title: Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing.
abstract: Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of T cells, B cells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management.
  
  
  
  We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting.
  
  
  
  The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions.
  
  
  
  Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7.
  
  
  
  High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.
authors.affiliation: Baylor Genetics, Houston, Tex.
authors.forename: Hui
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex.
authors.forename: Victor Wei
authors.initials: VW
authors.lastname: Zhang
authors.affiliation: Department of Pediatrics, Section of Immunology, Allergy, and Rheumatology, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex; Center for Human Immunobiology, Immunology, Allergy and Rheumatology, Texas Children's Hospital, Feigin Center, Houston, Tex; Baylor-Hopkins Center for Mendelian Genomics of the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex; Norwegian National Unit for Newborn Screening, Oslo University Hospital, Oslo, Norway.
authors.forename: Asbjørg
authors.initials: A
authors.lastname: Stray-Pedersen
authors.affiliation: Department of Pediatrics, Section of Immunology, Allergy, and Rheumatology, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex; Center for Human Immunobiology, Immunology, Allergy and Rheumatology, Texas Children's Hospital, Feigin Center, Houston, Tex.
authors.forename: Imelda Celine
authors.initials: IC
authors.lastname: Hanson
authors.affiliation: Department of Pediatrics, Section of Immunology, Allergy, and Rheumatology, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex; Center for Human Immunobiology, Immunology, Allergy and Rheumatology, Texas Children's Hospital, Feigin Center, Houston, Tex.
authors.forename: Lisa R
authors.initials: LR
authors.lastname: Forbes
authors.affiliation: Division of Allergy and Immunology, University of Texas Southwestern Medical Center, Children's Medical Center, Dallas, Tex.
authors.forename: M Teresa
authors.initials: MT
authors.lastname: de la Morena
authors.affiliation: Department of Pediatrics, Section of Immunology, Allergy, and Rheumatology, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex; Center for Human Immunobiology, Immunology, Allergy and Rheumatology, Texas Children's Hospital, Feigin Center, Houston, Tex.
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Chinn
authors.affiliation: Baylor Genetics, Houston, Tex.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Gorman
authors.affiliation: Children's Hospital and Clinics of Minnesota, Minneapolis, Minn.
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Mendelsohn
authors.affiliation: Pediatric Infectious Diseases and Immunology, Children's Hospitals and Clinics of Minnesota, St Paul, Minn.
authors.forename: Tamara
authors.initials: T
authors.lastname: Pozos
authors.affiliation: Baylor-Hopkins Center for Mendelian Genomics of the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Wiszniewski
authors.affiliation: Department of Pediatrics, Section of Immunology, Allergy, and Rheumatology, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex; Center for Human Immunobiology, Immunology, Allergy and Rheumatology, Texas Children's Hospital, Feigin Center, Houston, Tex.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Nicholas
authors.affiliation: University of Mississippi Medical Center, Jackson, Miss.
authors.forename: Anne B
authors.initials: AB
authors.lastname: Yates
authors.affiliation: University of Mississippi Medical Center, Jackson, Miss.
authors.forename: Lindsey E
authors.initials: LE
authors.lastname: Moore
authors.affiliation: Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.
authors.forename: Knut Erik
authors.initials: KE
authors.lastname: Berge
authors.affiliation: Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.
authors.forename: Hanne
authors.initials: H
authors.lastname: Sorte
authors.affiliation: Department of Pediatrics, Division of Pediatric Allergy/Immunology and Pulmonology, University of Iowa Carver College of Medicine, Iowa City, Iowa.
authors.forename: Diana K
authors.initials: DK
authors.lastname: Bayer
authors.affiliation: Department of Pediatrics, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia.
authors.forename: Daifulah
authors.initials: D
authors.lastname: ALZahrani
authors.affiliation: Division of Immunology, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Mass.
authors.forename: Raif S
authors.initials: RS
authors.lastname: Geha
authors.affiliation: Baylor Genetics, Houston, Tex.
authors.forename: Yanming
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Baylor Genetics, Houston, Tex.
authors.forename: Guoli
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, Section of Immunology, Allergy, and Rheumatology, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex; Center for Human Immunobiology, Immunology, Allergy and Rheumatology, Texas Children's Hospital, Feigin Center, Houston, Tex.
authors.forename: Jordan S
authors.initials: JS
authors.lastname: Orange
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex; Baylor-Hopkins Center for Mendelian Genomics of the Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex; Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Tex.
authors.forename: James R
authors.initials: JR
authors.lastname: Lupski
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Tex. Electronic address: ljwong@bcm.edu.
authors.forename: Lee-Jun
authors.initials: LJ
authors.lastname: Wong
mesh_terms: D000293:Adolescent; D002648:Child; D005260:Female; D014644:Genetic Variation; D006801:Humans; D008297:Male; D057089:Pathology, Molecular / Q000592:standards / Q000639:trends; D017422:Sequence Analysis, DNA; D016511:Severe Combined Immunodeficiency / Q000175:diagnosis* / Q000235:genetics*
keywords: Severe combined immunodeficiency; molecular diagnostics; next-generation sequencing; severe combined immunodeficiency newborn screening