Targeted next-generation sequencing revealed a novel homozygous mutation in the LRBA gene causes severe haemolysis associated with Inborn Errors of Immunity in an Indian family.

ABSTRACT Objectives LPS-responsive beige-like anchor protein (LRBA) deficiency abolishes LRBA protein expression due to biallelic mutations in the LRBA gene that lead to autoimmune manifestations, inflammatory bowel disease, hypogammaglobulinemia in early stages, and variable clinical manifestations. Materials and methods Mutational analysis of the LRBA gene was performed in Indian patients using targeted Next Generation Sequencing (t-NGS) and confirmed by Sanger sequencing using specific primers of exons 53. Then, bioinformatics analysis and protein modeling for the novel founded mutations were also performed. The genotype, phenotype correlation was done according to the molecular findings and clinical features. Results We report an unusual case of a female patient born of a consanguineous marriage, presented with severe anaemia and jaundice with a history of multiple blood transfusions of unknown cause up to the age of 5 yrs. She had hepatosplenomegaly with recurrent viral and bacterial infections. Tests for hemoglobinopathies, enzymopathies, and hereditary spherocytosis were within the normal limits. The t-NGS revealed a novel homozygous missense variation in exon 53 of the LRBA gene (chr4:151231464C > T; c.7799G > A) (p.C2600Y), and the parents were heterozygous. The further immunological analysis is suggestive of hypogammaglobulinaemia and autoimmune haemolytic anaemia. The bioinformatics tools are suggestive of deleterious and disease-causing variants. Conclusion This study concludes the importance of a timely decision of targeted exome sequencing for the molecular diagnostic tool of unexplained haemolytic anaemia with heterogeneous clinical phenotypes.


Introduction
Lipopolysaccharide (LPS)-responsive beige-like anchor protein (LRBA) deficiency, is one of the causes of Inborn Errors of Immunity (IEI), and presenting with autoimmunity and/or auto-inflammation phenotype comes under the group of common variable immunodeficiency (CVID-8; OMIM #614700) [1][2][3]. LRBA is ubiquitously expressed and regulates endosomal trafficking in endocytosis of ligand-activated receptors. In humans, it encodes the LRBA protein, associated with autophagy or self-digestion, which leads to antigen deficiency [4]. LRBA protein acts as signaling enzymes (PKA and PKC) with an A-kinase anchoring protein (AKAP) in organelles and membranes [5]. LRBA also regulates the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) expressions on the post-translational stage. It has the potential to suppress receptor and immune checkpoints [6]. The clinical manifestations are not well known, but humoral immune deficiencies can lead to IgA deficiency, and some have hypogammaglobulinaemia [7]. In most patients, recurrent infections are presented in early childhood. Most likely respiratory infections are more severe, which ultimately affect growth and difficulty to maintain weight, and some also develop various types of autoimmune haemolytic anaemia, idiopathic thrombocytopenic purpura, and also chronic diarrhea. In some cases, it is reported that LRBA deficiency can cause interstitial lung disease (ILD). The variable phenotypic symptoms are reported in LRBA deficiency. The testing for LRBA deficiency should be considered in cases without overt immunodeficiency. The low serum IgG and IgA and normal may find on Immunologic analysis. A normal or decreased B cells were seen in peripheral blood smear with less count of switched memory B cells and CD4 + regulatory T cells (Tregs). Bone marrow transplantation is the most valuable therapeutic option, but some patients may benefit from immunoglobulin replacement therapy. The IG prophylaxis (IV or SC) can be used regularly at a minimum of 500-600 mg/dL to maintain plasma levels [8,9].
The LRBA gene is located on chromosome 4 at 4q31.3. It is a 2863 codon spanning into 57 exons. So far, a total of 167 mutations have been reported in the Human Gene Mutation Database (HGMD®) (http:// www.biobase-international.com/product/hgmd), which includes 82 missense/nonsense mutations, 18 splicing mutations, 30 Small deletions, 14 Small insertions, 2 Small indels, 15 Gross deletions, 4 Gross insertions, 1 Complex rearrangement and 1 Regulatory modification [10,11]. Mutations in the LRBA are spread throughout the gene, affecting structural rearrangements due to the large numbers of mutations in exons. The discovery of the next-generation sequencing (NGS) technology plays an essential role in diagnosing several unexplained haemolytic anaemias and rare variable immunological disorders [12]. We report an unusual report of a female patient born of a consanguineous marriage, presented with severe anemia and jaundice with a history of multiple blood transfusions of unknown cause till 5yrs of age. Targeted Next-generation sequencing (t-NGS) technology identified a novel mutation in the LRBA gene. Her initial haematological and IEI work-up was normal. However, a timely decision to perform NGS helped diagnose underlying IEI due to LRBA deficiency in the patient who was transfusion-dependent and had a significant family history.

Clinical history
Written informed consent was taken for blood collection and DNA analysis from the patient's parents as a patient is a minor and for the publication of this report, as per the protocol of the institutional ethics review board of NIIH Mumbai.The clinical history of the patient was obtained from the previous medical records. She was referred to our Institute with major complaints of low haemoglobin (Hb-6.7 g/dl), yellowish discoloration of eyes and sclera. There was a history of jaundice on 3rd day of life, which was relieved with phototherapy. Parents also observed that child had weakness and used to be irritable intermittently. For 2 years, she had received ten units of packed red cells transfusions. Her peripheral blood smear revealed anisopoikilocytes and a few microspherocytes and a few nRBC. The patients' Coombs test (DAT) was strongly positive. The reticulocyte count was 40%, and the indirect bilirubin level was 6.15 mg/dl (total bilirubin level was 7.14 mg/dl).

Haematological, biochemical, immunological analysis
Routine laboratory haematology investigations, which include complete blood count, red cell indices, reticulocyte count, and morphological examination of peripheral blood smears, were performed as per the Dacie & Lewis [13]. The red cell enzymopathies (G6PD, PK, GPI, P5'N, and HK) were evaluated by the method described by Beutler at 340 nm at 37 0 C for 10 min. on a spectrophotometer (Analytical JENA, Germany) [14]. Eosin-5'-maleimide (EMA) binding assay by flow cytometry was performed to rule out Hereditary Spherocytosis [15]. Coombs test (DAT), PNH work-up, and lymphocyte subset analysis assay was done by methods described in standard operating procedures for immunophenotyping of haematolymphoidneoplasms. Serum IgA, IgG, and IgM levels in this patient were measured by nephelometric assays on the Behring Nephelometer Analyzer (BNA) [16]. All the analytical procedures and examinations were done as per the Helsinki Declaration of 1975.

DNA extraction and molecular characterization
Peripheral blood was collected from patients, parents, and healthy controls after taking informed consent. DNA extracted from whole blood by the protocol mentioned in our previous published paper. The Library for targeted next-generation sequencing (t-NGS) analysis was generated using Illumina's TruSeq Custom Amplicon v1.5 kit (FC 130 1001). We used 250 ng genomic DNA and followed the protocol as per the technical manual. The DNA samples were pooled and loaded at 20 pM on MiSeq using a v3 600 cycle reagent kit and sequencing 2×301 paired-end reads (Illumina, San Diego, CA, USA). Total coverage was between 80-100X on the Illumina MiSeq sequencing platform. The updated number of genes included in the t-NGS panel was listed in the supplementary Table (Table-S1). The accession numbers of genes listed in this panel were derived from the Ensemble Genome Browser (www.ensembl.org) and Single Nucleotide Polymorphism database (dbSNP at www.nchi.nlm.nih. gov/SNP) [17] The alignment of the sequences was done with the human reference genome (GRCh37/ hg19) using the BWA program and analyzed using Picard and GATK version 3.6. The pathogenic mutations identified were annotated in OMIM, ClinVar, GWAS, and HGMD databases accordingly. A variant identified by these bioinformatics tools was further confirmed by sequencing independent PCR products. We further verified in the 1000 Genomes, https://www.internationalgenome.org/and Human Gene Mutation Databasehttp://www.hgmd.cf.ac.uk/ ac/index.php to avoid polymorphic variations.

Results
A five-year-old Indian female child, born of third degree consanguineous marriage, fifth by birth order, of the Muslim community, hailing from interior parts of West Bengal (India), was referred to our laboratory. We report an unusual report of this patient presented with severe anaemia and jaundice with a history of multiple blood transfusions of unknown cause until the age of 5 yrs. She was complaining of yellowish discoloration of sclera and skin at intervals. She had highcolored urine and abdominal distension for one month. Parents also observed that child had weakness and used to be irritable intermittently. From her previous case history, it was found to have microspherocytes, anisocytosis, and increased reticulocyte count (>30%) and hepatosplenomegaly at the age of 2 yrs. She had a history of recurrent bacterial infections. She was initially following up with a local general physician for anaemia and was received a blood transfusion for her low haemoglobin of 6.7gm/dL. Later she was referred to a higher center because of underlying haematological conditions. Since the age of 2 years, she had received ten units of packed red cells transfusions. The last transfusion was received three months back before referring to NIIH Mumbai. There was a history of jaundice on 3rd day of life, which was relieved with phototherapy. However, history of receiving exchange transfusion with significant family history was recorded. One of her firstborn elder siblings' brothers was expired of similar complaints at the age of 9 days. Another younger (sibling) brother died at five years of age (unknown reason). Her family pedigree chart is shown in Figure 1A.
On examination child's general condition was fair. She was febrile and showed a mild pallor and moderate icterus. Her abdominal examination revealed splenomegaly (3 cm below costal margin); later confirmed on ultrasonography (USG). There were no palpable lymph nodes on examination. Other systemic investigations were within normal limits. Her work-up for red cell membrane disorders, enzymopathies, and inherited haematological diseases were ruled out. Peripheral blood smear showed anisocytosis, poikilocytosis, microcytosis, polychromasia, and left to shift of polymorphs. The hematological, biochemical, and molecular data are shown in Table 1. The Hb-electrophoresis of the index cases and her mother was normal. Haemoglobinopathies assays were normal in the patient and her parents. The most common red cell enzymes (PK, G6PD, GPI, and P5N) are within the normal limit, whereas LDH activity was increased to 3144 U/L ( Table 1). The EMA binding assay for red cell membrane protein defect was showed normal mean channel fluorescence (MCF) . There were few nRBCs, and reticulocyte count was increased to 40%.  Table 2). The patients' Coombs test (DAT) was strongly positive. Considering underlying autoimmune hemolysis, she was prescribed a low dose of oral steroids, and the least incompatible packed cell unit was transfused under constant medical supervision in view of positive DAT. The immunological analysis for affected individuals with an inborn error of immunity and severe transfusion history are shown in Table 2. Also, with suspicion of underlying inborn errors of immunity, a screening work-up was done that included LSSA, Immunoglobulin levels, and NBT. All reports were within normal limits except borderline IgG (4.3 g /dl) was detected. Her PNH work-up was carried on FLAER, and it was normal. Her autoimmune work-up showed the presence of anti-ANA antibodies (weak +). All her laboratory investigations are shown in Table 2.

Molecular and bioinformatics analysis
Since all biochemical investigations were normal, molecular analysis was recommended. Because of inadequate response, pediatrics Immune deficiency (PID) was suspected, and targeted clinical exome sequencing was performed. The t-NGS results revealed a novel homozygous missense variation in exon 53 of the LRBA gene (chr4:151231464C > T; c.7799G > A)  . This automated server-generated ten prospective models based on the amino acid sequence. For our study, we picked the template ID-6rxz.1.A and Swiss Protein databank viewer and PyMol software were used to study the effect of mutation on the structure and function of the protein (http://www.pymol.org/ and https:// spdbv.vital-it.ch/). The modeled structure is composed of a single chain of helices and loops. Swiss PBD viewer allowed visualizing the wild type residue Cys2600 along with Mutated type Tyr2600. We hypothesize that the phenyl ring of tyrosine hinders the side chains of neighboring amino acid Q2599 and disturbs the stability of the protein. (Figure 1C and 1D)

Discussion
LRBA is present in all cells but highly expressed in immune effectors cells. It is a cytosolic protein mostly present in lysosomes, trans-Golgi apparatus, endoplasmic reticulum, and endocytosis vesicles [18]. LRBA plays an important role in the polarization of vesicle trafficking and is also involved in the number of cellular processes [19]. This patient was initially referred to us to rule out the cause of unexplained haemolytic anaemia. The preliminary examination of the child revealed mild pallor, moderate icterus, and had hepatosplenomegaly. Her reticulocyte count was very high (40%), and her lactate dehydrogenase (LDH) enzyme level was significantly elevated indicates severe haemolysis. All the possible causes of haemolytic anaemia were ruled out. The HPLC for haemoglobinopathies was first investigated, followed by rare red cell enzymopathies including G6PD deficiency, pyruvate kinase (PK) deficiency, glucose phosphate isomerase (GPI) deficiency, pyrimidine 5' nucleotidase (P5'N) deficiency, and also perform EMA test for hereditary spherocytosis. The child had normal haematological and immunological work-up. Her weakly positive autoimmune work-up and significant family history were important clues of underlying immunodeficiency for us to carry out exome sequencing. The result of t-NGS revealed novel (p.Cys2600Tyr) homozygous mutation confirmed LRBA deficiency, and both the parents were heterozygous for the same mutation as there was a history of consanguinity. Most of the reported patients of LRBA deficiency are mainly due to homozygous or compound heterozygous mutations. In a few patients, large structural rearrangements affecting LRBA protein also have been reported [20,21]. In this case, we confirmed that homozygous mutation was the result of consanguineous marriages within the family. Also, immune dysregulation was confirmed by immunoglobulin assay and had recurrent infections in childhood. This particularly respiratory infection may be due to autoimmune haemolytic anaemia. A low IgG level suggests hypogammaglobulinaemia, and the molecular study confirmed LRBA protein deficiency. This is a rare report of molecular characterization of LRBA protein deficiency identified novel (p.Cys2600Tyr) mutation in the Indian population. So far, only one mutation has been reported previously in Indian patients (p.Val1695Leu) [22]. In 2011, Yong et al. identified first time the genetic basis for this syndrome. They stated that it is a common variable immunodeficiency-8 (CVID) with autoimmunity due to partial loss-of-functional mutations in the LRBA protein [23]. So far, no specific treatment is available to treat LRBA deficiency, but sirolimus and glucocorticoids are used as part of treatment. Some clinicians recently started using a CTLA4-fusion protein, Abatacept, to treat CVID [24]. In Many reported cases of LRBA deficiency, the allogeneic hematopoietic stem cell transplantation (HSCT) is preferably used as an effective therapeutic option. However, there is an overall survival rate is 70.8% that underwent HSCT [25]. The most common clinical manifestations reported in the recent publications on LRBA deficiency are almost the same as 82% of cases showed autoimmunity followed by 63% enteropathy, 57% splenomegaly, and 47% pneumonia. There was a similar history of death in this family also. One of her elder firstborn brothers was expired of similar complaints at the age of 9 days, and the other younger brother (sibling) died at five years of age (unknown reason). History of sibling death due to autoimmune manifestations of unusual severity in the consanguineous family should be considered for LRBA deficiency.
Other clinical presentations like sideroblastic anaemia, developmental delay, pan hypogammaglobulinaemia, and low B cells were also reported in LRBA deficiency, which disrupts the protein function and damages multi-organs [26]. This index case was noted to have hypogammaglobulinaemia and has been treated with regular blood transfusion as she had severe anaemia warranting transfusions, highlighting the heterogeneity of congenital anaemia. In Indian laboratories, next-generation sequencing is very rarely used for genetic diagnosis for hereditary haemolytic anaemia such as haemoglobinopathies, RBC enzymopathy, and membrane disorder whereas, whole-exome sequencing is used to diagnose Common Variable Immunodeficiency (CVID) in developed countries [27,28]. Next-generation sequencing is beneficial for the diagnosis of transfusion dependant hemolytic anaemias. The targeted gene panel has a higher success rate than those of whole-exome sequencing or genome sequencing. These gene panels are customized for specific genetic diseases, considering the high specificity and sensitivity of the entire gene with the maximum coverage because of the limited numbers of the gene in the customized NGS panel. This targeted NGS panel can provide a diagnosis to 80% of cases. However, this report is mainly based on their clinical history, type of mutation, and phenotype-genotype correlations. This is the second case of novel mutation causing LRBA deficiency from the Indian subcontinent to the best of our knowledge.

Conclusions
We identified a homozygous missense variant in LRBA, which likely explains inborn Errors of Immunity with severe haemolytic anaemia in this family compared to similar data from the literature. Our study concludes that the importance of a timely decision of targeted exome sequencing is a successful molecular diagnostic tool for unexplained haemolytic anaemia with heterogeneous clinical phenotypes.

Ethics approval and consent to participate
This study was approved of the Institutional Ethical Committee of ICMR-National Institute of Immunohaematology, Mumbai. All procedures performed in studies involving human participants were performed per the ethical standards of the institutional review board of the Institute and followed the 1964 Helsinki declaration. We obtained written informed consent from all individual members included in the study. In a minor, we received written consent from the parents or legal guardians.

Consent for publication
We obtained written informed consent to identify images or other personal or clinical details from all the participants. In the case of a minor, we obtained written informed consent for publications identifying pictures or additional clinical information from the parents or legal guardians.