The molecular profile in patients with polycythemia vera and essential thrombocythemia is dynamic and correlates with disease's phenotype.

Introduction: Polycythemia vera (PV) and essential thrombocythemia (ET) are diseases driven by canonical mutations in JAK2, CALR, or MPL gene. Previous studies revealed that in addition to driver mutations, patients with PV and ET can harbor other mutations in various genes, with no established impact on disease phenotype. We hypothesized that the molecular profile of patients with PV and ET is dynamic throughout the disease. Methods: In this study, we performed a 37-gene targeted next-generation sequencing panel on the DNA samples collected from 49 study participants in two-time points, separated by 78-141 months. We identified 78 variants across 37 analyzed genes in the study population. Results: By analyzing the change in variant allele frequencies and revealing the acquisition of new mutations during the disease, we confirmed the dynamic nature of the molecular profile of patients with PV and ET. We found connections between specific variants with the development of secondary myelofibrosis, thrombotic events, and response to treatment. We confronted our results with existing conventional and mutation-enhanced prognostic systems, showing the limited utility of available prognostic tools. Discussion: The results of this study underline the significance of repeated molecular testing in patients with PV and ET and indicate the need for further research within this field to better understand the disease and improve available prognostic tools.

Novel Tools for Comprehensive Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants.

Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor (LDLR) gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in the discovery of more than 3000 variants of the LDLR gene, but not all of them are clinically relevant. Therefore, functional studies of selected variants are needed for their proper classification. Here, a single-cell, kinetic, fluorescent LDL uptake assay was applied for the functional analysis of LDLR variants in a model of an LDLR-deficient human cell line. An LDLR-defective HEK293T cell line was established via a CRISPR/Cas9-mediated luciferase-puromycin knock-in. The expressing vector with the LDLR gene under the control of the regulated promoter and with a reporter gene has been designed to overproduce LDLR variants in the host cell. Moreover, an LDLR promoter-luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the LDLR gene, which can serve as a simple tool for screening and testing new HMG CoA reductase-inhibiting drugs for atherosclerosis therapy. The data presented here demonstrate that the obtained LDLR-deficient human cell line HEK293T-ldlrG1 and the dedicated pTetRedLDLRwt expression vector are valuable tools for studying LDL internalization and functional analysis of LDLR and its genetic variants. Using appropriate equipment, LDL uptake to a single cell can be measured in real time. Moreover, the luciferase gene knock-in downstream of the LDLR promotor allows the study of promoter regulation in response to diverse conditions or drugs. An analysis of four known LDLR variants previously classified as pathogenic and benign was performed to validate the LDLR-expressing system described herein with the dedicated LDLR-deficient human cell line, HEK293T-ldlrG1.

Genomic Profiling Identifies Putative Pathogenic Alterations in NSCLC Brain Metastases.

Introduction: Brain metastases (BM) severely affect the prognosis and quality of life of patients with NSCLC. Recently, molecularly targeted agents were found to have promising activity against BM in patients with NSCLC whose primary tumors carry "druggable" mutations. Nevertheless, it remains critical to identify specific pathogenic alterations that drive NSCLC-BM and that can provide novel and more effective therapeutic targets. Methods: To identify potentially targetable pathogenic alterations in NSCLC-BM, we profiled somatic copy number alterations (SCNAs) in 51 matched pairs of primary NSCLC and BM samples from 33 patients with lung adenocarcinoma and 18 patients with lung squamous cell carcinoma. In addition, we performed multiregion copy number profiling on 15 BM samples and whole-exome sequencing on 40 of 51 NSCLC-BM pairs. Results: BM consistently had a higher burden of SCNAs compared with the matched primary tumors, and SCNAs were typically homogeneously distributed within BM, suggesting BM do not undergo extensive evolution once formed. By comparing focal SCNAs in matched NSCLC-BM pairs, we identified putative BM-driving alterations affecting multiple cancer genes, including several potentially targetable alterations in genes such as CDK12, DDR2, ERBB2, and NTRK1, which we validated in an independent cohort of 84 BM samples. Finally, we identified putative pathogenic alterations in multiple cancer genes, including genes involved in epigenome editing and 3D genome organization, such as EP300, CTCF, and STAG2, which we validated by targeted sequencing of an independent cohort of 115 BM samples. Conclusions: Our study represents the most comprehensive genomic characterization of NSCLC-BM available to date, paving the way to functional studies aimed at assessing the potential of the identified pathogenic alterations as clinical biomarkers and targets.

Secondary chronic myeloid leukemia in a patient with CALR and ASXL1-mutated primary myelofibrosis.

Development of secondary CML has only been casually described, with few reports attempting to analyze and explain the mechanisms behind this phenomenon. Reported cases vary with regard to presumed pathogenesis and clinical characteristics, but similarities can be observed. This report presents the case of a patient diagnosed with CALR and ASXL1-mutated primary myelofibrosis who developed CML 13 years after the initial diagnosis. In contrast with previously reported cases, this patient did not have JAK2 or ABL1 gene mutations, and also exhibited primary resistance to tyrosine kinase inhibitor (TKI) treatment. Here, we analyze the molecular evolution of CML and describe successful treatment with concomitant therapy including a TKI and JAK inhibitor. This report aims to deepen clinical experience and further broaden knowledge about chronic myeloproliferative neoplasms.

New Approach to Paediatric Mastocytosis: Implications of KIT D816V Mutation Detection in Peripheral Blood.

is missing (Short communication).

Haplotypes of butyrylcholinesterase K-variant and their influence on the enzyme activity.

Butyrylcholinesterase (BChE) is a serine hydrolase widely distributed throughout the body. It provides protection against administrated or inhaled poisons by hydrolyzing or sequestering the toxic compounds. The most frequent genetic variant of BCHE gene - K variant (p.A539T) is located in the C-terminal tetramerization domain, outside of the catalytic center. Many studies tried to reveal the nature of the lower activity of BChE K-variant but results and conclusions were often contradictory. The aim of this study is to estimate K allele frequency and its coexisting alterations in BCHE gene in a population of 162 individuals, as well as, assess influence on the enzyme activity in serum. We present three haplotypes of BChE-K variant, two of them coexist in strong linkage disequilibrium with alterations in 5'UTR (rs1126680), intron 2 (rs55781031) or in exon 2 (rs1799807). We demonstrate a negative role of these alterations on enzyme activity. By oneself BCHE-K (with no other alterations in BCHE gene) haplotype exhibits wild type enzyme activity. Based on our previous and presented results we conclude that SNPs localized outside the coding sequence, in 5'UTR or/and in intron 2 of BCHE gene, but not solely in K-variant alteration (p.A539T) itself, are responsible for reduced enzyme activity.

Synergy between the alteration in the N-terminal region of butyrylcholinesterase K variant and apolipoprotein E4 in late-onset Alzheimer's disease.

While the life expectancy of the population has increased, Alzheimer's disease (AD) has emerged as one of the greatest health problems of old age. AD is characterized by neuronal loss and cognitive decline. In the AD brain, there is a decrease in levels of acetylcholinesterase (AChE) and an increase in the levels of the related enzyme butyrylcholinesterase (BChE), that accumulate in plaques and tangles. Apolipoprotein E (ApoE) is a major cholesterol carrier and plays an important role in maintaining lipid homeostasis. APOE-ε4 constitutes the most important known genetic risk factor for late-onset AD. It has been proposed that the BCHE-K allele (Ala539Thr) acts in synergy with the APOE-ε4 allele to promote risk for AD. However, there is insufficient evidence to support a correlation. Most studies focused only on the coding regions of the genes. In this study, we analyzed sequence regions beyond the BCHE coding sequence. We found synergy between APOE-ε4 and SNPs localized in 5'UTR (rs1126680) and in intron 2 (rs55781031) of the BCHE-K allele (rs1803274) in 18% of patients with late-onset AD (n = 55). The results show that the coexistence of the APOE-ε4 allele and 3 SNPs in the BCHE gene is associated with a highly elevated risk of late-onset AD. SNP (rs1126680) in 5'UTR of the BCHE gene is located 32 nucleotides upstream of the 28 amino acid signal peptide. Mass spectrometry analysis of the BChE protein produced by SNP (rs1126680) showed that the mutation caused an in frame N-terminal extension of 41 amino acids of the BChE signal peptide. The resultant variant with a 69 amino acid signal peptide, designated N-BChE, may play a role in development of AD.

Detection of BRCA1/2 mutations in circulating tumor DNA from patients with ovarian cancer.

Approximately 25% of patients with ovarian cancer harbor a pathogenic BRCA1/2 mutation that has been associated with favorable responses for targeted therapy with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors compared to wild-type individuals. The overall frequency of germline and somatic BRCA1/2 alterations is estimated at 13-15% and 3-10%, respectively. A high incidence of BRCA1/2 somatic variants significantly increases the number of patients eligible for treatment with PARP1 inhibitors. Here, we assessed circulating tumor DNA (ctDNA) from 121 patients with ovarian cancer for BRCA1/2 mutational analysis by next generation sequencing. A total number of patients carrying the pathogenic BRCA1/2 variants was 30/121 (24.8%), including 22 and 7 individuals with exclusively germline or somatic mutations, respectively and one patient with variants of both origin. Among this cohort, more than one known pathogenic BRCA1 and/or BRCA2 alterations were identified in 7/30 individuals. The most recurrent mutations were detected in the BRCA1 gene: c.5266dupC (p.Gln1756Profs*74) with the frequency of ~18%, followed by c.3756_3759del (p.Ser1253Argfs*10) and c.181T>G (p.Cys61Gly). In seven (5.8%) patients, coincidence of two or more BRCA1/2 pathogenic mutations have been identified. Our results clearly demonstrate that the detection of both germline and somatic BRCA1/2 mutations in ctDNA from ovarian cancer patients is feasible and may be a valuable complementary tool for identification of somatic alterations when the standard diagnostic procedures are insufficient. Finally, ctDNA can potentially allow to monitor the efficacy of PARP1 inhibitors and to detect a secondary reversion BRCA1/2 mutations.

Detection of somatic BRCA1/2 mutations in ovarian cancer - next-generation sequencing analysis of 100 cases.

The overall prevalence of germline BRCA1/2 mutations is estimated between 11% and 15% of all ovarian cancers. Individuals with germline BRCA1/2 alterations treated with the PARP1 inhibitors (iPARP1) tend to respond better than patients with wild-type BRCA1/2. Additionally, also somatic BRCA1/2 alterations induce the sensitivity to iPARP1. Therefore, the detection of both germline and somatic BRCA1/2 mutations is required for effective iPARP1 treatment. The aim of this study was to identify the frequency and spectrum of germline and somatic BRCA1/2 alterations in a group of Polish patients with ovarian serous carcinoma. In total, 100 formalin-fixed paraffin-embedded (FFPE) ovarian serous carcinoma tissues were enrolled to the study. Mutational analysis of BRCA1/2 genes was performed by using next-generation sequencing. The presence of pathogenic variants was confirmed by Sanger sequencing. In addition, to confirm the germline or somatic status of the mutation, the nonneoplastic tissue was analyzed by bidirectional Sanger sequencing. In total, 27 (28% of patient samples) mutations (20 in BRCA1 and 7 in BRCA2) were identified. For 22 of 27 patients, nonneoplastic cells were available and sequencing revealed the somatic character of two BRCA1 (2/16; 12.5%) and two BRCA2 (2/6; 33%) mutations. Notably, we identified six novel frameshift or nonsense BRCA1/2 mutations. The heterogeneity of the detected mutations confirms the necessity of simultaneous analysis of BRCA1/2 genes in all patients diagnosed with serous ovarian carcinoma. Moreover, the use of tumor tissue for mutational analysis allowed the detection of both somatic and germline BRCA1/2 mutations.

Efficacy of clinical diagnostic criteria for familial hypercholesterolemia genetic testing in Poland.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH), which leads to premature cardiovascular events, still remains underrecognized and undertreated in most countries. Untreated FH individuals aged 20-39 years are at 100-fold higher risk of mortality from coronary heart disease compared to those of a general population. Therefore, special efforts should be implemented to diagnose FH patients at early stages of life. The aim of this study was to evaluate the efficacy of the revised Dutch Lipid Clinic Network (DLCN) criteria proposed by the Polish Lipid Experts Forum to select index FH patients for DNA mutational analysis in Poland. METHODS: The study included 193 unrelated adult patients (mean age 48 ± 13 years) with clinical diagnosis of FH based on the revised DLCN score, tested sequentially for mutations in LDLR and APOB genes using bidirectional Sanger sequencing and MLPA techniques. The cut-off points of the clinical FH criteria score were assessed by ROC statistics to identify patients with the highest probability of carrying an FH-causing mutation. RESULTS: The causal heterozygous LDLR or APOB mutation was identified in 41% (80/193) of probands. Adults aged <40 years were more likely to carry an FH-causing mutation compared to subjects aged ≥40 years (65% vs. 33%; p < 0.001). LDL-C thresholds for the molecular diagnosis of FH were 5.79 mmol/l for individuals aged<40 and 6.7 mmol/l for subjects ≥40 years old. The threshold values of the clinical diagnostic score for efficient selection of patients for genetic testing were 5 and 7 points for individuals aged <40 and ≥40 years, respectively. CONCLUSIONS: The study validated the efficacy of proposed clinical FH criteria for the disease diagnosis in Poland. The clinical criteria score thresholds for positive FH molecular diagnosis differ depending on age (<40 and ≥40 years). We propose that in the healthcare systems with limited genetic testing resources individuals younger than 40 years, who fulfill the clinical criteria for possible, probable or definite FH should qualify for the FH mutation testing. The index patients aged ≥40 years with clinical diagnosis of probable or definite FH should also qualify for the genetic testing.

Activity and polymorphisms of butyrylcholinesterase in a Polish population.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 3'UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (Fluoride-Number) values outside the reference range (DN < 78 and FN < lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 5'UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity ≥6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes.

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

Noninvasive assessment of endothelial function and vascular parameters in patients with familial and nonfamilial hypercholesterolemia.

INTRODUCTION: Endothelial dysfunction is one of the markers of atherosclerosis. OBJECTIVES: The aim of the study was to evaluate endothelial function by assessing flow-mediated dilation (FMD) and to measure the parameters of brachial arterial stiffness in patients with familial hypercholesterolemia (FH) and those with high low-density lipoprotein (LDL) cholesterol levels without FH mutations (nonfamilial hypercholesterolemia - non-FH). PATIENTS AND METHODS: The study involved 60 patients (mean age, 41.9 ±7.7 y) without documented cardiovascular events and clinical symptoms of cardiovascular diseases: 21 patients with elevated plasma LDL cholesterol levels and genetically confirmed FH, 19 patients with elevated LDL cholesterol levels and without FH mutations, and 20 healthy controls. In each patient, ultrasound imaging was used to assess endothelium-dependent FMD and nitroglycerin-induced endothelium-independent dilation (EID) in the brachial artery. In addition, echo-tracking and photoplethysmography were used to assess the parameters of arterial stiffness. RESULTS: FMD was significantly lower in patients with FH (11.0% ±9.9% vs. 21.0% ±14.3%, P <0.01) and non-FH (14.2% ±10.1% vs. 21.0% ±14.3%, P <0.05) compared with controls. EID and arterial stiffness parameters were similar between the groups. CONCLUSIONS: Reduced FMD may suggest endothelial dysfunction. A lack of significant differences in arterial stiffness parameters may indicate that vascular remodeling is not advanced in patients with elevated LDL cholesterol levels. A lack of significant differences in FMD and arterial stiffness between patients with and without FH may indicate that FH mutation itself is not the main determinant of endothelial dysfunction and vascular remodeling in younger patients with hypercholesterolemia.