Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots.
BACKGROUND
Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program.


OBJECTIVE
This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening.


METHODS
DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available.


RESULTS
Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 μmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal.


CONCLUSIONS
TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail.
Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children's Hospital, Florence, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Florence, Italy.
Giancarlo
G
la Marca
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy.
Clementina
C
Canessa
Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children's Hospital, Florence, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Florence, Italy.
Elisa
E
Giocaliere
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy.
Francesca
F
Romano
Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children's Hospital, Florence, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Florence, Italy.
Sabrina
S
Malvagia
Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children's Hospital, Florence, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Florence, Italy.
Silvia
S
Funghini
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy.
Maria
M
Moriondo
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy.
Claudia
C
Valleriani
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy.
Francesca
F
Lippi
Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children's Hospital, Florence, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Florence, Italy.
Daniela
D
Ombrone
Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children's Hospital, Florence, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Florence, Italy.
Maria Luisa
ML
Della Bona
Zentrum fur Kinderheilkunde und Jugendmedizin, Centrum fuer Chronische, Immundefizienz-Universitaet Freiburg, Freiburg, Germany.
Carsten
C
Speckmann
Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany.
Stephan
S
Borte
Department of Allergy and Immunology, Children's Hospital Lund, Skane University Hospital, Lund, Sweden.
Nicholas
N
Brodszki
Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.
Andrew R
AR
Gennery
Division of Hematology and Oncology, Children's Hospital, Boston, Mass; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Mass.
Katja
K
Weinacht
Antalya Education and Research Hospital, Pediatric Immunology and Allergy, Antalya, Turkey.
Fatih
F
Celmeli
Pädiatrische Hämatologie und Onkologie, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck, Germany; Department of General Pediatrics, University Children's Hospital Muenster, Münster, Germany.
Julia
J
Pagel
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy.
Maurizio
M
de Martino
Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children's Hospital, Florence, Italy.
Renzo
R
Guerrini
Pediatric Rheumatology and Immunology, University Children's Hospital Muenster, Münster, Germany.
Helmut
H
Wittkowski
Department of Medicine, Duke University, Medical Center, Durham, NC.
Ines
I
Santisteban
Department of Medicine, Duke University, Medical Center, Durham, NC.
Pawan
P
Bali
Department of Pediatric Immunology and Allergy, Ankara University Medical School, Ankara, Turkey.
Aydan
A
Ikinciogullari
Department of Medicine, Duke University, Medical Center, Durham, NC.
Michael
M
Hershfield
Division of Immunology, Children's Hospital, Boston, Mass.
Luigi D
LD
Notarangelo
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy.
Massimo
M
Resti
Meyer Children's University Hospital, Florence, Italy; Department of Health Sciences, University of Florence, Florence, Italy. Electronic address: chiara.azzari@unifi.it.
Chiara
C
Azzari
D000293:Adolescent; D002675:Child, Preschool; D004260:DNA Repair; D003849:Deoxyguanosine / Q000032:analysis / Q000378:metabolism; D059788:Dried Blood Spot Testing; D005260:Female; D006151:Guanosine / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D007223:Infant; D007231:Infant, Newborn; D007288:Inosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D008214:Lymphocytes / Q000473:pathology; D008297:Male; D009154:Mutation; D015997:Neonatal Screening; D000081207:Primary Immunodeficiency Diseases; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency* / Q000235:genetics*; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D053719:Tandem Mass Spectrometry
D006151:Guanosine; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase; D003849:Deoxyguanosine; C012271:deoxyinosine
Purine nucleoside phosphorylase; T-cell receptor excision circle; delayed-onset; inherited disorder; late-onset; newborn screening; purine nucleoside phosphorylase–combined immunodeficiency; severe combined immunodeficiency; tandem mass spectrometry