et al2 clearly demonstrate that patientswith delayed-onset and late-onset ADA deficiency suffer signifi-cantly from the damaging consequences of their disease. Thosediagnosed as adults showed severe chronic lung disease and otherserious health problems and have not survived long after diagno-sis.5,6While ERT is a generally accepted treatment in ADA-SCIDto stabilize patients prior to stem cell transplantation, its use in pa-tients with a later onset of the disease is less well documented.7Although initial immune reconstitution can be observed in mostpatients receiving ERT, several develop neutralizing antibodiesagainst the bovine-derived ADA, which can completely reverseimmune recovery.8 Fortunately, in P2 and other patients, it hasbeen possible to overcome this problem by increasing thedose.9 In 2 adult sisters,6 immune recovery was minimal underERTand one of them died because of chronic lung disease. How-ever, more favorable results have been reported in other patients.7The data from P2 are encouraging, also regarding the lung diseaseobserved in some ADA-deficient patients.4 We believe that ERTalso has an important role in patients diagnosed beyond infancy,mainly to prevent the development of comorbidities that increasethe risk for stem cell transplantation.Since therapeutic success relies on the early initiation oftreatment prior to the occurrence of chronic organ damage, weaddressed the question whether the diagnosis of late-onset ADAdeficiency couldbe facilitatedbyusingnewborn screeningmethodsrecently introduced for SCID.10We therefore obtained dried bloodspot punches from the original Guthrie card from P2. Copy num-bers of T-cell receptor excision circles (TRECs) repeatedly provedto bewithin normal limits at birth, even though lymphopenia (1288/mL) was already documented at age 2 months. ADA deficiencywould have therefore been missed in P2 by currently establishedpopulation-based newborn screenings for SCID, which are basedon TREC analysis.10 Notably, the analysis of dried blood spotpunches revealed markedly reduced copy numbers of kappa-deleting recombination excision circles (KRECs)—despite thepatient’s later documented ability toproduce a significant repertoireof specific antibodies. In a screening setting, the phenotype of P2with normal TRECs and reduced KRECs would have rather pre-dicted a B-cell and not a T-cell deficiency.11 In addition, we alsoperformed tandemmass spectrometry for the detection of elevatedadenosine metabolites12 and identified that increased levels of des-oxiadenosine and adenosine were already present at birth.Our data indicate that late-onset ADA deficiency may bemissed when screening is based on TREC analysis only. In ourpatient, KREC analysis was more helpful, indicating that de-creased KREC copy numbers at birth should also raise suspicionof combined immunodeficiencies. Notably, mass spectrometryshowed markedly increased desoxiadenosine and adenosinelevels, indicating that this technique may also be useful innewborn screening to identify leaky ADA variants, particularlysince spectrometry can be performed at a negligible cost.12 Pro-spective analysis in ongoing screening programs will be neces-sary to prove this hypothesis. It must be considered, however,that screening will also identify patients with partial ADA defi-ciency,1 who will not develop symptoms of immunodeficiencyand do not require treatment. Determination of lymphocytecounts, total adenine deoxyribonucleotides in red blood cells,and genetic analysis will be helpful in evaluating the individualprognosis, since there is a strong correlation between ADA gen-otype and both metabolic and clinical phenotype.1Carsten Speckmann, MDa,bCarla Neumann, MDcStephan Borte, MDd,eGiancarlo la Marca, PharmScf,gJ€orn Oliver Sass, Dr.rer.nataElisabeth Wiech, PhDhPaul Fisch, MDhKlaus Schwarz, MDb,iBernd Buchholz, MDjMichael Schlesier, PhDb,kKerstin Felgentreff, MDa,b1TABLE I. Clinical phenoype and laboratory investigations in P1 and P2Clinical phenotype P1 P2Ethnicity Caucasian CaucasianAutoimmunity AIHA, eczema, asthma, colitis AsthmaIntensity of autoimmunnity Severe (AIHA) ModerateOnset of autoimmunity (y) 6 4Infection profile Sinopulmonary Otitis, pulmonaryIntensity of infections Severe ModerateOnset of infections (y) 3 3Radiological findings Bronchiectasis AlveolitisTreatment CSA, steroids, SCIG, antibiotics ERTAge at diagnosis (y) 21 (late-onset as defined in Arredondo-Vega et al1) 7 (delayed-onset as defined in Arredondo-Vega et al1)P1 P2 Normal rangeLaboratory investigationsLymphocytes/mL <500 900 1000-5300Eosinophils (% of leucocytes) 24 8 5T cellsCD31/mL 236 880 800-3500CD41/mL 110 156 300-1400CD81/mL 154 574 200-900%gamma/delta TCR1CD31 <10 59 <10%CD45RA of CD41 9.7 44 48-68B cellsCD191/mL 6 13 100-500Natural killer cellsCD3-CD161CD561/mL 17 20 90-600ImmunoglobulinsIgG (g/L) (prior SCIG) 10.8 7.8 7-16IgA (g/L) 0.95 0.41 0.7-4IgM (g/L) 0.23 0.27 0.4-2.3IgE (kU/L) 11,200 204 <100Specific IgG 1Tetanus 1hb, tetanus, hib, measles, mumps 12Measles 2Diptheria, pertussis, rubella, 1Isohemagglutinins 1 2 1Autoantibodies RBCs, neutrophils PEG-ADA 1ProliferationPHA Low* Low/normal after ERTAnti-CD3/CD28 Low* Low/normal after ERTTetanus Low* NDADA mutation c.637G>T c.396dupA(p.Val213Phe) (p.Val133SerfsX38)c.78011G>A c.78011G>AADA activity (at time of diagnosis)In DBSS (mmol/h/mg protein) 0 0 26.4 6 10In erythrocytes (IU/L erythrocytes) <20 111 250-650RBC nucleotides %dAXP 6.9 7.1 <1Analysis of newborn DBSSTRECs (mL dried blood)11 NA 98 >15KRECs (mL dried blood)11 NA 1.3 >10Desoxyadenosine (mM)12 NA 0.7 <0.07Adenosine (mM)12 NA 10 <1.5Nucleotide numbering according to reference NM_000022.AIHA, Autoimmune hemolytic anemia; CSA, cyclosporine A; dAXP, total adenine deoxyribonucleotides; DBSS, dried blood spot punches; hb, hepatitis B; hib, Haemophilusinfluenzae; NA, not available; ND, not determined; PEG-ADA, polyethylene glycol-modified adenosine deaminase; RBCs, red blood cells; SCIG, subcutaneous immunoglobulinsubstitution; TCR, T-cell receptor.*Determined by thymidine incorporation assay.Determined by carboxyfluorescein diacetate succinimidylester (CFSE) proliferation assay.J ALLERGY CLIN IMMUNOLnnn 20122 LETTER TO THE EDITORBodo Grimbacher, MDb,kInes Santisteban, PhDlPawan Bali, PhDlMichael S. Hershfield, MDlStephan Ehl, MDa,bFrom athe Centre for Pediatrics and Aldolescent Medicine and bthe Centre of ChronicImmunodeficiency, University of Freiburg Medical Centre, Freiburg, Germany; ctheRheumaeinheit, Medizinische Poliklinik, Klinikum der Universit€at M€unchen, Lud-wig-Maximilian Universit€at M€unchen, Munich, Germany; dthe Immuno DeficiencyCenter Leipzig at Hospital St Georg gGmbH Leipzig, Jeffrey Modell Diagnosticand Research Center for Primary Immunodeficiencies, Leipzig, Germany; etheFIG 1. The course of lymphocyte subsets in relation to enzyme activity andmetabolites in P2. Top panel: thecourse of lymphocyte subsets determined by flow cytometry before and after the initiation of ERT. Black lineindicates CD41 cells, spotted gray line CD81 cells, gray line CD191 cells, and gray dashed line CD16/561cells. Bottom panel: ADA activity (black dashed line) and total adenine deoxyribonucleotides (dAXP) levels(gray line) as determined by the analysis of dried blood spots from the time point of diagnosis (210) until 54weeks after the initiation of ERT with polyethylene glycol–modified adenosine deaminase. ‘‘0’’ indicates thetime point when ERT was started. The dose was increased at week 18 as indicated.J ALLERGY CLIN IMMUNOLVOLUME nnn, NUMBER nnLETTER TO THE EDITOR 3Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, Ger-many; fthe Department of Pharmacology, University of Florence, Florence, Italy; gtheMass Spectrometry Lab, Meyer Children’s University Hospital, Florence, Italy; htheInstitute for Pathology, University of Freiburg Medical Centre, Freiburg, Germany;ithe Institute for Transfusion Medicine, University Hospital Ulm and Institute forClinical Transfusion Medicine and Immunogenetics, Ulm, Germany; jthe UniversityChildren’s Hospital, Medical Faculty Mannheim, Heidelberg University, Heidelberg,Germany; kthe Department of Rheumatology and Immunology, University of Frei-burg Medical Centre, Freiburg, Germany; and lthe Department of Medicine, DukeUniversity Medical Center, Durham, NC E-mail: carsten.speckmann@uniklinik-freiburg.de.Supported by the Deutsche Forschungsgemeinschaft (SFB620 TPA4 [to S.E.]), the Ger-man Federal Ministry of Education and Research (BMBF 01 EO 0803), and theBMBF PtJ-Bio 0315883 grant to the Translational Centre for Regenerative Medicine.Disclosure of potential conflicts of interest: M.S.H., I.S., and P.B. received research sup-port from Sigma Tau Pharmaceuticals. The rest of the authors declare that they haveno relevant conflicts of interest.REFERENCES1. Arredondo-Vega FX, Santisteban I, Daniels S, Toutain S, Hershfield MS. Adeno-sine deaminase deficiency: genotype-phenotype correlations based on expressedactivity of 29 mutant alleles. Am J Hum Genet 1998;63:1049-59.2. Felgentreff K, Perez-Becker R, Speckmann C, Schwarz K, Kalwak K, Markelj G,et al. Clinical and immunological manifestations of patients with atypical severecombined immunodeficiency. Clin Immunol 2011;141:73-82.3. Arredondo-Vega FX, Santisteban I, Richard E, Bali P, Koleilat M, Loubser M, et al.Adenosine deaminase deficiency with mosaicism for a ‘‘second-site suppressor’’ ofa splicing mutation: decline in revertant T lymphocytes during enzyme replace-ment therapy. Blood 2002;99:1005-13.4. Somech R, Lai Y, Grunebaum E, Le Saux N, Cutz E, Roifman C. Polyethyleneglycol-modified adenosine deaminase improved lung disease but not liver diseasein partial adenosine deaminase deficiency. JAllergyClin Immunol 2009;124:848-50.5. Ozsahin H, Arredondo-Vega FX, Santisteban I, Fuhrer H, Tuchschmid P, JochumW, et al. Adenosine deaminase deficiency in adults. Blood 1997;89:2849-55.6. Shovlin CL, Simmonds HA, Fairbanks LD, Deacock SJ, Hughes JM, Lechler RI,et al. Adult onset immunodeficiency caused by inherited adenosine deaminasedeficiency. J Immunol (Baltimore, Md: 1950) 1994;153:2331-9.7. Gaspar HB, Aiuti A, Porta F, Candotti F, Hershfield MS, Notarangelo LD. How Itreat ADA deficiency. Blood 2009;114:3524-32.8. Lainka E, Hershfield MS, Santisteban I, Bali P, Seibt A, Neubert J, et al. Polyeth-ylene glycol-conjugated adenosine deaminase (ADA) therapy provides temporaryimmune reconstitution to a child with delayed-onset ADA deficiency. Clin DiagnLab Immunol 2005;12:861-6.9. Chaffee S, Mary A, Stiehm ER, Girault D, Fischer A, Hershfield MS. IgG antibodyresponse to polyethylene glycol-modified adenosine deaminase in patients withadenosine deaminase deficiency. J Clin Invest 1992;89:1643-51.10. Lipstein EA, Vorono S, Browning MF, Green NS, Kemper AR, Knapp AA, et al.Systematic evidence review of newborn screening and treatment of severe com-bined immunodeficiency. Pediatrics 2010;125:e1226-35.11. Borte S, von D€obeln U, Fasth A, Wang N, Janzi M, Winiarski J, et al. Neonatalscreening for severe primary immunodeficiency diseases using high-throughputtriplex real-time PCR. Blood 2012;119:2552-5.12. Azzari C, la Marca G, Resti M. Neonatal screening for severe combined immuno-deficiency caused by an adenosine deaminase defect: a reliable and inexpensivemethod using tandem mass spectrometry. J Allergy Clin Immunol 2011;127:1394-9.doi:10.1016/j.jaci.2012.04.004

Delayed-onset adenosine deaminase deficiency: Strategies for an early diagnosis.

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Delayed-onset adenosine deaminase deficiency: Strategies for an early diagnosis.

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