PMC5590079_01
A). Pedigree of the family
B). Flow cytometric immunophenotyping of peripheral blood lymphocytes gated on side scatter/CD45 plot. In CD19/CD3 plot, arrow depicts the count of B lymphocytes (CD19+) and T lymphocytes (CD3+).
C). Flow cytometric estimation of BTK protein expression on CD14+ monocytes in control and proband.
D). Heat map showing mutation spectrum corresponding to BTK protein structure and hotspots are marked with black diamond symbol. BTK domains (PH, TH, SH3, SH2 and Kinase) are marked in respective colors. The exons encoding specific region of domain and amino acids span for each domain is represented in schematic of BTK. A red triangle represents the novel variation p.F442fsX2, which lies in exon 14 and BTK kinase domain. The widely studied p.Y551 is also marked with black arrow. Pairwise alignment between wild type and truncated protein sequence is performed by using EMBOSS online software.
E). The chromatogram depicts capillary sequencing results of c.1325dupT (p.F442fsX2) (marked with asterisks).
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A five year old boy of north Indian origin, born out of a non-consanguineous marriage ( Figure 1A) presented to the hospital with headache, fever and a history of recurrent infections requiring hospitalization. The patient (III.1) has only CD3+ lymphocytes as observed on CD19/CD3 dot plot and there was complete absence of CD19+ cells (0.02%) ( Figure 1B). The staining for BTK protein on CD14+ monocytes showed decreased (20.71%) expression of BTK in proband as compared to control (72.07%) ( Figure 1C). Further in silico analysis suggested that the mutation causes Isoleucine at 443 residue in BTK to be replaced by Histidine and introduces a premature stop codon at 444 residue, which lies in the kinase domain of the BTK protein ( Figure 1D). Analyzer (Applied Biosystems, USA). Analysis revealed that the mutation was homozygous in child (III.1), heterozygous in mother (II.3) and absent in father (II.2) and maternal grandmother (I.4) ( Figure 1E).