The prevalence of CALR mutations in a cohort of patients with myeloproliferative neoplasms

INTRODUCTION: To investigate the prevalence of calreticulin (CALR) mutations in JAK2- and MPL-non-mutated patients with suspected myeloproliferative neoplasm (MPN) from a large MPN clinic and confirm a diagnosis of MPN.

METHODS: JAK2/MPL-non-mutated patients from the Belfast City Hospital (BCH) with either of the MPNs - ET or MF - and diagnosed between 1988 and 2014 were selected for CALR screen. All cases were validated according to the WHO 2008 classification for MPNs. Statistical analysis was performed with Minitab 16 Statistical Software package. Exon 9 of CALR was amplified by PCR using genomic DNA, and mutations were detected by fragment analysis.

RESULTS: Of the 62 JAK2/MPL-non-mutated MPN patients screened, 57 had ET and 5 had MF; 34 patients (53.1%) carried CALR mutations. Three of 5 MF patients were CALR positive. Thirty-one ET patients (54.3%) harboured CALR mutation, whereas 26 (45.7%) were classified as 'triple negatives'.

CONCLUSION: Detection of CALR mutations in a cohort of JAK2/MPL-non-mutated patients with suspected MPN confirmed the diagnosis of MPN in around 53% of cases. This is lower than initially reported, but similar to subsequent studies. However, a sizable cohort of patients remains lacking a specific molecular marker.

Clinical end points for drug treatment trials in BCR-ABL1-negative classic myeloproliferative neoplasms: consensus statements from European LeukemiaNET (ELN) and Internation Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT)

The discovery of somatic mutations, primarily JAK2V617F and CALR, in classic BCR-ABL1-negative myeloproliferative neoplasms (MPNs) has generated interest in the development of molecularly targeted therapies, whose accurate assessment requires a standardized framework. A working group, comprised of members from European LeukemiaNet (ELN) and International Working Group for MPN Research and Treatment (IWG-MRT), prepared consensus-based recommendations regarding trial design, patient selection and definition of relevant end points. Accordingly, a response able to capture the long-term effect of the drug should be selected as the end point of phase II trials aimed at developing new drugs for MPNs. A time-to-event, such as overall survival, or progression-free survival or both, as co-primary end points, should measure efficacy in phase III studies. New drugs should be tested for preventing disease progression in myelofibrosis patients with early disease in randomized studies, and a time to event, such as progression-free or event-free survival should be the primary end point. Phase III trials aimed at preventing vascular events in polycythemia vera and essential thrombocythemia should be based on a selection of the target population based on new prognostic factors, including JAK2 mutation. In conclusion, we recommended a format for clinical trials in MPNs that facilitates communication between academic investigators, regulatory agencies and drug companies.

A HuGE Review and Meta-Analyses of Genetic Associations in New Onset Diabetes after Kidney Transplantation

PURPOSE: New onset diabetes after transplantation (NODAT) is a serious complication following solid organ transplantation. There is a genetic contribution to NODAT and we have conducted comprehensive meta-analysis of available genetic data in kidney transplant populations.

METHODS: Relevant articles investigating the association between genetic markers and NODAT were identified by searching PubMed, Web of Science and Google Scholar. SNPs described in a minimum of three studies were included for analysis using a random effects model. The association between identified variants and NODAT was calculated at the per-study level to generate overall significance values and effect sizes.

RESULTS: Searching the literature returned 4,147 citations. Within the 36 eligible articles identified, 18 genetic variants from 12 genes were considered for analysis. Of these, three were significantly associated with NODAT by meta-analysis at the 5% level of significance; CDKAL1 rs10946398 p = 0.006 OR = 1.43, 95% CI = 1.11-1.85 (n = 696 individuals), KCNQ1 rs2237892 p = 0.007 OR = 1.43, 95% CI = 1.10-1.86 (n = 1,270 individuals), and TCF7L2 rs7903146 p = 0.01 OR = 1.41, 95% CI = 1.07-1.85 (n = 2,967 individuals).

CONCLUSION: Evaluating cumulative evidence for SNPs associated with NODAT in kidney transplant recipients has revealed three SNPs associated with NODAT. An adequately powered, dense genome-wide association study will provide more information using a carefully defined NODAT phenotype.

Compositonal Analysis of Potentially harmful Elements in Soils: Implications for Epidemiology and Kidney Disease

A substantial proportion of aetiological risks for many cancers and chronic diseases remain unexplained. Using geochemical soil and stream water samples collected as part of the Tellus Project studies, current research is investigating naturally occurring background levels of potentially toxic elements (PTEs) in soils and stream sediments and their possible relationship with progressive chronic kidney disease (CKD). The Tellus geological mapping project, Geological Survey Northern Ireland, collected soil sediment and stream water samples on a grid of one sample site every 2 km2 across the rural areas of Northern Ireland resulting in an excess of 6800 soil sampling locations and more than 5800 locations for stream water sampling. Accumulation of several PTEs including arsenic, cadmium, chromium, lead and mercury have been linked with human health and implicated in renal function decline. The hypothesis is that long-term exposure will result in cumulative exposure to PTEs and act as risk factor(s) for cancer and diabetes related CKD and its progression. The ‘bioavailable’ fraction of total PTE soil concentration depends on the ‘bioaccessible’ proportion through an exposure pathway. Recent work has explored this bioaccessible fraction for a range of PTEs across Northern Ireland. In this study the compositional nature of the multivariate geochemical PTE variables and bioaccessible data is explored to augment the investigation into the potential relationship between PTEs, bioaccessibility and disease data.

Optimising the management of polycystic kidney disease

Polycystic kidney disease (PKD) is the most common inherited renal disorder that results in chronic kidney disease. Clinical features include visible haematuria, loin pain, UTI and hypertension. The typical clinical course is a progressive increase in the number and size of renal cysts associated with gradual loss of kidney function (falling eGFR).

Novel Genetic Determinants of Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a devastating diabetes complication, with known heritability not fully revealed by previous genetics studies. We performed the largest genome-wide association study of type 1 DKD to date, in a 13-cohort consortium of 15,590 individuals of European ancestry genotyped on the Illumina HumanCoreExome Beadchip, which allows exploration of coding variation in addition to genomic markers.

As prior work has shown that different characterizations of the DKD phenotype highlight distinct genetic associations, we investigated a spectrum of DKD definitions based on proteinuria and renal function criteria. Controls were DKD-free after a minimum of 15 years diabetes duration; cases had diabetes for at least 10 years prior to DKD diagnosis. We also performed a quantitative trait analysis of estimated glomerular filtration rate in all participants.

Our top finding was a missense mutation in COL4A3, rs55703767 (Asp326Tyr); the minor allele is common in Europeans (20%) and East Asians (13%) but not Africans (2%). This SNP had a genome-wide significant association with traditionally defined DKD (macroalbuminuria or end-stage renal disease [ESRD], (OR= 0.79, P=1.9×10-9), and a suggestive association with macroalbuminuria (OR= 0.79, P=1.6×10-6) and ESRD (OR= 0.79, P=4.5×10-5) individually. Though its PolyPhen score is 0.3 (benign), this SNP has been implicated as a splice site disruptor.

The COL4A3 gene encodes the alpha 3 subunit of Type IV collagen, the major structural component of basement membranes. Pathogenic mutations in COL4A3 have been identified in thin basement membrane nephropathy, familial focal segmental glomerulosclerosis, and Alport syndrome. A proxy (r2=0.6) for rs55703767 had no significant associations in the CKDGen consortium, suggesting its pathogenicity occurs solely in the setting of hyperglycemia.

By significantly increasing sample size we have discovered a novel locus underlying DKD risk, paving the way for better understanding of pathology, prevention, and treatment.

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.