Damaging BTK Variant Demonstrated by Carrier, Allele-Specific BTK Expression in B Cells and Monocytes

  • Mary T. Bausch-Jurken
  • Mary Hintermeyer
  • Jeffrey Woodliff
  • Shaoying Chen
  • Amy Rymaszewski
  • James Verbsky
  • John RoutesEmail author
Letter to Editor

To the Editor:

With the increased use of next generation sequencing (NGS), ascertaining the clinical significance of a variant of unknown significance (VUS) is a difficult and common problem. In silico protein modeling and pathway analysis is currently not definitive, and in vitro or in vivo evaluation of the function of the mutant protein can be challenging, expensive and time consuming. We report a novel approach to define the pathogenicity of a VUS in the BTK gene that led to the diagnosis of XLA. This approach may be useful in defining the pathogenicity of VUS in other X-linked disorders.

We evaluated a 17-year-old (yo) male on IVIG previously diagnosed with CVID who presented with respiratory tract infections and hypogammaglobulinemia beginning in the first year of life. Laboratory evaluation of the proband demonstrated an absent/low levels of IgA/IgM, but with a normal level of IgE, and no B cells in the peripheral blood (Table 1). The IgG level prior to antibody replacement was...


X-linked agammaglobulinemia Bruton’s tyrosine kinase 



Bruton’s tyrosine kinase


Fluorescence activated cell sorting


Next generation sequencing


Variant of uncertain significance


X-linked agammaglobulinemia



We thank Emma Cook for her editorial expertise and Erin Hammelev for IRB assistance.

Compliance with Ethical Standards

Conflict of Interest

Dr. Routes reports a grant and independent contractor from CSL Behring, a grant from Baxalta, grant from Bio Products Laboratory Limited, outside the submitted work. All other authors declare that they have no conflict of interest.

Supplementary material

10875_2019_591_MOESM1_ESM.docx (19 kb)
ESM 1 (DOCX 18 kb)
10875_2019_591_MOESM2_ESM.pdf (96 kb)
Supplementary Figure 1 A. Position of the variant in BTK (c. 1004T>A) (top) and with p.V335D mutation in the protein (bottom). Primers for RT-PCR cDNA amplification are highlighted in yellow, with the nucleotide substitution indicated in red. PH, pleckstrin homology; TH, TEC homology domain; SH, src homology. B. Outline of the FACS and RNA sequencing of B cells and monocytes in carrier. (PDF 96 kb)
10875_2019_591_MOESM3_ESM.pdf (54 kb)
Supplementary Figure 2 Flow cytometry histograms measuring BTK expression in the proband and the sister. Expression was measured in CD14+ monocytes and CD19+ B cells. The isotype controls for each subject sample is presented across the top row, whereas the subject results are shown across the bottom row. Quantification of the expression is included in Table 1. (PDF 54 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Asthma, Allergy and Clinical Immunology, Department of PediatricsMedical College of WisconsinMilwaukeeUSA
  2. 2.Asthma, Allergy and Clinical ImmunologyChildren’s Hospital of WisconsinMilwaukeeUSA
  3. 3.Division of Rheumatology, Department of PediatricsMedical College of WisconsinMilwaukeeUSA
  4. 4.Department of Pediatrics, Children’s Hospital of WisconsinMedical College of WisconsinMilwaukeeUSA

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