**4.** *Btk* **genes and function**

X-linked agammaglobulinemia is caused by mutations in the gene encoding a cytoplasmic protein tyrosine kinase, called Bruton's tyrosine kinase (Btk), in honor of the discoverer of the disorder, Colonel Ogden Bruton, MD. Btk is signal transduction molecule downstream of pre-B-cell receptor (PreBCR) and the B-cell receptor (BCR). It is a key regulator for B-lymphocyte precursors to differentiate into B cells in bone marrow. Mutation in Btk results in the defective production and function of the enzyme. In a healthy person, the enzyme is activated by the pre-B-cell receptor, and it delivers biochemical signals that prompt the B cells to divide or mature and survive. Therefore, patients with defective Btk have almost complete absence of B cells and plasma cells due to arrest in maturation beyond pre-B cell [5–9].

The gene for this enzyme was identified in 1993 by two independent groups [18, 19]. It is located on the Xq21.3-Xq22, the long arm of the X chromosome [18–24]. Btk belongs to a distinct family of protein kinases. Tec, Itk, Bmx, and Txk are other members of this family. The protein contains five regions, PH, TH, SH3, SH2, and kinase domains, and any of these domains may be affected by mutations causing XLA [23, 24]

Since 1993, the number of genetic studies has increased. XLA is a variable disease in certain patients [25, 26]. The types of mutations causing XLA include missense, nonsense, point, frameshift, slice site, deletion, insertion, and premature stop codon mutations [27]. In general, missense mutations account for 40% of all mutations, whereas nonsense mutations account for 17%, deletions account for 20%, insertions account for 7%, and slice site accounts for 16%. This distribution is similar to those listed in Immunodeficiency mutation database [28].

In a study conducted on 56 patients, mutations affecting the *Btk* gene were demonstrated in 51 patients. It was shown that 22 mutations were missense [29]. In another study, the number of missense mutations was found to be higher [30]. In the study by Chan et al., 12 patients were evaluated, and 3 deletion mutations, 8 nucleotide substitution mutations, and 1 insertion– deletion mutation were detected [31]. In the study carried out in Central European patients with agammaglobulinemia, the point mutations were observed to be more frequent [32]. The first study in 16 Turkish XLA patients was done by Wang et al. [33], seven novel mutations were identified: 2 missense and 4 deletions resulting in frameshift and premature stop codon, novel mutations were determined in 7 cases; 2 missense, 1 nonsense, and 4 deletion mutations were detected. In the last update, lists of the online BTKbase, 1155 entries have been compiled from 974 unrelated families with 602 unique molecular events [32]. The genetic profile of XLA has been studied in 122 patients from 109 families in Eastern and Central European (ECE) countries in 2009 [17]. BTK sequence analysis revealed 98 different mutations, in which 46 of them were reported for the first time. The mutations included single nucleotide changes in the coding exons (35 missense and 17 nonsense), 23 splicing defects, 13 small deletions, 7 large deletions, and 3 insertions.

We conducted a study in Istanbul University, Cerrahpaşa Medical School, Children's Hospital, to determine the BTK mutation in a total of 19 Turkish boy from 18 unrelated families with recurrent infections, almost no CD19(+) B cells and agammaglobulinemia (Table 1) [34].


**Table 1.** Clinical data of 18 male patients with agammaglobulinemia

BTK gene mutations were determined within 10 patients. The types of the mutations were 3 missense, 4 frameshift and premature stop codon; 2 splice site; and 1 point. Missense mutations were determined in the three patients (patients 2, 4, and 17). In one patient (patient 17), a novel amino acid substitution was determined within the TH domain in exon 6 (c. 491G > A) (p.G164D), which was not included previously in the ESID database. A novel point mutation within the PH domain in exon 2 (c. 49A > T) (p.K17X) was detected in Patient 7. This mutation as well has not been defined previously in the ESID database. The frameshift and premature stop codon mutations were observed to be frequent, followed by missense mutations (Table 2). Although four of the patients have features relevant with a clinical and immunological diagnosis of XLA, a BTK gene mutation could not be determined. In such cases, other autoso‐ mal recessive gene defects (µ heavy chain, surrogate light chain λ5, Igα and Igβ signaling molecules, and B-cell linker adaptor protein (BLNK)) should be investigated [35–40]. These genes map proteins involved in maturation of pro-B cells into pre-B cells. These defects have also been shown to result in agammaglobulinemia and an absence of circulating B cells, which cannot be clinically distinguished from XLA.


**Table 2.** *Btk* mutations identified in 10 XLA patients

In the light of these studies, gene defect has to be defined for the accurate diagnosis of XLA, carrier detection, and prenatal diagnosis.

#### **5. Genetic counseling**

evaluated, and 3 deletion mutations, 8 nucleotide substitution mutations, and 1 insertion– deletion mutation were detected [31]. In the study carried out in Central European patients with agammaglobulinemia, the point mutations were observed to be more frequent [32]. The first study in 16 Turkish XLA patients was done by Wang et al. [33], seven novel mutations were identified: 2 missense and 4 deletions resulting in frameshift and premature stop codon, novel mutations were determined in 7 cases; 2 missense, 1 nonsense, and 4 deletion mutations were detected. In the last update, lists of the online BTKbase, 1155 entries have been compiled from 974 unrelated families with 602 unique molecular events [32]. The genetic profile of XLA has been studied in 122 patients from 109 families in Eastern and Central European (ECE) countries in 2009 [17]. BTK sequence analysis revealed 98 different mutations, in which 46 of them were reported for the first time. The mutations included single nucleotide changes in the coding exons (35 missense and 17 nonsense), 23 splicing defects, 13 small deletions, 7 large

We conducted a study in Istanbul University, Cerrahpaşa Medical School, Children's Hospital, to determine the BTK mutation in a total of 19 Turkish boy from 18 unrelated families with recurrent infections, almost no CD19(+) B cells and agammaglobulinemia (Table 1) [34].

> **IgM (mg/dL)**

**P 1** 4 <166 0 <128 3 (–) (–) **P 2** 1.5 29 0 <29 0 (–) (+) **P 3** 3 0 0 32 - (–) (+) **P 4** 3.5 250 0 31 0 (–) (–) **P 5a** 3.5 - - - 0 (+) (–) **P 5b** 3 months 21 3 <18 0 (+) (–) **P 6** 1.5 611 179 55 2 (–) (+) **P 7** 1.5 157 <25 <18 0 (–) (–) **P 8** 8 307 28 121 0 (–) (+) **P 9** 6 <145 0 28 0 (–) (–) **P 10** 5 83 4 12 0 (–) (–) **P 11** 4 447 6 4 0 (–) (+) **P 12** 2 <140 <6 <16 0 (–) (–) **P 13** 9 <153 0 <2 0 (–) (+) **P 14** 5 <143 <23 70 0 (+) (–) **P 15** 7 681 188 25 2 (+) (–) **P 16** 3 180 <28 <29 0 (–) (–) **P 17** 8 302 152 8 0 (–) (–) **P 18** 1 315 60 33 2 (–) (–)

**CD19+ B** **Family history** **Consanguinity**

**cells**

deletions, and 3 insertions.

220 Immunopathology and Immunomodulation

**diagnosis**

**IgG (mg/dL)**

**Table 1.** Clinical data of 18 male patients with agammaglobulinemia

**IgA (mg/dL)**

**Patient no. Age at**

XLA is inherited in sex-linked diseases (x-linked). As the defects are connected with the Xchromosome and the inheritance is recessive, only male infants are affected. If a boy inherits a defective gene, since he does not have a healthy gene from his father, the boy may have the disease. Women who carry a mutant allele of the *Btk* gene on one of their chromosomes are carrier of the disease. Therefore, mothers, sisters, and maternal aunts should be investigated for carrier status because they are obligate carriers. Brothers, uncles, or nephews of the mother must be questioned for this disorder. The family history of XLA is nonexistent in approxi‐ mately 50% of patients. Some of these patients (15–20%) with XLA may have a de novo mutation in *Btk* gene, and their mothers are not carriers. If the mutant *Btk* allele is known previously in the family, carrier testing for at-risk female relatives or prenatal diagnosis is possible [41–43].

#### **6. Immunology of XLA**

B cells arise from hematopoietic stem cells in the bone marrow. B cells begin to generate and express B-cell receptors (BCRs) (Fig. 1). The entire developmental process of B cells occurs within the bone marrow. A common lymphoid progenitor (CLP) gives rise to pro-B lympho‐ cytes, which next develop into pre-B lymphocytes and then to B lymphocytes. Stimulated B cells may further differentiate into plasma cells that synthesize and secrete immunoglobulins. Mutation in Bruton tyrosine kinase causes arrest in the development of B lymphocyte at the early stage of large pre-B-cell (CD19+ cytoplasmic µ+ ) stage in the bone marrow (Fig. 2). This defect is leaky, resulting in a few immature B cell. B-cell developmental defects in bone marrow lead to a marked decrease or absence of fully mature B lymphocytes in peripheral blood, absent or few follicles, and germinal centers in lymphoid organs. Plasmocytes are absent, and reticuloendothelial tissue and lymphoid organs (tonsils, spleen, Peyer plaques, and lymph nodes) are poorly developed. Therefore, secondary lymphoid organs such as lymph nodes and tonsils are reduced in size. The consequence of decreased immunoglobulin-producing B cell is diminished in all serum immunoglobulin isotypes, resulting in inability to produce anti‐ bodies against protein and polysaccharide antigens. The percentage of T cell is increased, and T cell functions are intact. These patients have the ability to control viral and fungal infections because of intact cell-mediated immunity. The thymus is in normal size and architecture.

Antibodies are produced by plasma cells that are terminally differentiated B cells. When B lymphocytes identify and interact with a specific antigen in the body, it is triggered to mature into a plasma cell that is able to produce specific antibodies. Plasmocytes produce nine antibody isotypes: immunoglobulins G (IgG1, IgG2, IgG3, and IgG4), immunoglobu‐ lins M (IgM), immunoglobulins A (IgA1 and IgA2), immunoglobulins D (IGD), and immunoglobulins E (IgE). Antibodies are soluble molecules that bind to antigens to render them harmless by agglutination and neutralization or "tag" the antigens to facilitate destruction and removal by phagocytes and via activating complement components. Antibodies are an important component of humoral immune responses and integral part of body's defense mechanism against bacteria. During the first 6–9 months of life, infants with XLA are protected from infections by transferred maternal IgG antibodies. Reduced maternal antibodies by 6–9 months of age and failures in humoral immunity leave the affected XLA patient with a reduced ability to resist infections and increased susceptibili‐
