Phylogeny of Tec Family Kinases: Identification of a Pre-Metazoan Origin of Btk, Bmx, Itk, Tec, Txk and the Btk Regulator SH3BP5

It is generally considered mammals and birds have five Tec family kinases (TFKs): Btk, Bmx (also known as Etk), Itk, Tec, and Txk (also known as Rlk). Here, we discuss the domains and their functions and regulation in TFKs. Over the last few years, a large number of genomes from various phyla have been sequenced making it possible to study evolutionary relationships at the molecular and sequence level. Using bioinformatics tools, we for the first time demonstrate that a TFK ancestor exists in the unicellular choanoflagellate Monosiga brevicollis, which is the closest known relative to metazoans with a sequenced genome. The analysis of the genomes for sponges, insects, hagfish, and frogs suggests that these species encode a single TFK. The insect form has a divergent and unique N-terminal region. Duplications generating the five members took place prior to the emergence of vertebrates. Fishes have two or three forms and the platypus, Ornithorhynchus anatinus, has four (lacks Txk). Thus, not all mammals have all five TFKs. The single identified TFK in frogs is an ortholog of Tec. Bmx seems to be unique to mammals and birds. SH3BP5 is a negative regulator of Btk. It is conserved in choanoflagellates and interestingly exists also in nematodes, which do not express TFKs, suggesting a broader function in addition to Btk regulation. The related SH3BP5-like protein is not found in Nematodes

Bruton tyrosine kinase in immunodeficiency and in B-cell malignancy

BTK inhibitors have induced high response rates in the treatment of leukemias and lymphomas. Ibrutinib is the first-in-class US Food and Drug Administration (FDA)-approved BTK covalent inhibitor to treat chronic lymphocytic leukemia (CLL). However, a sub-group of patients develops resistance to ibrutinib therapy. The most common resistance mechanism is substitution of cysteine 481 to serine of BTK, the residue to which ibrutinib binds irreversibly. Few other amino acid replacements at this site had been characterized. In paper I, we therefore performed functional analysis of all the possible amino acid replacements at C481 site due to a single nucleotide change. We also included threonine substitution because of its structural similarity to serine. BTK with cysteine-to-threonine substitution retains catalytic activity and cause ibrutinib resistance. Thus, we identified a new potential resistant variant, BTK C481T for BTK irreversible inhibitors. For the replacement with arginine, phenylalanine, tryptophan or tyrosine, BTK enzymatic activity was completely ablated, while glycine substitution still showed some kinase activity, but to a much lower extent. CLL patients receiving ibrutinib treatment show rapid mobilization of tumor cells from lymph nodes (LN) to peripheral blood (PB). However, the detailed mechanism of ibrutinibinduced tumor cell redistribution has not been clarified. In paper II, we tried to explore this observation by analyzing changes in plasma inflammation-related biomarkers, transcriptional levels in CLL cells, B-cell activation and migration markers, and PB mononuclear cell populations as early as 9h after ibrutinib treatment. We compared the changes between before and at 6 time points after treatment initiation in LN and PB. We observed a significant downregulation of 10 plasma inflammation-related markers, mainly chemokines but not CLL-derived within 9 hours. RNA-sequencing data showed significant and continuous expression changes of genes related to B-cell receptor (BCR) signaling and CLL biology. We conclude that ibrutinib rapidly blocks an ongoing inflammatory response and in particular influences LN CLL cells. Loss-of-function (LOF) mutations of BTK lead to a severe block in B lineage development, as seen in X-linked agammaglobulinemia (XLA). In paper III, we analyzed a large number of XLA patients, including 108 previously unreported cases. The tolerance to single amino acid replacements was predicted for full-length BTK. Moreover, we compared these germline XLA-causing mutations with those acquired in leukemia and lymphoma. Based on published cases and those reported to a mutation database, BTK mutation spectrum in more than 10,000 BTK-independent tumors was compared to the BTK-dependent malignancies, CLL and mantle cell lymphoma (MCL), and also to BTK-potentially-dependent malignancies, like diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and germinal center (GC)-derived B-cell lymphoma. This analysis for the first time identifies BTK to be a potential tumor suppressor in a subset of DLBCL and FL. Therefore, whether BTK inhibitors, which show highly efficient therapeutic effects in CLL and MCL, might promote tumorigenesis in a subset of other B cell malignancies remains an open question

Tools and annotations for variation

Since the finishing of the Human Genome Project, many next-generation (NGS) or high-throughput sequencing platforms have emerged. One of the applications of NGS technology, variant discovery, can serve as a basis for precision medicine. Large sequencing projects are generating huge amounts of genetic variation data, which are stored in databases, either large central databases such as dbSNP, or gene- or disease-centered locus-specific databases (LSDBs). There are many variation databases with many different formats and varying quality. Apart from storage and analysis pipeline capacity problems, the interpretation of the variation is also an issue. Computational methods for predicting the effects of variants have been and are being developed, since experimental assessment of variation effects is often not feasible. Benchmark datasets are needed for the development and for performance assessment of such prediction methods.We studied quality related aspects of variant databases and benchmark datasets. The online tool called VariOtator was developed to aid in the consistent use of the Variation Ontology, which was specifically developed to describe variation. Standardization is one aspect of database quality; the use of an ontology for variant annotation will contribute to the enhancement of it.BTKbase is a locus-specific database containing information on variants in BTK, the gene involved in X-linked agammaglobulinemia (XLA), a primary immunodeficiency. If available, phenotypic data, i.e. the variant effects, are also provided. Statistics on variants and variation types showed that there is a wide spectrum of variants and variation types, and that the distribution of protein variants in the different BTK domains is not even.The VariSNP database containing datasets with neutral (non-pathogenic) variants was generated by selecting variants from dbSNP and filtering for variants found in the ClinVar, PhenCode and SwissProt databases. Variants in these three databases are considered to be disease-related. The VariSNP database contains 13 datasets following the functional classification of dbSNP, and is updated on a regular basis.To study the sensitivity to variation in different protein and disease groups, we predicted the pathogenicity of all possible single amino acid substitutions (SAASs) in all proteins in these groups, using the well-performing prediction method PON P2. Large differences in the proportions of harmful, benign and unknown variants were found, and distinctive patterns of SAAS types were found, both in the original and variant amino acids.Representativeness is one quality aspect of variation benchmark datasets, and relates to the representation of the space of variants and their effects. We studied the coverage and distribution of protein features, including structure (CATH) and enzyme classification (EC), Pfam domains and Gene Ontology terms, in established benchmark datasets. None of the datasets is fully representative. Coverage of the features is in general better in the larger datasets, and better in the neutral datasets. At the higher levels of the CATH and EC classifications, all datasets were unbiased, but for the lower levels and other features, all datasets were biased

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults

<p>Abstract</p> <p>Background</p> <p>X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Bruton's tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood.</p> <p>Methods</p> <p>Flow cytometric analysis of the peripheral monocytes using the anti-BTK antibody was used to characterize a 27 year old male patient with mild hypogammaglobulinemia (IgG, 635 mg/dl; IgM, 11 mg/dl; IgA, <5 mg/dl). He had suffered from frequent pneumonia since age 25 but had no history of frequent infections in his childhood or in adolescence. Sequencing of the BTK cDNA obtained from an Epstein–Barr virus-transformed B lymphoblastoid cell line derived from the bone marrow of the patient was performed to confirm a genetic defect.</p> <p>Results</p> <p>Flow cytometric analysis of cytoplasmic BTK protein in peripheral monocytes indicated that the patient presents a rare case of adult-onset XLA and that his mother is an XLA carrier. Sequencing of the BTK gene revealed a deletion of AG in the codon for Glu605 (AGT), resulting in an aberrant stop codon that truncates the BTK protein in its kinase domain.</p> <p>Conclusions</p> <p>This case suggests that some XLA cases may remain undiagnosed because they only show mild hypogammaglobulinemia and they lack repeated infections in childhood. Flow cytometric analysis is a powerful method to screen these patients.</p

X-linked agammaglobulinemia diagnosed late in life: case report and review of the literature

<p>Abstract</p> <p>Background</p> <p>Common variable immune deficiency (CVID), one of the most common primary immunodeficiency diseases presents in adults, whereas X-linked agammaglobulinemia (XLA), an inherited humoral immunodeficiency, is usually diagnosed early in life after maternal Igs have waned. However, there have been several reports in the world literature in which individuals have either had a delay in onset of symptoms or have been misdiagnosed with CVID and then later found to have mutations in Bruton's tyrosine kinase (BTK) yielding a reclassification as adult-onset variants of XLA. The typical finding of absent B cells should suggest XLA rather than CVID and may be a sensitive test to detect this condition, leading to the more specific test (Btk mutational analysis). Further confirmation may be by mutational analyses.</p> <p>Methods</p> <p>The records of 2 patients were reviewed and appropriate clinical data collected. BTK mutational analysis was carried out to investigate the suspicion of adult-presentation of XLA. A review of the world literature on delayed diagnosis of XLA and mild or "leaky" phenotype was performed.</p> <p>Results</p> <p>2 patients previously diagnosed with CVID associated with virtual absence of CD19⁺B cells were reclassified as having a delayed diagnosis and adult-presentation of XLA. <b>Patient 1</b>, a 64 yr old male with recurrent sinobronchial infections had a low level of serum IgG of 360 mg/dl (normal 736–1900), IgA <27 mg/dl (normal 90–474), and IgM <25 mg/dl (normal 50–415). <b>Patient 2</b>, a 46 yr old male with recurrent sinopulmonary infections had low IgG of 260 mg/dl, low IgA <16 mg/dl, and normal IgM. Mutational analysis of BTK was carried out in both patients and confirmed the diagnosis of XLA</p> <p>Conclusion</p> <p>These two cases represent an unusual adult-presentation of XLA, a humoral immunodeficiency usually diagnosed in childhood and the need to further investigate a suspicion of XLA in adult males with CVID particularly those associated with low to absent CD19⁺B cells. A diagnosis of XLA can have significant implications including family counseling, detecting female carriers, and early intervention and treatment of affected male descendents.</p

A novel gene mutation, c.82delC (p.Arg28 Alafs5), in a Korean family with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a hereditary humoral immunodeficiency that results from Bruton’s tyrosine kinase (BTK) gene mutations. These mutations cause defects in B-cell development, resulting in the virtual absence of these lymphocytes from the peripheral circulation. Consequently, this absence leads to a profound deficiency of lg all isotypes, and an increased susceptibility to encapsulated bacterial infections. A 15-month-old Korean boy presented with recurrent sinusitis and otitis media after 6 months of age, and had a family history of 2 maternal uncles with XLA. Laboratory tests revealed a profound deficiency of Ig isotypes, and a decreased count of CD19+ B cells in the peripheral circulation. Based on his family history and our laboratory test results, he was diagnosed with XLA. We performed BTK gene analysis of peripheral blood samples obtained from family members to confirm the diagnosis. Mutational analysis revealed a novel hemizygous frameshift mutation (c.82delC, p.Arg28Alafs*5), in the BTK gene. His mother and maternal grandmother were heterozygous carriers of this mutation and his two maternal uncles were hemizygous at the same position. After XLA diagnosis, intravenous immunoglobulin (400 mg/kg, monthly) treatment was initiated; recurrent sinusitis and otitis media were subsequently brought under control. To our knowledge, this is the first reported case of a Korean pedigree with a novel mutation in the BTK gene

Bruton’s Disease

Bruton’s disease, in other terms X-linked agammaglobulinemia (XLA), is the first reported primary immunodeficiency in 1952, caused by a single genetic defect. The development of B cell is under control of signals transmitted by the B-cell antigen receptor (BCR) complex. Lyn, Syk, and Bruton’s tyrosine kinase (BTK) are cytoplasmic protein tyrosine kinases. XLA is caused by mutations in the Btk gene, and Btk mutations are responsible for 85% of all antibody deficiencies. Btk mutation interrupts the B-cell development at the pre-B-cell stage, resulting in the absence of B lymphocytes and plasma cells in peripheral blood and peripheral lymphoid tissues. Up till now, 380 unique mutations have been identified. Autosomal recessive forms of agammaglobulinemia also result in B-cell defects, but more severe bacterial infections are seen in XLA patients due to absolute block in early B-cell development. All serum immunoglobulin isotypes are decreased, and antibody production especially against vaccine antigens is impaired

Tubulointerstitial nephritis complicating IVIG therapy for X-linked agammaglobulinemia

BACKGROUND: Patients with X-linked agammaglobulinemia (XLA) develop immune-complex induced diseases such as nephropathy only rarely, presumably because their immunoglobulin (Ig) G concentration is low. We encountered a patient with XLA who developed tubulointerstitial nephritis during treatment with intravenous immunoglobulin (IVIG). CASE PRESENTATION: A 20-year-old man was diagnosed with XLA 3 months after birth and subsequently received periodic γ-globulin replacement therapy. Renal dysfunction developed at 19 years of age in association with high urinary β2-microglobulin (MG) concentrations. A renal biopsy specimen showed dense CD3-positive lymphocytic infiltration in the tubulointerstitium and tubular atrophy, while no IgG4-bearing cell infiltration was found. Fibrosclerosis and crescent formation were evident in some glomeruli. Fluorescent antibody staining demonstrated deposition of IgG and complement component C3 in tubular basement membranes. After pulse steroid therapy was initiated, urinary β2-MG and serum creatinine concentrations improved. CONCLUSION: Neither drug reactions nor collagen disease were likely causes of tubular interstitial disorder in this patient. Although BK virus was ruled out, IgG in the γ-globulin preparation might have reacted with a pathogen present in the patient to form low-molecular-weight immune complexes that were deposited in the tubular basement membrane