Jouni Väliaho

Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling

Summary:  Brutons tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X‐linked agammaglobulinemia (XLA) in humans and X‐linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B‐lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full‐length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype–phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering‐RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA‐, Xid‐, and Btk‐knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid‐ and Btk‐KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C‐missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

Mutations of the human BTK gene coding for Bruton tyrosine kinase in X-linked agammaglobulinemia

X‐linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations lists 544 mutation entries from 471 unrelated families showing 341 unique molecular events. In addition to mutations, a number of variants or polymorphisms have been found. Mutations in all the five domains of BTK cause the disease, the single most common event being missense mutations. Most mutations lead to truncation of the enzyme. The mutations appear almost uniformly throughout the molecule. About one‐third of point mutations affect CpG sites, which usually code for arginine residues. The putative structural implications of all the missense mutations are provided in the database. BTKbase is available at http://www.uta.fi/imt/bioinfo. Hum Mutat 13:280–285, 1999.

PON-P: Integrated Predictor for Pathogenicity of Missense Variants

High‐throughput sequencing data generation demands the development of methods for interpreting the effects of genomic variants. Numerous computational methods have been developed to assess the impact of variations because experimental methods are unable to cope with both the speed and volume of data generation. To harness the strength of currently available predictors, the Pathogenic‐or‐Not‐Pipeline (PON‐P) integrates five predictors to predict the probability that nonsynonymous variations affect protein function and may consequently be disease related. Random forest methodology‐based PON‐P shows consistently improved performance in cross‐validation tests and on independent test sets, providing ternary classification and statistical reliability estimate of results. Applied to missense variants in a melanoma cancer cell line, PON‐P predicts variants in 17 genes to affect protein function. Previous studies implicate nine of these genes in the pathogenesis of various forms of cancer. PON‐P may thus be used as a first step in screening and prioritizing variants to determine deleterious ones for further experimentation. Hum Mutat 33:1166–1174, 2012.

Novel immunodeficiency data servers

The Internet contains scientific information in increasing amounts. It is possible to obtain the latest information, and Web services can easily be maintained and updated. We have set up three Internet services on immunodeficiencies. Immunodeficiency-related mutation infor mation is available in immunodeficiency mutation databases (IDbases). Currently 14 registries are distributed, including information about Bloom syndrome (BLMbase), X-linked agammaglobulinemia (BTKbase), X-linked and autosomal recessive chronic granulomatous diseases (CYBBbase for X-linked CGD, CYBAbase for p22(phox) deficiency, NCF1base for p47(phox) deficiency, NCF2base for p67(phox) deficiency), CD3gamma and CD3epsilon deficiencies (CD3Gbase, CD3Ebase), X-linked hyper-IgM syndrome (CD40Lbase), T-B+ severe combined immunodeficiency (JAK3base), V(D)J recombination defects (RAG1base, RAG2base), X-linked lymphoproliferative syndrome (SH2D1Abase), and ZAP-70 deficiency (ZAP70base). Information on laboratories analysing the genetic defects is collected to IDdiagnostics registry. Due to the rareness of immunodeficiencies there are very few laboratories performing genetic diagnostics. Such laboratories are listed in IDdiagnostics and physicians can use the registry to find a suitable laboratory for their diagnostic needs. Immunodeficiency Resource (IDR) is a comprehensive integrated knowledge base for all the information on immunode ficiencies, including clinical, biochemical, genetic, structural and computational data and analyses. All three services are available at http: //www.uta.fi/imt/bioinfo/.

IDR knowledge base for primary immunodeficiencies

BackgroundThe ImmunoDeficiency Resource (IDR) is a knowledge base for the integration of the clinical, biochemical, genetic, genomic, proteomic, structural, and computational data of primary immunodeficiencies. The need for the IDR arises from the lack of structured and systematic information about primary immunodeficiencies on the Internet, and from the lack of a common platform which enables doctors, researchers, students, nurses and patients to find out validated information about these diseases.DescriptionThe IDR knowledge base, first released in 1999, has grown substantially. It contains information for 158 diseases, both from a clinical as well as molecular point of view. The database and the user interface have been reformatted. This new IDR release has a richer and more complete breadth, depth and scope. The service provides the most complete and up-to-date dataset. The IDR has been integrated with several internal and external databases and services. The contents of the IDR are validated and selected for different types of users (doctors, nurses, researchers and students, as well as patients and their families). The search engine has been improved and allows either a detailed or a broad search from a simple user interface.ConclusionThe IDR is the first knowledge base specifically designed to capture in a systematic and validated way both clinical and molecular information for primary immunodeficiencies. The service is freely available at http://bioinf.uta.fi/idr and is regularly updated. The IDR facilitates primary immunodeficiencies informatics and helps to parameterise in silico modelling of these diseases. The IDR is useful also as an advanced education tool for medical students, and physicians.

Characterization of All Possible Single-Nucleotide Change Caused Amino Acid Substitutions in the Kinase Domain of Bruton Tyrosine Kinase

Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease‐causing variations among the human protein kinases, still it is just 10% of all the possible single‐nucleotide substitution‐caused amino acid variations (SNAVs). In the BTK kinase domain (BTK‐KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease‐causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty‐seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms.

IDR: the ImmunoDeficiency Resource

The ImmunoDeficiency Resource (IDR), freely available at http://www.uta.fi/imt/bioinfo/idr/, is a comprehensive knowledge base on immunodeficiencies. It is designed for different user groups such as researchers, physicians and nurses as well as patients and their families and the general public. Information on immunodeficiencies is stored as fact files, which are disease- and gene-based information resources. We have developed an inherited disease markup language (IDML) data model, which is designed for storing disease- and gene-specific data in extensible markup language (XML) format. The fact files written by the IDML can be used to present data in different contexts and platforms. All the information in the IDR is validated by expert curators.

Online registry of genetic and clinical immunodeficiency diagnostic laboratories, IDdiagnostics.

Primary immunodeficiencies (IDs) are caused by inherited genetic defects leading to intrinsic defects in cells of the immune systems. Most IDs are rare diseases and can be difficult to diagnose because similar symptoms characterize several disorders. Mutation detection is the most reliable method in such cases. These tests are not available at most centers and physicians can have difficulties in finding laboratories that could analyze the genetic defects because certain genes are possibly analyzed by just one laboratory. The IDdiagnostics registry has been established to provide information for physicians and other health care professionals. The database at http://bioinf.uta.fi/IDdiagnostics contains currently information for the analysis of defects in 30 ID-related genes. Another part of IDdiagnostics is a database of clinical tests. Laboratories performing these analyses, either gene or clinical tests, are asked to submit their information to the database by using a printed form or electronic submission at http://bioinf.uta.fi/cgi-bin/submit/IDClini.cgi. The clinical test database contains information about tests for clinical data, immune status, and studies of function, antibody response, cell function, enzyme assays, clinical function, and apoptosis assays. Both the services are freely available and regularly updated. The services aim at increasing the awareness of IDs and helping to obtain exact and early diagnosis.

Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1

BackgroundSTAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE) 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood.ResultsHere, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1.ConclusionsOur results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.

Structure-Function Effects in Primary Immunodeficiencies

Several immunodeficiency‐related genes have been identified and a large number of mutations in these genes. Currently, a genetic defect has been determined in more than 2000 patients. Only recently has it become possible to address structure‐function effects of these mutations in the corresponding proteins. The consequences of mutations in structure are discussed for Btk in X‐linked agammaglobulinemia (XLA), Jak3 in T−B+ severe combined immunodeficiency (SCID), p47phox and p67phox in autosomal chronic granulomatous disease (CGD) and SH2D1 A in X‐linked lymphoproliferatine disease (XLP). The experimental and homology modelling derived structures were used to analyze mechanisms related to these diseases.