Typical SCID and Newborn Screening for SCID and T Cell Defects Jennifer Puck, MD [email protected] Department of Pediatrics University of California San Francisco and Benioff Children’s Hospital Smith Cardiovascular Research Institute San Francisco, California Disclosure Spouse employed by InVitae, Inc., a clinical gene sequencing company. NBS Case: term infant, 0 TREC, 473 Actin Low total lymphocytes T- B+ NK+ No naïve T cells Severe Combined Immunodeficiency, SCID, Before Newborn Screening • Rare; many genes. • Healthy at birth, but get recurrent, severe infections, failure to thrive. • High mortality in first year. • Absent or low T cells. • Absent or non-functional B cells. • Possible cure by bone marrow transplant from a healthy matched donor (first in 1968). David Veer (1971-1984), Texas Children’s Hospital h8p://www.wired.com/news/images/full/BuBBleBoy3_f.jpg Disclosures and Gratitude State of CA, NewBorn screening immunology expert Baxter Inc, ImmunogloBulin clinical trial Research Support from: Jeffrey Modell Foundaon, Primary Immunodeficiency Center NIH NICHD, NIAID DHHS Maternal and Child Health Bureau CDC NewBorn Screening Branch, Division of Lab Sciences TREC: T Cell Receptor Excision Circle, a Biomarker for Thymic T Cell Production • Normal diverse T cell repertoire is made By cung and pasQng alternate DNA secQons of receptor genes • Excised DNA forms T Cell Receptor Excision Circles (TRECs) as a Byproduct. • TRECs can Be detected By PCR. TCRD locus Vα V δRec Dδ Jδ Cδ ΨJα Jα Cα J Dδ δ // // // // Cδ TCRA locus // TREC PCR across joint SCID Newborn Screening, 2008: Wisconsin 2008 Screening Not screening DHHS Sec. Advis. Commiee: SCID nominated for newborn screening SCID Newborn Screening, December 2009: 3 states, Navajo Nation, Puerto Rico 2008 2009 2009 Screening 2009 Not screening CDC and JMF Grants for Pilot SCID Screening in response to Sec. Advis Commiee refusal due to no proven screening test SCID Newborn Screening, December 2010: 5 states, Navajo Nation, Puerto Rico 2008 2009 2010 2010 2009 Screening 2009 Not screening DHHS Sec. Advis. Commiee approves SCID as a disease for newborn screening. California and New York add 750,000 births/year. SCID Newborn Screening, December 2011: 6 states, Navajo Nation 2008 2009 2011 2010 2011 2010 2009 2010, Screening stopped Not screening Louisiana, Puerto Rico stopped when pilot funds ran out SCID Newborn Screening, December 2012: 11 states, Navajo Nation 2008 2009 2011 2010 2011 2012 2010 2012 2009 2012 2012 2010, Screening stopped 2012 Not screening SCID Newborn Screening, December 2013: 17 states, Navajo Nation 2013 2008 2010 2009 2013 2011 2011 2013 2013 2013 2013 2012 2010 2012 2009 2012 2012 2010, Screening 2008-2012 stopped Screening 2013-2015 2012 Not screening 11 NBS programs pool data from 3 million infants screened. Kwan et al., JAMA 2014 SCID Newborn Screening, December 2014: 27 states, Navajo Nation 2014 2014 2013 2014 2008 2010 2009 2013 2011 2014 2013 2011 2014 2013 2014 2013 2013 2012 2014 2014 2010 2012 2014 2009 2014 2012 2012 2010, Screening 2008-2012 stopped Screening 2013-2015 2012 Not screening US FDA approved SCID screening assay (PerkinElmer) SCID Newborn Screening, December 2015: 28 states, Navajo Nation 2014 2014 2013 2014 2008 2010 2009 2013 2011 2014 2013 2011 2014 2013 2014 2013 2013 2012 2014 2014 2010 2012 2014 2009 2015 2015 2015 2014 2012 2012 2010, Screening 2008-2012 stopped Screening 2013-2015 2012 Not screening SCID Newborn Screening, September, 2016: 41 states, Navajo Nation and Puerto Rico 2014 2016 2016 2014 2013 2014 2016 2008 2016 2016 2016 2010 2009 2013 2011 2014 2013 2011 2014 2013 2014 2013 2013 2012 2014 2014 2016 2010 2012 2014 2016 2009 2015 2015 2016 2016 2015 2014 2012 2016 2012 2010, Screening 2008-2012 stopped Screening 2013-2015 2012 2016 Screening, 2016 Not screening >85% of USA Births; 93% expected By end of 2016 IniQal TREC TREC >22 Normal assay, DNA TRECs copies/uL of TREC >22 ≤22 blood PosiQve / Repeat β-Acn TREC OK ABnormal with TREC ≤5 California β-Acn β-Acn DAF Bad Regular NBS β-Acn OK TREC NICU Incomplete TREC 6-22 β-Acn Bad Algorithm DAF CBC, lymphocyte subsets by NBS Program Repeat TREC ≤22 Low TREC DAF or Heel x2 at one contract Lab; Incomplete Sck interpreted by Immuno TREC >22 Normal Consultants Not normal: <1,500 T cells or absent Normal: ≥1,500 naïve CD4/CD45RA T cells – Refer to CD3 T cells with Immunodeficiency Center naïve cells IniQal TREC TREC >22 Normal assay, DNA TRECs copies/uL of TREC >22 ≤22 blood PosiQve / Repeat β-Acn TREC OK ABnormal with TREC ≤5 California β-Acn β-Acn DAF Bad Regular NBS β-Acn OK TREC NICU Incomplete TREC 6-22 β-Acn Bad Algorithm DAF CBC, lymphocyte subsets by NBS Program Repeat TREC ≤22 Low TREC DAF or Heel x2 at one contract Lab; Incomplete Sck interpreted by Immuno TREC >22 Normal Consultants Not normal: <1,500 T cells or absent Normal: ≥1,500 naïve CD4/CD45RA T cells – Refer to CD3 T cells with Immunodeficiency Center naïve cells SCID Definition “Classic” SCID pre-NewBorn Screening: • No T cells; no specific anQBody, failure to thrive, thrush, recurrent infecQons. New definiQon with screening needs to Be Based on lab values: • “Typical” SCID: <300/uL autologous T cells; <10% of normal proliferaon to mitogens such as PHA; no producQon of specific anQBodies. Proven with null mutaon(s) in known SCID gene. • “Leaky” SCID (includes Omenn syndrome): 300-1500 T cells or more, But low to absent naïve CD45RA T cells, impaired PHA, hypomorphic mutaon(s) in SCID gene. Many SCID Genes, Distinct T, B, NK profiles • IL2RG (common γ chain, X-linked) T- B+ NK- • JAK3 (γ chain associated Janus kinase) T- B+ NK- • IL7R (IL-7 receptor α chain) T- B+ NK+ • CD45 (membrane tyrosine kinase) T- B+ NK+ • TCRD/E/Z ( TCR CD3 δ,ε,ξ chains) T- B+ NK+ • RAG1/RAG2 T- B- NK+ • DCLRE1C (Artemis) T- B- NK+ • LIG4 (DNA ligase IV) T- B- NK+ • PRKDC (DNA PKcs) T- B- NK+ • ADA (adenosine deaminase) T- B- NK- • AK2 (reticular dysgenesis, deafness) T- B+/- NK+ • TTC7A (multiple bowel atresias) T- B+/- NK+ • RMRP (cartilage hair hypoplasia) T- B+/- NK+ • FOXN1 (nude mouse) T- B+ NK+ • CORO1A (Coronin 1A) T- B+/- NK+ Primary Immune Deficiency Treatment Consortium (PIDTC) >40 USA, Canadian and European Centers from 2009 Uniform data collection Retrospective Study 6902: Cross-sectional evaluation Prospective Study 6901: Natural history, observational Identify factors affecting outcomes of HCT, ERT, or GT 5 year survival for Typical SCID depends more on infection than age at HCT • >3.5 months with active infection worse than any other group. • <3.5 months and >3.5 months with no infection had best survival; • >3.5 months with infection resolved not p<0.001 significantly worse than <3.5 months or uninfected. Pai et al, NEJM, 2014 371:434-46. PIDTC SCID Diagnosis by Screening vs. Infection, Family History % Diagnosed Genotypes of Typical and Leaky SCID Reports from Transplant California, with Centers, no Screening TREC Screening Duke University, European 3½ years, 1.7 ×106 infants centers (estimates) Unknown 3% RAG2 1% DCLRE1C 5% Unknown RMRP 1% 12% TTC7A 1% IL2RG 28% JAK3 7% IL2RG JAK3 6% 50% ADA RAG1 14% ADA 12% 18% IL7R IL7R 10% 15% RAG1 1% Overall Survival ~74% Overall Survival 94% 4 Years of California SCID Newborn Screening (2010-2014) Leaky SCID 2 million infants 3% Idiopathic Typical screened. TCL/Variant SCID SCID 12% 255 flow cytometry 4% 2nd tier tests. (1/8,000 infants) 109/255 had <1500 T Normal T Syndrome 13% cells/uL (43%). cells by flow 1/55,000 Typical and 57% Leaky SCID. Secondary 1/180,000 Idiopathic 5% T Cell Lymphopenia Preterm 6% Non-SCID Conditions: Get Early Diagnosis, Avoid Live Vaccines MulQsystem syndromes with variable T cell deficiency 57% DiGeorge/chromosome 22q11.2 deleon 15% Trisomy 21 3% Ataxia telangiectasia 2% CHARGE syndrome Secondary T lymphopenia 25% Congenital cardiac anomalies 38% Other congenital anomalies 13% Vascular leakage, third spacing, hydrops 3% Neonatal leukemia 3-5% Maternal immunosuppressive medicaons Extreme preterm Birth—T cells Become normal over Qme Idiopathic T lymphopenia—few naïve T cells, no maternal cells, poor immune funcQon, no gene defect at outset (when solved move to correct category) Search for Gene Diagnosis Using Deep Sequencing ProBand DNA Filter to retain variants with: Good sequence quality Capture Fragment, exon add linkers Changes predicted in gene fragments product Sequence Absence in controls both ends of Locaon in gene or pathway of fragments interest, or in gene expressed in a Qssue of interest Raw data: short Presence in relaves according to end reads, expected inheritance paern; bi-direconal variants in same gene in similarly affected, unrelated individuals Align to reference sequence; List variants Damage to protein funcQon Initial Clinical Findings • Leaky SCID: 0 TRECs, low T cells, B+, NK+, poor PHA • Microcephaly • Hypertelorism • ABnormal nasal creases • Neonatal tooth • Decreased tone • Desquamang rash • HirsuQsm, loose skin with extra folds • ABsent corpus callosum Exome Trio: De novo BCL11b het missense N441K. Normal human cord blood CD34+ differentiation after transduction with lentiviruses Empty GFP, Patient mutant Bcl11b (N441K) + GFP, or Bcl11b-siRNA + GFP GFP GFP 25 mut 16 mut siRNA siRNA 14 * 20 12 * 10 15 * 8 10 6 * 4 %GFP+ CD19+cells 5 %GFP+ CD3+ cells 2 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Days in co-culture on OP9 monolayers Days in co-culture on OP9-DL1 monolayers * p < 0.05, GFP v/s mut and GFP v/s siRNA Mut Bcl11b and Bcl11-b siRNA do not affect B cell development, but abrogate T cell development on OP9 cells.