Clinical, immunological and genetic characterization of patients with X‐linked agammaglobulinemia in Costa Rica

X‐linked agammaglobulinemia is caused by mutations in the gene encoding Bruton tyrosine kinase. It produces an arrest in the maturation and differentiation of B cells with very low levels of all immunoglobulins isotypes. The aim of the study was to characterize the clinical, immunological and genetic defects in patients with XLA in Costa Rica. Sixteen cases were identified over a period of 30 years, a case every 2 years, approximately. Three patients were asymptomatic and diagnosis was made on family history. The average age of onset of symptoms was 1.46 years‐old (0.08–6.1). Six patients (44%) had onset of symptoms before 1 year of age and 12 (81%) patients before 5 years of age. The average age of diagnosis was 3.63 years‐old (0.17–13, SD 3.51 years‐old the average time between the onset of symptoms and the diagnosis was 2.5 years (2.5 months to 12 years, SD 3 years). The initial reason to study the patients was a recurrent infection, family history of XLA, arthritis and neutropenia. Four patients had pneumonia and two had suppurative lung disease. Nine patients had recurrent infections: acute otitis media, sinusitis, mastoiditis and recurrent diarrhoea. Three patients presented with arthritis. Neutropenia as an isolated event was not identified in any case. All patients receive monthly IVIG and no deaths were reported. Three new likely pathogenic/pathogenic variants in BTK gene have been described in our population. This is the first report of XLA Costa Rican patients and their BTK mutations.


| INTRODUCTION
X-linked agammaglobulinemia (XLA, OMIM # 300300) is a primary immunodeficiency (PID) caused by mutations in the gene encoding Bruton tyrosine kinase (BTK). It produces a maturation and differentiation impairment of B cells, that results in absence of B cells in peripheral blood and very low levels or absence of all immunoglobulins isotypes, leading to the inability to produce specific antibodies. [1][2][3][4] Patients also have susceptibility to bacterial and enteroviral infections. They present with recurrent sinusitis, bronchitis, otitis media, pneumonia and chronic diarrhoea. [2][3][4][5] It is not clear if the specific infections could vary from region to region, and Pseudomona infection has been reported in XLA at the time of diagnosis. [6][7] Chronic lung disease is a common sequelae. Since the description of the disease, the use of parenteral gammaglobulin replacement reduces morbidity, mortality and significantly improves quality of life. 2,6 BTK gene codes for a tyrosine kinase protein expressed in most haematopoietic cells. It is in Xq21.3-22 and contains 19 exons. Mutations have been described over the entire gene but most of them are in the tyrosine kinase domain. [8][9][10][11] A clear genotype-phenotype relationship has not been established. 12 The aim of this study was to characterize clinically, immunologically and genetically the patients with XLA in Costa Rica.

| METHODS
The study was approved by the HNN Bioethics and Research Unit (code CLOBI-HNN-003-2012 and CEC-HNN-024-2017) and the permission to publish was obtained from the Research Subarea. The diagnostic criteria of PAGID and ESID 4 were applied to clinically define XLA cases. Patients with other causes of hypogammaglobulinemia were identified and excluded. Each patient's clinical record was reviewed to obtain information on socio-demographic, clinical, immunological and genetic characteristics. Patients that met the criteria for XLA, or their parents, in the case of those under 18 years of age, signed the informed consent. Genomic DNA was extracted from blood leukocytes according to standard protocols. They were sampled for automated DNA extraction using platform MagNA Pure 2.0 (Roche Diagnostics). Standard PCR protocols, Sanger sequencing 10 and methods established by Danielian et al 13 were used to carry out BTK gene mutational analysis. PCR products were evaluated with a QIAxell system (Qiagen Company) and the mutations were evaluated by Sanger sequencing on an ABI 3130 Genetic Analyser at the University of Costa Rica and ABI 3500 at the National Children´s Hospital and Life Technologies, California, United States by using BigDye 3.1 chemistry. They were analysed amplifying all exons and the flanking intronic regions of BTK by either PCR and subsequent Sanger sequencing or Next Generation Sequencing using a commercial kit (Inmunodeficiencias-GeneSGKit® and GeneSystems® software from Sistemas Genómicos) and the MiSeq platform (Illumina Inc). Amplified sequences were compared to the BTK canonic transcript (NM_000061.3) by using the Basic Local Alignment Search Tool for nucleotides (BLASTn) software. Clinically relevant variants detected by NGS were confirmed by Sanger sequencing or MLPA techniques. Sanger sequencing was based on Big Dye Terminator cycle chemistry (Applied Biosystems), and analysed with an ABI 3500 capillary sequencer (Applied Biosystems). Sequence variants were described with respect to a canonical BTK transcript NM_000061.3.

| RESULTS
While 17 cases met the clinical case definition of XLA, after genetic testing only 16 patients had identified mutations on BTK gene. In a period of 31 years, there was approximately one case every 2 years. Fourteen patients came from the central urban area of Costa Rica and one case was born in Cuba but diagnosed in Costa Rica. The mean age of the patients was 17.41 years (5.13-32.25, SD 9.45).
With regard to family history of PIDs, two patients had a family history of a brother that died from a pulmonary infection; two pairs of siblings were found with XLA and an extended family had three affected members from two different generations. Furthermore, three patients were diagnosed based on their family history while being asymptomatic; one at 1 month of age and two at 2 months of age. For the remaining 13 patients, the average age of onset of symptoms was 1.46 years (0.08-6.1). Six patients (44%) had onset of symptoms before their first year of life and 12 (81%) patients before 5 years of age and only one after 5 years of age. This patient with later manifestations coincided with the onset of respiratory symptoms in his brother who died 15 days before his diagnosis.
The average age of diagnosis was 3.63 years (0.17-13, SD 3.51 years). Five patients (31%) were diagnosed within their first year of life and only six patients (38%) after 5 years of age. Regarding the 13 patients that had symptoms before the diagnosis, the average time between the onset of symptoms and the diagnosis was 2.5 years (2.5 months to 12 years, SD 3 years). Table 1 summarizes the data on age, onset of symptoms and diagnosis. Table 2 contains the most important symptoms and laboratory data that led to the diagnosis of XLA. Patients raised suspicious of XLA because they presented with recurrent infection, family history of XLA or family death due to infection, arthritis and neutropenia. The most common finding in 12 patients (75%) was infection; pneumonia and recurrent otitis media were the most relevant. Invasive pneumococcal infection presented in three patients and H. influenzae in one patient. An atypical presentation of ecthyma gangrenosum and septicemia due to Pseudomona aeruginosa was the initial presentation in one patient. In four patients (25%), arthritis was the most important manifestation; in one of them it was a single finding and in three patients, it was accompanied by a history of recurrent infection. The family history of death due to infection was relevant in two patients for the clinical suspicion of PID.
Neutropenia was present in six cases (38%) at the diagnosis, but not as an isolated event. Physicians were aware of the neutropenia only in one patient, it was not diagnosed in the other five patients. All patients had normal CD3, CD4, CD8 and CD16/56 lymphocytes and fulfilled the criteria of less than 2% of B cells. Immunoglobulin levels varied as seen in Table 2. One patient had normal IgG levels in two different measurements when diagnosed. Specific antibody responses were performed only in seven patients because initially this assay was not available in Costa Rica.
All patients receive monthly intravenous gammaglobulin replacement. Patients are receiving an average dose of 606 mg/kg per dose of IVIG (SD 206 mg/kg/dose) with average trough IgG levels of 903 mg/dL (SD 191 mg/dL). Half of the patients had infections even after gammaglobulin replacement, specially rhino-sinusitis. Serious adverse events were not reported with the use of IVIG.
The most frequent complications observed were recurrent or chronic upper respiratory tract infections (otitis media and sinusitis) and chronic diarrhoea. There were two patients with suppurative acne. One patient had a recurrent hematuria for which no germ was documented. Three patients have nutritional impairment even after IVIG. Chronic pulmonary disease is present in five patients (31%) and bronchiectasis in four (25%) patients. One patient has a degenerative neurological condition, where infections have been excluded. Malignancy, autoimmunity and mortality were not reported as complications in the patients during the study's period. Table 3 shows mutations in patients diagnosed with XLA. Nine mutations were previously described, including single nucleotide substitutions, intronic variants and deletions. 3,8,[14][15][16][17] Three novel mutations were found according to our knowledge: in exon 17 c.1738 A > T, in exon 14 c.1235 C > A and a duplication in exon 2 c.99dupC. Unfortunately, functional studies to prove abnormal protein function are not available in Costa Rica.

| DISCUSSION
It has been reported that XLA corresponds to 6-11% of all patients with PIDs and that the incidence is approximately 1 in 100 000 to 200 000 live births. 2,18 Table 4 summarizes the information from some studies with the incidence and prevalence according to the country that reports cases of XLA. In a report from Latin America, the minimal calculated incidence for XLA is reported in Honduras: 0.11 per 100 000 live births and the highest in Argentina: 1.68 per 100 000 live births. 1 In a previous study, Costa Rica reports nine patients with hypogammaglobulinemia and absence of T A B L E 1 Distribution according to family history, age of onset of symptoms and diagnosis of patients with XLA in Costa Rica

History of XLA, PIDs or deaths due to infection
Year of birth Age a (years)

Age of diagnosis (years)
Interval between the onset of Symptoms and diagnosis (years)  22 where XLA corresponds to 5.5% of all PIDs based on the present study. According to the literature, 50% of patients are diagnosed with a/hypogammaglobulinemia near 2 years of age although the specific diagnosis of XLA is made several years afterwards. The problem in Costa Rica is that immunoglobulin quantification is mainly done in central hospitals and many doctors from rural or remote areas do not routinely use this laboratory test.
In other series, patients with a family history of XLA are diagnosed significantly earlier (around 2 years) compared to those without a family history (around 5 years), and only 35% of patients who have a family member with XLA were diagnosed before the development of symptoms attributable to their immunodeficiency. 2,20,23 According to a study done in the United States, more than 40% of patients with PIDs are not diagnosed until the disease is in a late stage, even though many report serious and chronic health conditions before diagnosis such as sinusitis, bronchitis and pneumonia. 2 The importance of the recognition and management of PIDs is further accentuated in the analysis of the rate of pre-and post-diagnosis hospitalization. It is reported that 70% of patients have hospitalizations before diagnosis and only 48% after diagnosis. Multiple reviews emphasize the importance of early diagnosis of PIDs to prevent disease-related morbidity and hence mortality. [23][24][25] Recurrent infection or infection was presented as the pivot sign in these patients, and the presence of recurrent bacterial infections within the first years of life is also the most important characteristic described in most studies. 2,20,[26][27] They also presented arthritis as an initial manifestation that led to the diagnosis. It is worthwhile to note that neutropenia was not a guiding factor in the Costa Rican population.
The most common cause of morbidity is respiratory infections. Since 1956 there is a report of patients with agammaglobulinemia (both, congenital and acquired) and lung disease. It is important to note that many of these manifestations are prior to the onset of IVIG in these patients and in patients where the diagnosis is made more than The most frequent complications observed were recurrent acute and/or chronic otitis media, sinusitis, chronic diarrhoea; which are the most commonly reported in the literature. They are not quantified, nor documented in a standardized way that allows their statistical analysis. It is impossible to perform this analysis in the population studied with the data available in the patients' files This is a clear limitation of retrospective studies, which suggests that it is necessary to begin prospective studies of patients with PID should be started to determine the most common infections and agents in this population, especially to improve early clinical care.
In particular, one patient had a degenerative (noninfectious) neurological condition; there are reports of gross deletion that not only involved the BTK gene, but T A B L E 3 Genetic findings of XLA patients in Costa Rica

Patient
Nucleotide variation a Protein variation a ACMG classification and criteria 174 [29] a It corresponds to patients with hypogammaglobulinemia and absence of B cells.
b It corresponds to the minimum incidence in Honduras and the maximum in Argentina.

T A B L E 4
Summary of incidence of XLA reported in different countries or regions also TIMM8A genes that underlie the Mohr-Tranebjaerg syndrome (MTS). [31][32][33] The nucleotide variation for this patient is: Exon 15 c.1480C > T, not a deletion. His neurological condition is characterized by aggressive behaviour and loss of cognitive functions. It is of special concern that he has a younger brother with the same mutation, but he has not developed any neurological symptoms so far. It has been shown in multiple studies that gammaglobulin replacement decreases the frequency and severity of bacterial and viral infections. It also improves morbidity and mortality and decreases the incidence of pneumonia and hospital admissions due to infection. 2 So far, there are more than 1800 BTK mutations reported in BTKbase. 11 Mutations are distributed throughout the gene in coding and non-coding regions. Point mutations (∼65%), small insertions, deletions (∼25%), and large genomic DNA alterations (∼10%) have been described. They have also been described along the BTK domains. 3,34 Table 4 describes the mutations found in our patients.
Except for three cases, all pathogenic/likely pathogenic variants have been previously described by other clinical studies 3,8,17,[35][36][37][38][39][40][41] or specific databases. 34 Most of these mutations are missense variants (n = 6), followed by nonsense (n = 2), and one variant for splicing site, indels, in-frame deletion, frameshift deletion and large deletion. Seven out of 16 (44%) patients showed mutations in the kinase domain of BTK (BTK-KD) or SH1 domain, in line with previous studies. 2,3,17,42 It has been reported that the BTK-KD is highly susceptible to mutation due to several conserved amino acid residues, such as K430, E445, R641 and motifs that are tightly conserved throughout the eukaryotic protein kinase superfamily. Around 67% of single nucleotide amino acid variations in this region are predicted to be harmful mutations, associated with the complete phenotype of XLA. 35,43 The novel mutations described in this paper cannot be proved by functional assays because they are not available in Costa Rica. Patient 9 has changed an isoleucine for a phenylalanine in exon 17. This specific site was not previously described but in this exon, patients with atypical presentations such as HLH 44 and Pseudomonas infections 6 have been published. This could potentially affect the structure of the protein through the introduction of a benzene derivate, with differential physical-chemical properties. In addition, the nearby residues W581 and A582 are important in the structure and stability of protein binding to the catalytic site. 45,46 The BTK Base review shows mutations at positions 579 and 581, but not at this specific site. At site 579 Tóth 17 describes a Macedonian patient. It is proposed that nearby sites (residue 572 to 582) correspond to a cluster of amino acids critical for protein binding. In addition, the nearby sites 581 and 582 are important in the structure and stability of protein binding to the catalytic site. 43 Maniar et al 47 concluded that mutations in residues R562, W563 and A582 inactivate kinase activity.
Multiple mutations in exon 14, such as those in patient 10, have been described to disrupt a highly conserved glycine-rich motif of the ATP binding site. 46 Patient 10 presented a replacement of an original glutamine by a proline in the exon 14 (p.Gln412Pro), which confers conformational rigidity. Both mutations are in the SH1 domain that, as mentioned above, is highly susceptible to missense variants, and is a common mechanism of disease. Although this mutation has not been previously described, the site already has two patients with the W421X mutation. 34 The effects of these have not been significantly described either in the BTK expression or its function.
In patient 15, a cytosine duplication was detected in nucleotide 99 of exon 2, generating a frameshift mutation with a premature stop signal 8 residues downstream. Therefore, it is expected to result in an absent or interrupted protein product at amino acid 42 of 659.
This is the first genetic study conducted in Costa Rica and Central America for the diagnosis of PIDs, all other tests were performed elsewhere. This pioneering study provides relevant information and should set an example to start the diagnosis of PIDs by molecular techniques. Moreover, three new likely pathogenic variants in BTK gene have been described in the Costa Rican population.

AUTHOR CONTRIBUTIONS
Dra. Ivankovich developed this project as a thesis to graduate as Master, she participated in all parts of the study. Dra. Danielian helped in developing the molecular analysis. Dra. Atmella and Dra. Silva helped perform initial molecular analyses at the University of Costa Rica and Dr. Morera, Dr. Santamaria and Dra. Barboza developed the molecular analyses for the clinical setting at the Children´s Hospital. Dr. Porras was the mentor for the thesis and revised all the project. All author approved the manuscript.

ACKNOWLEDGMENTS
To my colleagues: Olga Arguedas, Gustavo Lazo, Iris Leiva, Dora Matus for their clinical work and to Henriette Raventós-Vorst for her reviews on the manuscript. To Natalia Porras for their English style review of the manuscript.

FUNDING INFORMATION
This project was part of the Master's degree Program of Dra. Ivankovich as part of the Center of Molecular and Cellular Biology, University of Costa Rica. San José, Costa Rica.