Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an extremely rare inherited primary immunodeficiency characterized by recurrent bacterial infections, decrease in number of mature B cells and low serum immunoglobulins. XLA is caused by mutations in the gene encoding Bruton's tyrosine kinase. We report a case of a young Indian boy suspected to have XLA. Immunophenotyping was performed for the affected child using CD20, CD19 and CD3 antibodies. Whole exome sequencing was performed using trio-based approach. The variants were further analyzed using capillary sequencing in the trio as well as maternal grandmother. Initial immunophenotyping in the affected child showed decreased count of CD19+ B cells. To strengthen the clinical findings and confirm the diagnosis of XLA, we performed whole exome sequencing. Our analysis identified a novel frameshift insertion (c.1325dupT) in the BTK gene, which was further validated by Sanger sequencing. Our approach shows the potential in using whole exome sequencing to pinpoint the molecular lesion, enabling timely diagnosis and genetic counseling, and potentially offering prenatal genetic testing for the family.

This article is included in the Rare diseases collection.

Introduction
Primary immunodeficiencies are congenital defects in the immune defence mechanisms of the host against invading pathogens. X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disorder (OMIM# 300755) characterized by recurrent infections causing pneumonia, conjunctivitis, gastrointestinal infections, otitis media and sinopulmonary infections 1 , which may require frequent hospitalization. The disease is extremely rare with an estimated prevalence of 3-6 per 1,000,000 males 2 and is inherited in an X-linked recessive manner. The disease arises from genetic defects, due to which the mature B lymphocytes are either low in number or completely absent in the bloodstream while also exhibiting a complete absence of serum immunoglobulins 1 .
The absence of immunoglobulins results in a compromised humoral immune response, which makes the affected individual extremely vulnerable to infections with encapsulated bacteria and enteroviruses. Molecular genetic studies have conclusively mapped the genetic locus of XLA to the gene encoding for the Bruton's tyrosine kinase (BTK) 3 . A comprehensive mutation database (BTKbase) lists over 700 unique mutations associated with XLA that affect the activity of BTK protein 4 .

Case report
A five year old boy of north Indian origin, born out of a nonconsanguineous marriage ( Figure 1A) presented to the hospital with headache, fever and a history of recurrent infections requiring hospitalization. The antenatal and perinatal periods were uneventful. His clinical history revealed that the child had been hospitalized for septicaemia and underwent treatment with intravenous (IV) antibiotics for 2 weeks at one year of age. Later, at 2.5 years, the child developed fever with swelling in right knee joint that was diagnosed as septic arthritis. The child was again hospitalized at 5 years of age for pyogenic meningitis. The culture of cerebrospinal fluid was found to be positive for Pseudomonas aeruginosa. On close examination, the tonsils were found to be absent and there was no peripheral lymphadenopathy. The immunoglobulin profile revealed serum level of IgG 50 mg/dl (200-700 mg/dl), IgA 5 mg/dl (40-200 mg/dl) and IgM 9 mg/dl (50-200 mg/dl). The hemogram showed hemoglobin concentration of 9.5 g/dl (11.5-15.5 g/dl), total leukocyte count 14800/cumm (6000-13500/cumm), platelet count 931000/cumm (150000-400000/cumm) and erythrocyte sedimentation rate ESR 18mm/1st hr (0-14 mm/1st hr).
The blood flow cytometric analysis of the affected child was performed to evaluate the status and count of mature B cells. The patient (III.1) has only CD3+ lymphocytes as observed on CD19/CD3 dot plot and there was complete absence of CD19+ cells (0.02%) ( Figure 1B). The staining for BTK protein on CD14+ monocytes showed decreased (20.71%) expression of BTK in proband as compared to control (72.07%) ( Figure 1C). This observation was consistent with the diagnosis of XLA 5 . The patient had no family history of immunodeficiency and no such characteristics were present in any other family members.
The blood samples were collected and processed for genomic DNA isolation by salting out method 6 . We performed the whole exome sequencing using trio-based approach (patient, mother and father). In brief, the whole-exome library was prepared using Nextera rapid capture expanded exome kit (Illumina Inc., USA) according to manufacturer's standard protocol. Sequencing was performed on Illumina Hiseq2500 platform (Illumina Inc., USA) with 130bp paired-end reads. Reads were trimmed using Trimmomatic v0.33 7 and aligned to reference genome hg19 (GRCh37) by Stampy v1.0.23 8 along with BWA v0.7.12-r1039 9 . PCR duplicates were marked using Picard tools v1.127. Variations were called using Platypus v0.7.9 10 and annotated using ANNOVAR 11 . Analysis revealed a novel frameshift insertion c.1325dupT in exon 14 of the BTK gene. The mutation was found to be homozygous in child and heterozygous in mother. The identified mutation c. 1325dupT has not yet been reported in the BTKbase 4 and absent in ExAC, 1000genome as well as internal control databases from South Asia and Middle East (http://clingen.igib.res.in/almena), which confirms the novelty of the variation. The mutation evaluation by SIFT Indel tool (http://sift.bii.a-star.edu.sg/www/ SIFT_indels2.html, 12) was predicted to be damaging and caused nonsense mediated decay (confidence score 0.858). Further in silico analysis suggested that the mutation causes Isoleucine at 443 residue in BTK to be replaced by Histidine and introduces a premature stop codon at 444 residue, which lies in the kinase domain of the BTK protein ( Figure 1D).
The variation was further validated by PCR amplification of region encompassing the variation using specific primer sets (Forward primer: 5'-CCCCAAATGCTACTGAGATGGT-3' and Reverse primer: 3'-CCTATTTCTACCCCAGTAGGGA-5') with the annealing temperature of 59°C using Brazilian taq polymerase (Invitrogen, USA) according to manufacturer instruction. PCR products were purified using Qiaquick PCR purification kit (QIAGEN, Germany). Capillary sequencing was performed using BigDye-terminator chemistry on 3130xl Genetic Analyzer (Applied Biosystems, USA). Analysis revealed that the mutation was homozygous in child (III.1), heterozygous in mother (II.3) and absent in father (II.2) and maternal grandmother (I.4) ( Figure 1E).

Discussion
XLA is a primary immunodeficiency disorder characterized by recurrent infections causing pneumonia, conjunctivitis, gastrointestinal infections, otitis media and sinopulmonary infections 1 . Whole exome sequencing has been increasingly used to identify mutations in rare genetic diseases mainly due to the speed, cost and amenability as compared to traditional capillary sequencing 13 . Recent reports have suggested the application of whole exome sequencing for mutation detection in a variety of primary immunodeficiency cases 14,15 .
In the present report, we performed whole exome sequencing using a trio-based approach for a child from an Indian family who presented to the clinic with the suspected diagnosis of XLA. The

Amendments from Version 1
The updated version of the manuscript has incorporated the reviewer's suggestions, a revised Figure 1, and patient's BTK expression profile. lack of readily available specific gene sequencing assays coupled with absence of a next-generation sequences (NGS) based targeted gene panels for XLA provided the impetus for attempting exome sequencing.

REVISED
Our exome sequencing analysis revealed a novel frameshift inser tion c.1325dupT in exon 14 of the BTK gene. The mutation was found to be homozygous in patient and heterozygous in unaffected mother, which was further validated by capillary sequencing. This confirmed the X-linked inheritance and carrier status of the mother for the mutation. The mutation was found to be absent in unaffected father and maternal grandmother. The identified muta tion c.1325dupT was found to be novel and damaging due to --truncation of the BTK at 444 residue of kinase domain. The flow cytometric analysis for BTK stained monocytes shows decreased expression of BTK in proband as compared to control ( Figure 1C). The mutation excludes functionally well characterized active site residue Y551 of the protein. Additionally, nonsense mutation at the codon Y425X, E441X, Q459X and Q497X is known to cause loss of kinase activity of BTK, which has been previously demonstrated using in vitro kinase activity assay in Japanese individuals 16 . Since c.1325dupT (p.F442fsX2) lies in the vicinity of the above mentioned well studied codon positions, the effect of the mutation is expected to be damaging to BTK. Currently the patient is on intravenous immunoglobulin replacement therapy (15 g every 3-4 weekly) and is responding well. We could not avail the RNA samples to perform transcript analysis or functional studies. 1.

2.
3. XLA is a rare disease which gave this case report high value for being indexed. However I have some concerns regarding the report as below:

Open Peer Review
Since the patient was suspected with XLA and BTK gene is well known as the cause of the condition. Why Whole Exome Sequencing (WES) was conducted for this case? Why not conduct targeted sequencing for BTK since that would be much cheaper and easier to analyse. Since WES was conducted, I think authors should provide other results from WES analysis such as variant analysis, etc.
The results in Figure 1D for heterogeneous needs further explanation. It seems like the heterogeneous is not having two peaks at the asterisks position, but having two peaks at 5 positions downstream to the asterisks.
I would like to suggest evidence to be provided for the truncated BTK gene due to the mutation (Reverse transcriptase-PCR).
I would like to suggest the authors to referred to other database with bigger number of samples 4. I would like to suggest the authors to referred to other database with bigger number of samples such as Exome Aggregation Consortium and 1000 Genomes database (not only BTKbase).
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author The variant found in the index case was also checked in the ExAC and not found to be reported there.
No competing interests were disclosed.
The whole exome sequencing generated a total of 67437 variants in the patient, and 2046 and 891 variants for the mother and father respectively. Analysis of the variants are summarized in . Table 1 Analysis revealed four variants in genes related to agammaglobulinemia. Out of which three were with an allele frequency quite common in the Indian population. The BTK variant was previously not described in the variant frequency databases. The variants are summarized in . Table 2 2. The results in Figure 1D for heterogeneous needs further explanation. It seems like the heterogeneous is not having two peaks at the asterisks position, but having two peaks at 5 positions downstream to the asterisks.
The asterisk position represents the site where 'A' base is inserted. The frameshift Clarification 2: starts to be noticed after 1 peak due to AAA repeats. Please see . Figure   3. I would like to suggest evidence to be provided for the truncated BTK gene due to the mutation (Reverse transcriptase-PCR).
We could not avail the RNA sample for the case for further studies. We have Clarification 3: added a sentence in the discussion to highlight this caveat. Ample evidence suggests that the nonsense mutation at the codon Y425X, E441X, Q459X and Q497X is known to cause loss of kinase activity of BTK, which has been previously demonstrated using kinase activity assay in vitro in Japanese individuals (Hashimoto et al. 1996) . Since c.1325dupT (p.F442fsX2) lies in the vicinity of the above mentioned well studied codon positions, the effect of the mutation may be damaging to BTK. There is bit correction in the total number of variations mentioned in comment box (See