Clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis.

BACKGROUND: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the "Vasculitis and Inflammation Panel" (VIP) for AID and vasculitis. METHODS: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. RESULTS: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%). CONCLUSIONS: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation ofLyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn

Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lynup/up) on erythropoiesis and Epo receptor signaling. Adult Lynup/up mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn+/up mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn+/up erythroid cell lines and primary CD71+ Lynup/up erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated- binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn+/up cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects

Out-of-pocket expenses experienced by rural Western Australians diagnosed with cancer

© 2018 Springer-Verlag GmbH Germany, part of Springer Nature Purpose: Out-of-pocket expenses (OOPE) can have a significant impact on patients’ experiences of cancer treatment. This cross-sectional study sought to quantify the OOPEs experienced by rural cancer patients in Western Australia (WA), and determine factors that contributed to higher OOPE. Methods: Four hundred people diagnosed with breast, lung, colorectal or prostate cancer who resided in selected rural regions of WA were recruited through the WA Cancer Registry and contacted at least 3 months after diagnosis to report the medical OOPE (such as surgery or chemotherapy, supportive care, medication and tests) and non-medical OOPE (such as travel costs, new clothing and utilities) they had experienced as a result of accessing and receiving treatment. Bootstrapped t tests identified demographic, financial and treatment-related factors to include in multivariate analysis, performed using log-linked generalised linear models with gamma distribution. Results: After a median 21 weeks post-diagnosis, participants experienced an average OOPE of AU$2179 (bootstrapped 95$1873–$2518), and 45 (11%) spent more than 10% of their household income on these expenses. Participants likely to experience higher total OOPE were younger than 65 years (p = 0.008), resided outside the South West region (p = 0.007) and had private health insurance (PHI) (p < 0.001). Conclusions: Rural WA cancer patients experience significant OOPE following their diagnosis. The impact these expenses have on patient wellbeing and their treatment decisions need to be further explored

Csk-binding protein controls red blood cell development via regulation of Lyn tyrosine kinase activity

Erythropoiesis is controlled principally through erythropoietin (Epo) receptor signaling, which involves Janus kinase 2 (JAK2) and Lyn tyrosine kinase, both of which are important for regulating red blood cell (RBC) development. Negative regulation of Lyn involves C-Src kinase (Csk)-mediated phosphorylation of its C-terminal tyrosine, which is facilitated by the transmembrane adaptor Csk-binding protein (Cbp). Although Cbp has significant functions in controlling Lyn levels and activity in erythroid cells in vitro, its importance to primary erythroid cell development and signaling has remained unclear. To address this, we assessed the consequence of loss of Cbp on the erythroid compartment in vivo and whether Epo-responsive cells isolated from Cbp-knockout mice exhibited altered signaling. Our data show that male Cbp−/− mice display a modest but significant alteration to late erythroid development in bone marrow with evidence of increased erythrocytes in the spleen, whereas female Cbp−/− mice exhibit a moderate elevation in early erythroid progenitors (not seen in male mice) that does not influence the later steps in RBC development. In isolated primary erythroid cells and cell lines generated from Cbp−/− mice, survival signaling through Lyn/Akt/FoxO3 was elevated, resulting in sustained viability during differentiation. The high Akt activity disrupted GAB2/SHP-2 feedback inhibition of Lyn; however, the elevated Lyn activity also increased inhibitory signaling via SHP-1 to restrict the Erk1/2 pathway. Interestingly, whereas loss of Cbp led to mild changes to late RBC development in male mice, this was not apparent in female Cbp−/− mice, possibly due to their elevated estrogen, which is known to facilitate early progenitor self-renewal

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival

Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn-/- erythroblasts had attenuated ex vivo expansion, and late stage Lyn-/-erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn-/-erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKa/ß (glycogen synthase kinase a/ß) phosphorylation and diminished p27Kip1 induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro-and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling