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Newborn Screening for SCID. Francisco A. Bonilla, MD, pHd , FAAAAI BOSTON CHILDREN’S HOSPITAL HARVARD MEDICAL SCHOOL. Disclosures. Albany Medical College Honorarium Baxter, Inc. Consultant The Cowen Group, Inc. Consultant CSL Behring, Inc. Consultant, research support
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Newborn Screening for SCID Francisco A. Bonilla, MD, pHd, FAAAAI BOSTON CHILDREN’S HOSPITAL HARVARD MEDICAL SCHOOL
Disclosures • Albany Medical College • Honorarium • Baxter, Inc. • Consultant • The Cowen Group, Inc. • Consultant • CSL Behring, Inc. • Consultant, research support • Gerson-Lehrman Group, Inc. • Consultant • Grand Rounds Health • Consultant • Green Cross / American Research Group, Inc. • Data safety monitor • Immune Deficiency Foundation • Medical Advisory Board • Consultant Immunologist Program • Octapharma, Inc. • Data safety monitor • UpToDatein Medicine • Royalties
The immune system Cellular Humoral Phagocytic cells Complement NK cells Innate Adaptive T cells Antibody (B cells)
TREC and KREC • T cell receptor • Recombination • Excision • Circle • Kappa chain • Recombination • Excision • Circle
T cell receptor recombinationexcision circle (TREC) Ponchel et al. BMC Biotechnology2003; 3:18
TRECs in T cell development Thymus Recent thymic emigrants Peripheral expansion
TRECs and thymic output with age Douek et al., Nature 1998; 396:690.
SCID • Failure to thrive, chronic diarrhea • Disseminated infections with low-grade pathogens • Opportunistic infections, “non pathogens” • Alymphocytosis/lymphopenia • Very low T cell function
Absolute Lymphocyte Count Distributions in SCID (25 newborns with SCID and 14 healthy newborns at birth) Kalman L, Lindegren ML, Kobrynski L, Vogt R, Hannon H, Howard JT, Buckley R. Genet Med. 2004 Jan-Feb;6(1):16-26.
T cell receptor recombinationexcision circle (TREC) Ponchel et al. BMC Biotechnology2003; 3:18
Guthrie card 3 mm punch, elute DNA Quantitative PCR, measure TREC Compare TREC number to standard curve and amplification of “housekeeping” gene (quality control) Low TREC = T cell lymphopenia (decreased thymic output) Newborn screening using TRECs
Distribution of TREC and RNAseP copy number (copies/μL blood) in the non-NICU populations • Fail RNAse P quality control, request new specimen • TRECs ≥ 252 and pass quality control = screen negative • TRECs < 252 and pass quality control = screen positive, send specimen for flow cytometry • T cells < 2,500/mm3 or naïve CD4 or CD8 < 50%, possible SCID, T cell functional test • Any abnormality on flow, follow as indicated Gerstel-Thompson et al., ClinChem2010; 56:1466.
Evaluation protocol in Massachusetts Flow Cytometry: CD3, CD4, CD8, CD16/56, CD19 naïve/memory T cell panel Clinic Referral for functional testing if: total T cell number <2500/mL % naïve T cells <50% any other abnormality Genotyping/Karyotyping if: Suspected SCID Suspected DiGeorge, other Referral to Transplant Center for: SCID
Case definition • SCID • (<300 autologous T cells/mL AND PHA <10% of lower range of normal) • Leaky SCID/Omenn syndrome • (300-1500 autologous T cells/mL, no maternal T cells, • PHA 10-30% of lower range of normal) • Variant SCID • (300-1500 autologous T cells/mL with impaired function, • no maternal cells, no genotype known • Syndromes with T cell impairment • (DiGeorge syndrome, CHARGE, CHH, Jacobsen, Down, etc.) • Secondary T cell lymphopenia • (heart disease, chylothorax, etc.) • Preterm Dvorak et al. J ClinImmunol 2013; 33:1156
T cell memory panel CD4 CD8 CD45RA CCR7
Recent thymic emigrant panel CD4 cells only CD45RA CD31
Proliferation assay Culture PBMCs with mitogen Add 3H thymidine Harvest cells on filter disk Measure radioactivity
Stimuli • Mitogens – plant lectins, stimulate 50-80% of T cells • Phytohemagglutinin • Concanavalin A • Pokeweed mitogen • Antigens – precursor frequency about 10-5-10-3 • Tetanus toxoid • Diphtheria toxoid
Proliferation • Positive to both mitogens and antigens – fully functional • Negative to both mitogens and antigens – fully non-functional • Positive mitogen response with negative antigen response – partially non-functional • Positive antigen response with negative mitogen response – does not occur • Antigen test is more sensitive • Mitogen test is more specific
11 states, DIFFERENT PROTOCOLS IN DIFFERENT STATES 3,030,083 infants screened Overall, 41.8:100,000 = 1:2,392 = 0.04% of pts. screened referred for flow cytometry (total 1,265) 411 cases of non-SCID T cell lymphopenia (32.5%) 52 cases SCID/leaky SCID/Omenn syndrome (4.1%) Incidence is 1:58,000 (95% CI 1:46,000-80,000) Overall survival 87% (45/49 = 92% of infants receiving SCT, gene therapy or enzyme replacement0 SCID NBS: national experience Kwan etal. JAMA 2014; 312:729.
SCID and leaky SCID/Omenn, n=52 Kwan etal. JAMA 2014; 312:729.
2 – busulfan toxicity (RAG1, 1 – diaphragm defect (Tet. 12p, Pallister-Killian syndrome) 1 – post transplant CMV present at diagnosis (unknown defect) 1 – not treated, severe congenital anomalies (unknown defect) 1 – post transplant respiratory illness present at diagnosis (genetic testing incomplete) 1 – not treated, died before transplant (genetic testing incomplete) SCID/leaky SCID/Omenn deaths Kwan etal. JAMA 2014; 312:729.
Primary T cell lymphopenia, n=136 Kwan etal. JAMA 2014; 312:729.
Secondary T lymphopenia, n=117 Kwan etal. JAMA 2014; 312:729.
Delayed diagnosis of SCID is costly – transplantation in the first months of life has lower cost (1/3, -$240,000) and improved outcomes Cost of screening test $4.22 Assuming incidence of 1:75,000, remains cost effective up to per test cost of $15.00 True incidence closer to 1:50,000, cost effective up to $20.00 Cost effectiveness of SCID screening Chan et al., Mol Genet Metab2011; 104:383
Challenges of SCID newborn screening • Harmonization of methods • Case definition • Validation of diagnosis • Clinical care network • Diagnostic dilemmas • Is there a role also for KRECs?
Validation of diagnosis • Newborn screening for SCID is a 2-tier process • Second tier must be based on immunological tests • with extremely high sensitivity and excellent specificity • These tests must be readily available and must be • supported by robust interpretation and counseling • States that have implemented newborn screening • for SCID do not use yet a common set of assays • to validate the diagnosis • Different formats are currently used for counseling
TREC is a marker of thymus function Cost effective and efficient for high volume Not cost effective and efficient for low volume Flow cytometry is more common for measure of thymus function for PIDD diagnosis Should be assessed whenever T cell dysfunction is suspected TRECs in immunodeficiency diagnosis
KRECs in B cell development Bone marrow Naïve B cells Peripheral expansion: only occurs during antibody response
Marker of B cell development Completely analogous to TREC KRECs absent in infants with agammaglobulinemia Classification of CVID? (other antibody deficiencies?) KRECs
TRECs and KRECs in CVID C A B D Kamae et al. J Allergy ClinImmunol 2013;131:1437
TRECs and KRECs in CVID Kamae et al. J Allergy ClinImmunol 2013;131:1437
TREC screening for SCID is a BREAKTHROUGH OF MONUMENTAL PROPORTIONS • New newborn screen, highly efficient, cost effective • Multiplex PCR • KREC, what’s next? • TREC/KREC could become part of diagnosis/management Summary and conclusions