The Genetics of Ankylosing Spondylitis and Axial Spondyloarthritis

Ankylosing spondylitis (AS) and spondyloarthritis are strongly genetically determined. The long-standing association with HLA-B27 is well described, although the mechanism by which that association induces AS remains uncertain. Recent developments include the description of HLA-B27 tag single nucleotide polymorphisms in European and Asian populations. An increasing number of non-MHC genetic associations have been reported, which provided amongst other things the first evidence of the involvement of the IL-23 pathway in AS. The association with ERAP1 is now known to be restricted to HLA-B27 positive disease. Preliminary studies on the genetics of axial spondyloarthritis demonstrate a lower HLA-B27 carriage rate compared with AS. Studies with larger samples and including non-European ethnic groups are likely to further advance the understanding of the genetics of AS and spondyloarthritis. © 2012.

Integrin-mediated Adhesion of Endothelial Cells Induces JAK2 and STAT5A Activation: Role in the Control of c-fos Gene Expression

Integrin-mediated adhesion induces several signaling pathways leading to regulation of gene transcription, control of cell cycle entry and survival from apoptosis. Here we investigate the involvement of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in integrin-mediated signaling. Plating primary human endothelial cells from umbilical cord and the human endothelial cell line ECV304 on matrix proteins or on antibody to β1- or αv-integrin subunits induces transient tyrosine phosphorylation of JAK2 and STAT5A. Consistent with a role for the JAK/STAT pathway in regulation of gene transcription, adhesion to matrix proteins leads to the formation of STAT5A-containing complexes with the serum-inducible element of c-fos promoter. Stable expression of a dominant negative form of STAT5A in NIH3T3 cells reduces fibronectin-induced c-fos mRNA expression, indicating the involvement of STAT5A in integrin-mediated c-fos transcription. Thus these data present a new integrin-dependent signaling mechanism involving the JAK/STAT pathway in response to cell–matrix interaction.

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events. (Blood. 2009; 113: 646-648)

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis

Background The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. Methods We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. Results We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. Conclusions JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

Two routes to leukemic transformation after a JAK2 mutation-positive myeloproliferative neoplasm

Acute myeloid leukemia (AML) may follow a JAK2-positive myeloproliferative neoplasm (MPN), although the mechanisms of disease evolution, often involving loss of mutant JAK2, remain obscure. We studied 16 patients with JAK2-mutant (7 of 16) or JAK2 wild-type (9 of 16) AML after a JAK2-mutant MPN. Primary myelofibrosis or myelofibrotic transformation preceded all 7 JAK2-mutant but only 1 of 9 JAK2 wild-type AMLs (P = .001), implying that JAK2-mutant AML is preceded by mutation(s) that give rise to a "myelofibrosis" phenotype. Loss of the JAK2 mutation by mitotic recombination, gene conversion, or deletion was excluded in all wild-type AMLs. A search for additional mutations identified alterations of RUNX1, WT1, TP53, CBL, NRAS, and TET2, without significant differences between JAK2-mutant and wild-type leukemias. In 4 patients, mutations in TP53, CBL, or TET2 were present in JAK2 wild-type leukemic blasts but absent from the JAK2-mutant MPN. By contrast in a chronic-phase patient, clones harboring mutations in JAK2 or MPL represented the progeny of a shared TET2-mutant ancestral clone. These results indicate that different pathogenetic mechanisms underlie transformation to JAK2 wild-type and JAK2-mutant AML, show that TET2 mutations may be present in a clone distinct from that harboring a JAK2 mutation, and emphasize the clonal heterogeneity of the MPNs.

A novel zebrafish jak2a V581F model shared features of human JAK2 V617F polycythemia vera

Objective : The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2^V617F) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2^V617F (referred herewith jak2a^V581F) by site-directed mutagenesis and examined its relevance as a model of human PV.

Materials and Methods : Zebrafish embryos at one-cell stage were injected with jak2a^V581F mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.

Results : Injection with jak2a^V581F mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp+ population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% ± 0.08%; jak2a^V581F injected: 5.71% ± 0.07%, coinjecting jak2a^V581F mRNA and stat5.1 morpholino: 4.66% ± 0.13%; p < 0.01). jak2a^V581F mRNA also upregulated gata1 (1.83 ± 0.08 fold; p = 0.005), embryonic α-hemoglobin (1.61 ± 0.12 fold; p = 0.049), and β-hemoglobin gene expression (1.65 ± 0.13–fold; p = 0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a^V581F mRNA significantly reduced erythropoietin gene (0.24 ± 0.03 fold; p = 0.006) and protein expression (control: 0.633 ± 0.11; jak2a^V581F mRNA: 0.222 ± 0.07 mIU/mL; p = 0.019).

Conclusion : The zebrafish jak2a^V581F model shared many features with human PV and might provide us with mechanistic insights of this disease.

Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34(+) hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34(+) cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34(+) cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Functional interplay of SP family members and nuclear factor Y is essential for transcriptional activation of the human Calreticulin gene

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between −5.2 and −6.1 kb in the 5′-flanking hiNOS promoter DNA, which contains multiple nuclear factor κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

A single phosphotyrosine residue of the prolactin receptor is responsible for activation of gene transcription.

Members of the cytokine/growth hormone/prolactin (PRL) receptor superfamily are associated with cytoplasmic tyrosine kinases of the Jak family. For the PRL receptor (PRLR), after PRL stimulation, both the kinase Jak2 and the receptor undergo tyrosine phosphorylation. To assess the role of tyrosine phosphorylation of the PRLR in signal transduction, several mutant forms of the PRLR in which various tyrosine residues were changed to phenylalanine were constructed and their functional properties were investigated. We identified a single tyrosine residue located at the C terminus of the PRLR to be necessary for in vivo activation of PRL-responsive gene transcription. This clearly indicates that a phosphotyrosine residue in the cytoplasmic domain of a member of the cytokine/growth hormone/PRL receptor superfamily is directly involved in signal transduction.

The Calreticulin gene and myeloproliferative neoplasms

The Philadelphia negative myeloproliferative neoplasms include polycythaemia vera (PV), essential thrombocytopenia (ET) and primary myelofibrosis (PMF). Patients with these conditions were mainly thought to harbour JAK2V617F mutations or an Myeloproliferative leukaemia (MPL) substitution. In 2013, two revolutionary studies identified recurrent mutations in a gene that encodes the protein calreticulin (CALR). This mutation was detected in patients with PMF and ET with non-mutated JAK2 or MPL but was absent in patients with PV. The CALR gene encodes the calreticulin protein, which is a multifactorial protein, mainly located in the endoplasmic reticulum in chromosome 19 and regulates calcium homeostasis, chaperones and has also been implicated in multiple cellular processes including cell signalling, regulation of gene expression, cell adhesion, autoimmunity and apoptosis. Somatic 52 bp deletions and recurrent 52 bp insertion mutations in CALR were detected and all resulted in frameshift and clusters in exon 9 of the gene. This review will summarise the current knowledge on the CALR gene and mutation of the gene in pathological conditions and patient phenotypes.

Lymphotoxin-beta receptor and RANK signaling in TEL-JAK2-induced T-cell leukemia

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

No evidence for association of ataxia-telangiectasia mutated gene T2119C and C3161G amino acid substitution variants with risk of breast cancer

Background There is evidence that certain mutations in the double-strand break repair pathway ataxia-telangiectasia mutated gene act in a dominant-negative manner to increase the risk of breast cancer. There are also some reports to suggest that the amino acid substitution variants T2119C Ser707Pro and C3161G Pro1054Arg may be associated with breast cancer risk. We investigate the breast cancer risk associated with these two nonconservative amino acid substitution variants using a large Australian population-based case–control study. Methods The polymorphisms were genotyped in more than 1300 cases and 600 controls using 5' exonuclease assays. Case–control analyses and genotype distributions were compared by logistic regression. Results The 2119C variant was rare, occurring at frequencies of 1.4 and 1.3% in cases and controls, respectively (P = 0.8). There was no difference in genotype distribution between cases and controls (P = 0.8), and the TC genotype was not associated with increased risk of breast cancer (adjusted odds ratio = 1.08, 95% confidence interval = 0.59–1.97, P = 0.8). Similarly, the 3161G variant was no more common in cases than in controls (2.9% versus 2.2%, P = 0.2), there was no difference in genotype distribution between cases and controls (P = 0.1), and the CG genotype was not associated with an increased risk of breast cancer (adjusted odds ratio = 1.30, 95% confidence interval = 0.85–1.98, P = 0.2). This lack of evidence for an association persisted within groups defined by the family history of breast cancer or by age. Conclusion The 2119C and 3161G amino acid substitution variants are not associated with moderate or high risks of breast cancer in Australian women.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.