1. Novel IL7R Mutation in a T-B+NK+ SCID Infant Y Chang 1, P Sriaroon 2, GA Jervis 1, J Shi 1, MJ Sutcliffe 1, A Petrovic 1, JW Leiding 2 1 All Children’s Hospital, St. Petersburg, FL 2 University of South Florida Morsani College of Medicine, Department of Pediatrics, Division of Allergy and Immunology Tampa, FL Objectives Abstract Alamut Visual Software Results  Lymphocyte surface expression of IL7R was dramatically reduced by flow cytometry confirming the variant identified as a pathogenic mutation.  The infant remained asymptomatic in protective isolation.  She received a haploidentical bone marrow transplant without conditioning at 11 weeks and is well.  IL7R gene is composed of 8 exons (dark green) with the 5’ and 3’ un-translated regions (UTR) (light green)  Exons 1-5 encode the extracellular portion of the receptor, Exon 6 encodes the transmembrane region and Exons 7-8 encode the intracellular tail  The transcript is 4619 bps long and contains 460 amino acids  C.707-2A>G in the acceptor splice site of intron 5 causes altered splicing which generates a soluble protein lacking exon 6 (transmembrane domain of IL7R (CD127) protein) IL7R Gene Spicing Site Variant in IL7R Gene was Detected Through Next Generation Sequencing Laboratory Findings Description of Novel IL7R Variant Confirmation and Treatment Abnormal acceptor splicing sites predicted by Alamut Visual software  Flow cytometry revealed profound lymphopenia with extremely low CD3 T lymphocytes and presence of B and NK cells  Lymphocyte mitogen proliferation showed essentially absent response to PHA  Laboratory findings consistent with T-B+NK+ SCID T and B Cell Enumeration by Flow Cytometry Patient’s results Normal range Absolute lymphocyte 1,900 2,500-10,500 (cells/μL) CD3 T cells 82500-5500 CD4 T cells 3 1600-4000 CD8 T cells 6560-1700 CD56 NK cells 785170-1100 CD19 B cells 639300-2000  Novel homozygous variant of IL7R c.707-2A>G was detected at acceptor site of intron 5  Homozygous variant is consistent with LOH region on SNP array (as opposed to compound heterozygous variant)  Variant was detected using a custom designed 180 gene Primary Immunodeficiency Panel (Haloplex, Aligent Technologies ) An 8 day old female of Guatemalan parents presented with zero TRECs detected by the newborn screening program of the State of Florida. Laboratory testing results showed extremely low T cells numbers, normal B cell and IgM levels, and normal NK cell counts. The proliferation response to PHA was essentially absent, and the response to PWM was decreased but detectable. The diagnosis of T- B+ NK+ SCID is indicated. Chromosomal microarray did not show 22q11 deletion but did reveal 51 Mb of regions of homozygosity. 15 OMIM genes associated with 27 AR conditions are within the LOH regions, including IL7R gene. Genomic DNA of the patient was sequenced with next generation sequencing technology. A panel of primary immunodeficiency genes including SCID genes was analyzed. A novel significant homozygous splicing site substitution in IL-7R gene was revealed (707- 2A>G). This variant is located in the acceptor splice site of intron 5 and likely results in skip of exon 6. Exon 6 encodes for transmembrane domain of IL7RA (CD127) protein. Flow cytometry confirmed that the lymphocyte surface expression of IL7RA was dramatically reduced in this patient. The infant remains asymptomatic for infections or fevers. Due to flat PHA response, patient is in protective isolation with infection prophylaxis. Allogeneic bone marrow transplant at 6 weeks of age is anticipated.  51 Mb of LOH regions (purple); Coefficient of inbreeding, F = 1/64 fifth degree parental relationship (second cousins or equivalent)  15 genes associated with 27 autosomal recessive conditions within LOH regions, including one SCID gene = IL7R; homozygous or compound heterozygous pathogenic mutations of IL7R associated with T-B+NK+ SCID Affymetrix SNP Array Result Schematic representation of the signal transduction initiated by IL7R through JAK3  Conformational changes resulting from the binding of IL7 (cytokine) to IL7R (receptor) induces auto- phosphorylation of JAK3 which can then phosphorylate IL7R  This leads to the recruitment of STAT and other adaptor proteins which are also phosphorylated by JAK3  Phosphorylated STAT factors migrate to the nucleus where they act as transcription factors of genes important for survival, proliferation and differentiation of lymphoid cells Int J Biochem Cell Biol. 2009 Dec:41(12):2376