Clinical and mutational features of Vietnamese children with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA, OMIM 300300), first described by Bruton in 1952, is a fully penetrant X-linked recessive disorder characterized by recurrent bacterial infections, profound hypogammaglobulinemia and marked decrease in the number of B cells in the presence or absence of positive family history. It occurs in approximately one in 200 000 individuals [1–5]. The gene responsible for XLA is the Bruton Tyrosine Kinase (BTK) gene mapped to the long arm of chromosome X in the region of Xq 21.3- q 22 [2, 4, 6–8]. The BTK gene is expressed in B cells and moncytes throughout their differentiation but not in T cells [9]. The gene contains 19 exons and encodes a protein with five functional domains: plekstrin homology (PH) domain, Tec homology (TH) domain, Src homology 3 (SH3) domain, Src homology 2 (SH2) domain and catalytic (SH1) domain [3–5]. According to the BTK database (http://rapid.rcai.riken.jp/RAPID/mutation?pid_id=AGID_8), at present 592 unique BTK mutations have been found in XLA patients. These mutations are found in both exons and introns throughout the gene and may result in complete absence of protein, or non functional proteins [4].

There have been very few reports of XLA from developing countries [10, 11]. In Vietnam, after many years in war and in low socio-economic conditions, we usually make a diagnosis of XLA based on clinical manifestations, family history, hypogammaglobulinemia, and low numbers of circulating B cells, but not genetic analysis. In this study, we report for the first time 4 Vietnamese boys with XLA, confirmed by mutation analysis of the BTK gene in an attempt to improve the diagnosis and management of XLA in Vietnam.

XLA is caused by a B-cell developmental defect. Being an arrest of differentiation of pre-B cells to mature B cells, pre-B cells are found in normal amounts while mature B cells are nearly undetectable, that result in a pronounced reduction of serum Ig of all classes. Therefore, affected boys suffer from recurrent bacterial and enteroviral infections after six months of life after maternal antibodies are no longer present in the infant’s circulation. Mutations in all five domains of the BTK gene have been found to cause XLA [6, 12–16]. In the present study, we report four Vietnamese boys with XLA and investigated the mutation characteristics of their BTK gene.

The clinical manifestations of our XLA patients were typical and similar to previous reports [5, 8, 10, 11], with severe recurrent infections (Table 1), low levels of mature B cells and serum immunoglobulin (Ig) (Table 2). Pneumonia, septicemia, otitis media were observed in all our patients. Bronchiectasis was present in patients 1 and 2, possibly reflecting the delay of diagnosis with a complication of acute pneumonia in their histories. Patients 1 and 4 presented with arthritis, which was treated as juvenile idiopathic arthritis before diagnosis of XLA; both patients had markedly inverted CD4⁺/CD8⁺ ratio (Table 1). However, a relationship between inverted CD4⁺/CD8⁺ ratio and arthritis is presently unclear. Similar to previous reports [8, 10, 11], all our patients were diagnosed with XLA late. The age of onset of infection was between 8 months and 6 years (mean 2.5 years). Nonetheless, the age of diagnosis of XLA ranged from 5 to 11 years old (mean 8 years), possibly reflecting the poor knowledge of health workers about XLA and insufficient equipment for diagnosis of XLA in Vietnam, such as flow cytometer. Moreover, the delayed diagnosis may be because the disease is rare and infections (such as pneumonia, sinusitis, and otitis media) are common in our pediatric population. As the estimated prevalence of XLA is 1/200 000 [8], it is suggested that many XLA cases in Vietnam may be undiagnosed. Because early initiation of immunoglobulin replacement therapy could prevent sequelae associated with infections, early diagnosis is of great importance. National networks and diagnostic guidelines for XLA may be helpful for us to improve these issues.

Our XLA patients began to be treated with prophylactic IVIG and appropriate antibiotics for acute and chronic infections. Patients 1 and 2 were free from serious infections with 400 and 300 mg/kg of IVIG replacement therapy every 4 weeks, respectively. Patient 3 developed serious infections frequently while he was receiving 300 mg/kg of IVIG every month but was well after the dose had been increased to 450 mg/kg. Patient 4 was well after 5 months on 400 mg/kg of IVIG therapy every month; however, his family decided to use traditional medicines instead of IVIG at age of six. Patient 4 was hospitalized again with multiple complications of serious infections; he died after 7 months despite reintroduction of treatment with IVIG and antibiotics. Our results suggest that IVIG replacement represents an important role to improve the prognosis of XLA [10, 11, 17].

Our patients were examined for the presence of BTK gene mutations. Their mutations were distributed in both coding and non-coding (data not shown) regions of the BTK gene, which were compatible with the range of mutation as previously described [3–5, 18, 19]. Three out of 5 BTK mutations (60%) were located on SH1 domain (Table 3). In accordance with previous reports [4, 5, 8, 20], their BTK mutations affected mainly SH1 domain; this may be explained by the length of SH1 domain. There was no correlation between clinical phenotype and the mutation’s site, suggesting that other factors are contributing to the phenotype and affecting the severity of disease, such as other components of the BTK mediated signaling pathway (μ heavy chain, λ5) and variants in proteins that function as part of the innate immune system [1, 2, 21]. Environmental and potentially epigenetic factors may also contribute to the phenotype. We have analyzed the XLA carrier status in mother of patient 3 and found the same mutation. Because of the variability in clinical phenotype, the diagnosis of XLA may be difficult in some cases with atypical phenotype or without family history of disease. In such cases, molecular genetic testing provides an important tool for XLA confirmation and may allow accurate carrier detection [2, 5, 8].

One out of 4 patients (25%) developed neutropenia during the course of the disease. Several previous studies have reported that about 10-27% of XLA patients might be associated with neutropenia including mild, moderate and severe neutropenia [22–25]. Patient 4 had two episodes of moderate neutropenia before and at the time of diagnosis of XLA (Table 2). The neutropenia durations of patient 4 were less than one week and resolved completely after two weeks of treatment with antibiotic and IVIG therapy suggesting the role of infections inducing neutropenia [22, 26]. This phenomenon can most probably be explained by reactive oxygen species- mediated apotosis of neutrophils triggered by the engagement of innate receptors and not by abnormal myeloid differentiation [27].

We describe four Vietnamese boys with typical phenotype of XLA but delayed diagnosis. Our patients were definitively diagnosed by genetic analysis. Their mutations are three novel and one recurrent BTK mutations. This is the first Vietnamese study describing clinical features and BTK mutations in patients with XLA. The delayed diagnosis may be attributable to insufficient awareness of this rare disease on the background of frequent infections even in the immunocompetent pediatric population in Vietnam.