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Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method.
Background: The opioid system is involved in the control of pain, reward, addictive behaviors and vegetative effects. Opioids exert their pharmacological actions through the agonistic binding at opioid receptors and variation in the coding genes has been found to modulate opioid receptor expression or signaling. However, a limited selection of functional opioid receptor variants is perceived as insufficient in providing a genetic diagnosis of clinical phenotypes and therefore, unrestricted access to opioid receptor genetics is required.
Methods: Next-generation sequencing (NGS) workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes related to the opioid receptor group (OPRM1, OPRD1, OPRK1, SIGMA1, OPRL1) on an Ion PGM™ Sequencer. A cohort of 79 previously studied chronic pain patients was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding. In-silico analysis was performed using SNP and Variation Suite® software.
Results: The amplicons covered approximately 90% of the target sequence. A median of 2.54 × 106 reads per run was obtained generating a total of 35,447 nucleotide reads from each DNA sample. This identified approximately 100 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19, including functional variants such as the OPRM1 rs1799971 SNP (118 A > G) as the most scientifically regarded variant or rs563649 SNP coding for μ-opioid receptor splice variants. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.
Conclusion: Results suggested that the NGS approach based on AmpliSeq™ libraries and Ion PGM sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of opioid receptor genes. The method includes the variants studied so far for functional associations and adds a large amount of genetic information as a basis for complete analysis of human opioid receptor genetics and its functional consequences.