Clinical characteristics and prenatal diagnosis in 22 families in Henan Province of China with X-linked agammaglobulinemia (XLA) related with Bruton’s tyrosine kinase (BTK) gene mutations.

Background: X-linked agammaglobulinaemia (XLA) is a rare immunodeficiency disease, and the main clinical symptoms is recurrent severe infections. BTK is the main disease-causing gene, and the genetic mode is X chromosome recessive inheritance. But the current mutations do not fully explain this disorder. Methods: We detected the percentage of CD19+ B cells and serum immunoglobulin (IgG, IgA, and IgM) levels by Flow cytometer and rate scatter immunoturbidimetry, and investigated the mutation profile of BTK gene through Sanger sequencing and Real- Time PCR in 22 XLA patients. Results: We described the clinical symptoms and investigated the genetic mutations in 22 XLA patients, and then identified six novel mutations of BTK gene, which included 2 missense mutations (c.23G>T and c.112T>C), 2 frameshift mutations (c.522_523insC and c.1060delA), 1 large deletion (deletion of exon 2 to 5), and 1 splice-site mutation (c.1631+2T>C). Prenatal diagnosis were performed in six families (F10, F11, F15, F18, F20 and F21). F10 was a male fetus without c.922_923delGA mutation; F15 was a male fetus without c.1631+1G>T splicing mutation; F20 was a female fetus without c.1931T>C mutation; and F21 was a male fetus without large deletion mutation. All four fetuses were less likely to become XLA patients in the future. Family 11 and Family 18 were male fetuses with c.1060delA and c.1684C>T mutations, respectively. The pregnant women in F18 chose to terminate the pregnancy, and the F11 chose to continue the pregnancy. Conclusion: It is noticeable that we confirmed the diagnosis of 22 XLA patients from 22 unrelated families and found six new pathogenic mutations. Prenatal diagnosis was performed in six families. Early genetic diagnosis and routine lifelong immunoglobulin replacement therapy can prevent and treat the infections of the XLA children

Background X-linked agammaglobulinaemia (XLA) (OMIM # 300755) is a rare immunodeficiency disease, which is caused by defective B cell development and extremely decreased numbers of mature B cells [1]. The main clinical symptoms of XLA is recurrent severe infections [2]. Its estimated incidence is approximately 1:250,000 and the responsible gene is Bruton's tyrosin kinase (BTK) [3,4]. The BTK gene is located at Xq21.3-Xq22 and include 37.5 kb that contain 19 exons. The BTK protein is a cytoplasmic tyrosine kinase and has five different functional domains, that is, the pleckstrin homology (PH), Tec homology (TH), Src homology 3 (SH3), SH2, and the kinase (TK) domains [5]. The N-terminal PH domain binds membrane phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and TH, SH3, and SH2 domains involved in protein-protein interactions. Y223 and Y551 represent two tyrosine phosphorylation sites in the SH3 and TK domain [6]. BTK activates many of major downstream signaling pathways, including the phosphoinositol-3 kinase (PI3K)-AKT pathway, phospholipase-C (PLC), protein kinase C, and nuclear factor kappaB (NF-kB) [7]. BTK also participates in the engagement to B-cell receptor (BCR) by antigens and induces a range of protein interactions and the recruitment of signaling molecules, resulting in B-cell survival, proliferation, differentiation, and the production of antibodies [8].

Patients and study design
From 2016 to 2019, 22 male XLA patients from 22 unrelated families in Henan province of China were enrolled in this study. XLA was diagnosed according to the diagnostic criteria for XLA developed by the Joint European Society for Immunodeficiencies Committee [9]. After determining the proband's BTK gene mutations, the fetal villi or amniotic fluid of the high risk pregnant women were performed for prenatal diagnosis. The mutation analysis of the fetal genome was carried out by DNA sequencing.
The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

Routine immunological analysis
The serum was separated from 3 ml of peripheral venous blood without anticoagulant treated. The immunoglobulin was tested by rate scatter immunoturbidimetry using Siemens BN II automatic protein analyzer. CD19+ was detected by FACSCanto II flow cytometer using 3 ml of EDTA-treated blood.

Genetic test
Genomic DNA was extracted from 2 ml of EDTA-treated peripheral venous blood of the proband and 4 their mother using Blood DNA Midi Kit D3494 (Omega Biotek, USA) through nucleic acid automatic extraction equipment (Eppendorf epMotion 5075 m, Germany). Amniotic fluid cell DNA was extracted and cleaned using QIAamp Blood DNA Midi Kit (250, Germany) and Genomic DNA Clean& Concentrator (Zymo Research, USA). The DNA sequence of BTK gene was obtained from the UCSC Genome Bioinformatics database (http://www.Genome.UCSC.edu) as the standard. PCR amplification was carried out using the related primers (Table S1) under the conventional PCR reaction conditions. The PCR product was confirmed using 2% agarose gel, and was purified using ABI BigDye3.1 kit for two-way sequencing. The sequencing product was separated using ABl3130xl gene sequencing machine. The sequencing results were compared by Chromas software in order to look for the genetic mutation loci. For novel mutations, sequencing of 100 alleles from normal controls was performed to rule out the possibility of polymorphisms. In addition, the large deletion mutation of the BTK gene was tested by QuantStudio 5 Real-Time PCR System (ABI, USA).

Clinical characteristics
Twenty-two families were enrolled in this study. The mean age of onset of XLA was 3 years, and the mean age of diagnosis was 7 years. At the time of diagnosis, the clinical infections are shown in Table   1. Of the types of infections, respiratory infection was the most common (n=19, 78.9%), followed by sinusitis, sepsis, otitis media and central nervous system infection.

Immunological features
As shown in Table 1, all patients exhibited very low percentage of CD19+ B cells and serum immunoglobulin (IgG, IgA, and IgM) levels at diagnosis. No patients received intravenous immunoglobulin (IVIG) substitution therapy before diagnosis. The percentage of CD19+ B cells in all patients was 0-1%, and ten of the sixteen patients had 0% B cell. There were 19 out of 21 patients that the concentration of serum IgG was less than 2 g/L. 2 out of 21 patients that the concentration of serum IgG was more than 2 g/L, but less than 5.66 g/L. The concentration of serum IgA (n=19) in all patients was less than 0.8 g/L. The concentration of serum IgM in all patients was less than 0.3 g/L except patient 2, patient 9 and patient 13.

BTK Mutation analysis
To confirm the diagnosis, mutation analysis of the BTK gene was performed ( Table 2) The mothers in the other six families were not tested because of death, divorce or subjective will. The pedigree of the above 22 families was shown in Table 2  Prenatal diagnosis There were 6 families (F10, F11, F15, F18, F20 and F21) who received prenatal diagnosis ( Figure 2 and Figure 3). It has been confirmed that the fetal villi or amniotic fluid have not been contaminated by the mother source. Family 10 was a male fetus without P. D308Lfs*14 mutation; Family 15 was a male fetus without c.1631+1G>T splicing mutation; Family 20 was a female fetus without P. F644S mutation; and Family 21 was a male fetus without large deletion mutation ( Figure 2). All four fetuses were less likely to become XLA patients in the future. The above four families chose to continue the pregnancy after genetic counseling. The umbilical cord blood was collected for genetic diagnosis after full-term delivery, and the results were consistent with the prenatal diagnosis. Telephone follow-up after 1 year was found that the general development of infants were normal. Family 11 and Family 18 were male fetuses with P. T354Pfs*49 and P. R562W mutations, respectively. After genetic counseling, the pregnant women in Family 18 chose to terminate the pregnancy, and the DNA 6 analysis of the abortion tissues was consistent with the prenatal diagnosis results. However, the pregnant women in Family 11 chose to continue the pregnancy.

Discussion
In this paper, we reviewed the clinical data of 22 male XLA patients from 22 unrelated Chinese families in Henan Province. Our results showed that all patients had typical clinical presentations including recurrent infections and hypogammaglobulinemia with few or absence B cells in the peripheral blood. Respiratory infection was the main clinical feature of the patients in this study, and it was consistent with the findings in other reports [10]. The mean age at diagnosis was 7 years, which is higher than previously reported series from the United States [11], but similar to the series reported from Other provinces of China [12].There was also a considerable delay in diagnosis, and especially Patient 6 was diagnosed after 17 years in this study. This could be because that the doctors were lack of awareness about the XLA in China.
The BTK gene is localized at Xq21.3-Xq22 and encompasses 37.5 kb that contain 19 exons. The first exon of this gene is a non-coding region, and the next 18 exons code the Btk protein [13]. As so far, 911 mutations of the BTK gene that are related with XLA have been found in the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=BTK). These mutations contain a variety of types, including missense mutations, nonsense mutations, splice site mutations, insertions, and deletions. The missense mutation is the most common type. It has shown that mutations can occur in the exons, introns, and promoters of the BTK gene [14,15]. Six novel mutations were found in our study ( Table 2). Three of these six novel mutations were point mutations, one was insertional mutagenesis and two were deletion mutations. Other mutations are recurrent mutations, which have been reported in other articles. Their mutation is the large deletions. This results in the loss of fragments in the peptide chain, and then the BTK protein lose the original function. The serious condition of these two patients has been 7 greatly alleviated after the diagnosis and IVIG treatment. Thus it can be seen that there is some correlation between genotype and phenotype in the XLA mutations in patients, and this is consistent with the previous report [16]. The age of onset and diagnosis of XLA was 5 years in Patient 5. His mother described he has no any sign of weakened immune systems before he was five years. He gets immunoglobulin injections twice a year, and is in reasonable health. The second male fetuses has the P. T354Pfs*49 mutation in BTK gene that is the same as his brother's in Family 11. This family chose to continue the pregnancy, considering the phenotype of the proband. Therefore, the different types and sites of mutations of BTK gene may lead to the different disease conditions in XLA.
XLA is the X-linked recessive genetic disease. The patient is mostly male and the carrier is female. It

Consent for publication
Informed consent for publication was obtained from all subjects or their parents.

Availability of data and material
The datasets generated and/or analysed during the current study are not publicly available.