Distinct Clinical Features and Novel Mutations in Taiwanese Patients With X-Linked Agammaglobulinemia

Abstract

Background: X-linked agammaglobulinemia (XLA) is caused by a mutation of the Bruton's tyrosine kinase (BTK) gene and is the most common genetic mutation in patients with congenital agammaglobulinemia. The aim of this study was to analyze the clinical features, genetic defects, and/or BTK expression in patients suspected of having XLA who were referred from the Taiwan Foundation of Rare Disorders (TFRD). Methods: Patients with recurrent bacterial infections in the first 2 years of life, serum IgG/A/M below 2 standard deviations of the normal range, and ≦2% CD19+B cells were enrolled during the period of 2004-2019. The frequency of infections, pathogens, B-lymphocyte subsets, and family pedigree were recorded. Peripheral blood samples were sent to our institute for BTK expression and genetic analysis. Results: Nineteen (from 16 families) out of 29 patients had BTK mutations, including 7 missense mutations, 7 splicing mutations, 1 nonsense mutation, 2 huge deletions, and 2 nucleotide deletions. Six novel mutations were detected: c.504G>T [p.K168N], c.895-2A>G [p.Del K290 fs 23^*], c.910T>G [p.F304V], c.1132T>C [p.T334H], c.1562A>T [p.D521V], and c.1957delG [Del p.D653 fs plus 45 a.a.]. All patients with BTK mutations had obviously decreased BTK expressions. Pseudomonas sepsis developed in 14 patients and led to both Shanghai fever and recurrent hemophagocytic lymphohistiocytosis (HLH). Recurrent sinopulmonary infections and bronchiectasis occurred in 11 patients. One patient died of pseudomonas sepsis and another died of hepatocellular carcinoma before receiving optimal treatment. Two patients with contiguous gene deletion syndrome (CGS) encompassing the TIMM8A/DDP1 gene presented with early-onset progressive post-lingual sensorineural Deafness, gradual Dystonia, and Optic Neuronopathy syndrome (DDON) or Mohr-Tranebjaerg syndrome (MTS). Conclusion: Pseudomonas sepsis was more common (74%) than recurrent sinopulmonary infections in Taiwanese XLA patients, and related to Shanghai fever and recurrent HLH, both of which were prevented by regular immunoglobulin infusions. Approximately 10% of patients belonged to CGS involving the TIMM8A/DDP1 gene and presented with the DDON/MTS phenotype in need of aggressive psychomotor therapy.

Keywords: Bruton's tyrosine kinase (BTK); Mohr-Tranebjaerg syndrome (MTS); TIMM8A/DDP1 gene; X-linked agammaglobulinemia (XLA); contiguous gene deletion syndrome (CGS); deafness-dystonia-optic neuronopathy syndrome (DDON).

Figure 1

After PBMC purified by centrifugation, we utilized FSC and SSC to locate the monocyte region and gated them by CD14+ in a representative patient (P15) with the BTK mutations showed an almost complete absence of BTK expression (1.1%) and a bimodal pattern in his carrier mother (38.9%) compared to the normal healthy control (85.7%).

Figure 2

RT-PCR amplification of cDNA included two designed two pairs: BTK1-BTKC1 for the coding region from exon 1 to exon 13 (product 1,277 b.p.); and BTC5-BTKC2 (1,253 b.p.) for the coding region from exon 11 to exon 19. Compared to the healthy control and mother of P15, only cDNA amplification by RT-PCR of the product from BTK1-BTKC1 was right detectable in P14, and the others were all undetectable. PMNs should express BTK, but much lower than PBMCs. We evaluated BTK expression in two cell lines of PBMCs and PMNs in P15. The two leukocyte components did not express any BTK after PCR-amplification in P15 (A). Therefore, each exon was amplified from genetic DNA. Exon 19 in the BTK gene in P14 and exons 6–19 in P15 were missing (B). The contiguous gene TIMM8A with two exons was amplified, but it revealed only non-specific products in exon 1 (GGA GTT GGA CGC CTG CCT CGC; CTT GAA TCC TGT CAT GAT GAA for exon 1, product 1,593 b.p.) and undetectable in exon 2 (GAA CCT GGC GGA GGT TAC AGT; CCT TGG AAT CAG CCC ATG CTA, product 1,742 b.p.). In the normal control, two white-arrows pointed at the correct locations (C). Two duplications were performed each.

Figure 3

Under the same condition for PCR amplification of candidate genes to predict carrier status, the concentration-density ratio between TIMMA8-exon 2 (total 4 locus in two X chromosome) and GADPH-exon 2 (total 2 locus in one chromosome 12) was 198.8/84.1 in the normal non-carrier female. However, in these two carrier-mothers, the concentration-density between TIMMA8-exon 2 (total 2 locus in one X chromosome) and GADPH-exon 2 (total 2 locus in chromosome 12) was 98.3/85.1 in mother P14 and 87.4/82.8 in mother P15, and both were certainly carriers and equal to a male (P14 father with one X chromosome) 102.8/93.3. This implied the half-dose existence of the TIMMA8 gene on X chromosome in their carrier-mothers compared to the normal healthy females.