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RNA sequencing reveals the consequences of a novel insertion in dedicator of cytokinesis-8  Shaheen Khan, PhD, Merin Kuruvilla, MD, David Hagin, MD, PhD,

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Presentation on theme: "RNA sequencing reveals the consequences of a novel insertion in dedicator of cytokinesis-8  Shaheen Khan, PhD, Merin Kuruvilla, MD, David Hagin, MD, PhD,"— Presentation transcript:

1 RNA sequencing reveals the consequences of a novel insertion in dedicator of cytokinesis-8 
Shaheen Khan, PhD, Merin Kuruvilla, MD, David Hagin, MD, PhD, Benjamin Wakeland, BSc, Chaoying Liang, BSc, Kasthuribhai Vishwanathan, MS, Richard A. Gatti, MD, Troy R. Torgersen, MD, PhD, Roshini S. Abraham, PhD, Edward K. Wakeland, PhD, Nicolai S.C. van Oers, PhD, M. Teresa de la Morena, MD  Journal of Allergy and Clinical Immunology  Volume 138, Issue 1, Pages e6 (July 2016) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Two siblings with PIDs have mutations in DOCK8. A, Family pedigree with 2 affected siblings (black filled) and an unaffected sister. B, Sequencing and filtering strategy used to identify candidate genes. C, Schematic representation of intronic-exonic organization of DOCK8 with a 2 nucleotide (CA) insertion detected at the splice junction preceding exon 27. D, The sequence read number is displayed as a read pile-up for P1 and P2. CA insertion noted in green. E, The regions of homozygosity on chromosome 9 are shown as green segments. The red line expands the area encompassing an extensive region of homozygosity unique to the affected siblings. CA, Cytosine-adenosine; P1, patient 1; P2, patient 2; SEQ, sequencing; SNP, single nucleotide polymorphism. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 RNA sequencing uncovers abnormal DOCK8 transcripts. A, RNA sequencing was used to assess the expression levels of DOCK8, LAMB2, and GPRC38 transcripts in the peripheral blood of the patients and family members. B, The percentage of aberrant reads in the region spanning the exon-intron boundaries was determined for the 2 patients, the unaffected sibling, and their father from the RNA-sequencing data. C, The RPKM values for DOCK8 transcripts were compared in peripheral blood and skin fibroblasts from the indicated patients and control. The presence and type of aberrant sequence read preceding exon 27 is shown for P1 (D), P2 (E), the unaffected sibling (F), and their father (G) and compared with that for 2 independently isolated normal controls (H and I). J, Flow cytometry analysis of lymphocytes isolated from the affected siblings (P1 and P2) is compared with that for normal controls. Cells were analyzed for forward and side scatter properties, and for the appearance of DOCK8 protein, as detected by intracellular staining. P1, Patient 1; P2, patient 2; RPKM, reads per kilobase per million. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Two siblings have similar clinical presentations including radiosensitivity. Dose-response curve (%SF) for fibroblasts of patient 1 (green X) scored in an intermediate range of radiosensitivity, when compared with fibroblasts from a wild-type control (blue diamonds), AT control (pink squares), and a Ligase-4 deficiency control (black triangle), studied in the same experiment. AT, Ataxia telangiectasia; IR, ionizing radiation; P1, patient 1; SF, survival fraction; WT, wild-type. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 DOCK8 is significantly reduced in CD4, CD8, NK, and B cells from patients with a 2-bp insertion in the intronic region of DOCK8. Histogram analysis of intracellular DOCK8 staining (green line) compared with unstained samples (red) and isotype controls (blue). Electronic gating was used to select CD4, CD8, and natural killer (CD56) cells and B cells (CD19), which were then analyzed for the intracellular levels of DOCK8. NK, Natural killer. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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