pyrosequencing of dock8  (Pyrosequencing Inc)

Journal: Clinical Immunology (Orlando, Fla.)

Article Title: Hypomorphic function and somatic reversion of DOCK8 cause combined immunodeficiency without hyper-IgE

doi: 10.1016/j.clim.2015.12.003

Figure Lengend Snippet: A novel compound heterozygous mutation in DOCK8 results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.

Article Snippet: Somatic reversion of DOCK8 in T cells was confirmed by pyrosequencing of DOCK8 ( D).

Techniques: Mutagenesis, Expressing, Sequencing, Control, Hybridization, Transformation Assay

Journal: Clinical Immunology (Orlando, Fla.)

Article Title: Hypomorphic function and somatic reversion of DOCK8 cause combined immunodeficiency without hyper-IgE

doi: 10.1016/j.clim.2015.12.003

Figure Lengend Snippet: Improvement of T cell proliferation over time, somatic reversion of DOCK8 in T cells and hypomorphic function of the truncated DOCK8 protein. Proliferation of PHA-stimulated, CFSE-labeled PBMCs of the patient at (A) 10 and (B) 15 years of age, and a healthy control. Depicted are percentages of CFSE low cells gated on CD3 + CD4 + or CD4 − or CD8 + T cells. (C) Sanger sequence trace showing somatic reversion of the single nucleotide duplication (c.6019dupT) resulting in expression of about 60% wild-type DOCK8 transcripts in the patient's CD3 + T cells. (D) “T” nucleotide phosphorescence ratios obtained by pyrosequencing DOCK8 of primary CD3 + CD4 + and CD3 + CD8 + T cells, primary CD19 + B cells and the EBV B cell line of the patient. (T/± 1 or 2) depicts the signal ratio of c.6018-19T to nucleotides 1 and 2 positions up and downstream. The PCR templates and pyrosequencing reactions were performed in triplicate. Each symbol represents the mean of the three ratio measurements at respective nucleotide positions. The bar represents the mean “T” nucleotide phosphorescence ratio of all 4 different nucleotide ratios in indicated cell populations. (E) Sanger sequence trace showing expression of solely mutated DOCK8 transcripts in EBV-transformed B cells of the patient. The duplicated T-nucleotide is indicated by the arrow and #. (F) Migration of EBV-transformed B cells of 5 different healthy controls (each symbol represents the mean of 3 independent experiments for each of the healthy control samples), the patient and a patient with a complete loss-of-function mutation in DOCK8 (DOCK8 null ). The bar of the healthy control samples represents the mean of the mean of each of the 5 healthy control samples. The bar for each of the patient samples represents mean and standard deviation of 3 independent experiments for each sample.

Article Snippet: Somatic reversion of DOCK8 in T cells was confirmed by pyrosequencing of DOCK8 ( D).

Techniques: Labeling, Control, Sequencing, Expressing, Transformation Assay, Migration, Mutagenesis, Standard Deviation