Carlos A. Saavedra-Matiz, MD NYSDOH-NBS Program DNA Lab NYMAC Albany, NY June 4-5, 2012 David Phillip Vetter (Sept 21, 1971 – Feb 22, 1984)Sept Feb

Immune System (IS) Function: Recognize and eliminate invading foreign organisms and molecules Peripheral Blood Lympochytes (IR or immune response): T Cells: Undergo differentiation in the thymus. Cellular IR B Cells: Undergo differentiation in the bone marrow. Humoral IR Lymphatic Organs: Primary: Thymus and bone marrow Secondary: Lymph nodes and spleen “An extremely efficient, genetically complex IS has evolved. Its development and function are regulated by genes of extraordinary diversity.” - Antibodies (Immunoglobulines) - Antigen-antibody binding - Antigen Receptors (TCR)

Multiple genotypes of SCID Gene defect Molecular pathogenesis % SCID cases * * T cells † † B cells Natural killer cells IL2RG (X-linked) Failure of IL-2 receptor (common) γ-chain signaling by cytokines IL-2, -4, - 7, -9, -15, Low/ absent PresentAbsent ADAAdenosine deaminase deficiency Low/ absent Absent IL7RFailure of signaling through IL-7 receptor Low/ absent Present JAK3Failure of Janus kinase 3 activation by common γ-chain Low/ absent PresentAbsent DCLRE1C ‡ ‡ Failure of antigen receptor rearrangement <5Absent Present RAG1 ‡ ‡ Failure of antigen receptor rearrangement <2Absent Present RAG2 ‡ ‡ Failure of antigen receptor rearrangement <2Absent Present CD45 Lack of a cell surface protein tyrosine phosphatase receptor (PTPRC), required for T- and B-cell activation by antigen 20 and Rare Low/ absent Present Low/ variable TCRDT-cell receptor δ chain deficiency Very rare Low/ absent Present LCK Lack of lymphocyte tyrosine kinase p56lck, required for T-cell development and activation Very rare Low/ absent Present FOXN1 Lack of forkhead box N1transcription factor, required for thymus and hair follicle development (ortholog of nude mouse) Very rare Low/ absent Present Deletion of Chr 22q11 Complete DiGeorge syndrome with thymus aplasia; part of DiGeorge- Velocardiofacial syndrome RareAbsentVariablePresent Currently unknown Unknown, including reticular dysgenesis 26 and congenital anomaly syndromes with SCID 27 and Each one rare Low/ absent Variable

SAMPLES ARE PUNCHED INTO 96-WELL PLATES Wadsworth Center

Duplex Amplification Plots TREC Amplification plotRNase P amplification plot ADA

From Sept 29, 2010 to April 30, 2012: 382,406 infants screened No false negative cases

Overall PPV = 18.7% Classic SCID PPV = 2.1%

SCIDLeaky SCID/ Omenn Sy Variant SCID Syndromes with T cell impairment Secondary T cell lymphopenia Pre term alone IL2RG No Known Mut DiGeorge (22q11.2 deletion) Intestinal lymphangiectasia JAK3 CHARGEAnasarca IL7R JacobsenGastroschisis RAG1 RAC2 defectThird-spacing RAG2 DOCK 8 deficiency Gastrointestinal atresia ADA Ataxia telangiectasia Cardiac surgery +/- thymectomy CD45 VACTERL Association Congenital heart defects DCLRE1C Barth SyndromeNeonatal leukemia AK2OtherTAR SyndromeChylothorax OtherDown SyndromeChyloperitoneum Und (IL-7 Signaling Defect > 300 T cell impairmen t Ectrodactilia ectrodermal dysplasia Hypoplastic left heart defect Multiple congenital anomalies Degenerative neuromuscular Presumed metabolic dis.

The Ion Torrent Personal Genome Machine (PGM TM ) sequencer enables researchers to obtain highly accurate sequence in record time.

doi: /nature10242 RESEARCH SUPPLEMENTARY INFORMATION Figure S1 Process overview

GeneGene c.DNA PosRef PosAmplicon SizeRefIon SNPFreq 1Freq 2Counts 1Counts 2Ion CoverageSanger SNP% Concor danceComments MCAD AA/G A/G100 MCAD TT/C T/C100 MCAD CG/C G/C100 MCAD CG G/C50 MCAD GG/A G50Polymorphic region MCAD TT0T/C50 MCAD GA A/G50 MCAD CG G/C50 MCAD AA/C A50Primer Region MCAD TT/A T50Primer Region MCAD AA/G A/G100p.V387V MCAD AA/G A/G100p.K395R GALC TC/T C/T100 GALC GG/C G/C100 GALC GG/C G/C100p.A5P GALC CA/C A/C100p.G9G GALC GA/G G50Polymorphic region GALC CT C0Polymorphic region GALC GG/A G/A100 GALC AA/G A/G100p.T96A GALC AG/A G/A100p.M101V GALC GA/G A/G100 GALC CC/T C/T100 GALC GA/G A/G100p.D232N GALC TT/C T/C100 GALC CT/C T/C100 GALC TC C/T50 GALC CC/T C/T100 GALC GG/A G/A100P.T524T (Cis-M101V) GALC CT/C T/C100 GALC AA/T A/T100p.V550V GALC CC/G C/G100Cis-M101V GALC AG/A G/A100p.A625T (Cis-M101V) VLCAD GA/G A/G100p.M1I VLCAD TG/T G/T100 VLCAD AT A50 VLCAD AC C100? VLCAD CT T/C50 VLCAD GG/A G/A100p.E534K VLCAD TT/C T/C100 PKU TC/T C/T100 PKU CC/T C/T100 PKU AG G100 PKU GA/G A/G100 PKU AA/G A/G100p.Q312Q PKU GG/A G/A100p.V245V PKU GC/G C/G100p.L383L GALT TA A100 GALT GG/T G/T100p.K285N GALT CC/T C/T100p.A320V GALT* AT T100