Is There Association between Altered Adrenergic System Activity and Microvascular Endothelial Dysfunction Induced by a 7-Day High Salt Intake in Young Healthy Individuals

This study aimed to test the effect of a 7-day high-salt (HS) diet on autonomic nervous system (ANS) activity in young healthy individuals and modulation of ANS on microvascular endothelial function impairment. 47 young healthy individuals took 7-day low-salt (LS) diet (3.5 g salt/day) followed by 7-day high-salt (HS) diet (~14.7 g salt/day). ANS activity was assessed by 24-h urine catecholamine excretion and 5-min heart rate variability (HRV). Skin post-occlusive reactive hyperemia (PORH) and acetylcholine-induced dilation (AChID) were assessed by laser Doppler flowmetry (LDF). Separately, mental stress test (MST) at LS and HS condition was conducted, followed by immediate measurement of plasma metanephrines’ level, 5-min HRV and LDF microvascular reactivity. Noradrenaline, metanephrine and normetanephrine level, low-frequency (LF) HRV and PORH and AChID significantly decreased following HS compared to LS. MST at HS condition tended to increase HRV LF/HF ratio. Spectral analysis of PORH signal, and AChID measurement showed that MST did not significantly affect impaired endothelium-dependent vasodilation due to HS loading. In this case, 7-day HS diet suppressed sympathetic nervous system (SNS) activity, and attenuated microvascular reactivity in salt-resistant normotensive individuals. Suppression of SNS during HS loading represents a physiological response, rather than direct pathophysiological mechanism by which HS diet affects microvascular endothelial function in young healthy individuals.     

No evidence of association of methylenetetrahydrofolate reductase polymorphism with occurrence of second neoplasms after treatment of childhood leukemia

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms have been associated not only with the risk for acute lymphoblastic leukemia (ALL) in adults and children, but also with increased methotrexate toxicity. The present study aimed to investigate whether MTHFR polymorphisms modify the risk for development of secondary malignancies in children treated for ALL with protocols that included high-dose methotrexate. MTHFR genotypes were determined in DNA samples isolated from archived bone marrow smears of 15 patients with a second malignancy and a matched control group of 30 patients who did not developed a second malignancy after the treatment for ALL. The frequencies of MTHFR C677T and A1298C genotypes in all patients were: C677T: CC 40%, CT 46.7% and TT 13.3% and A1298C: AA 46.7%, AC 44.4% and CC 8.9%. The relative risk for second malignancy was not significantly increased in ALL patients having at least one polymorphic C667T [odds ratio (OR) 1.51; 95% confidence interval (CI) 0.43 - 5.31] or one polymorphic A1298C allele (OR 1; 95% CI 0.29 - 3.46). Our study suggests that MTHFR polymorphisms are not associated with increased risk of second cancer in children treated with high-dose methotrexate.     

Clinical Impacts of Copy Number Variations in B-cell Differentiation and Cell Cycle Control Genes in Pediatric B-cell Acute Lymphoblastic Leukemia: a Single Centre Experience

BackgroundIKZF1 gene deletions have been identified as a poor prognostic factor in pediatric B-cell acute lymphoblastic leukemia (B-ALL), especially in the presence of co-occurring deletions (IKZF1^plus profile). This study aimed to determine the frequency of IKZF1 deletions and deletions in other B-cell differentiation and cell cycle control genes, and their prognostic impact in Slovenian pediatric B-ALL patients.Patients and methodsWe studied a cohort of 99 patients diagnosed with B-ALL from January 2012 to December 2020 and treated according to the ALL IC-BFM 2009 protocol. Eighty-eight bone marrow or peripheral blood samples were analysed for copy number variations (CNVs) using the SALSA MLPA P335 ALL-IKZF1 probemix.ResultsAt least one CNV was detected in more than 65% of analysed samples. The most frequently altered genes were PAX5 and CDKN2A/B (30.7%, 26.1%, and 25.0%, respectively). Deletions in IKZF1 were present in 18.2% of analysed samples and were associated with an inferior 5-year event-free survival (EFS; 54.8% vs. 85.9%, p = 0.016). The IKZF1^plus profile was identified in 12.5% of the analysed samples, and these patients had an inferior 5-year EFS than those with deletions in IKZF1 only and those without deletions (50.8% vs. 75.0% vs. 85.9%, respectively, p = 0.049). Overall survival (OS) was also worse in patients with the IKZF1^plus profile than those with deletions in IKZF1 only and those without deletions (5-year OS 76.2% vs. 100% vs. 93.0%, respectively). However, the difference between the groups was not statistically significant.ConclusionsOur results are in concordance with the results obtained in larger cooperative clinical trials. Copy number variations analysis using the SALSA MLPA kit is a reliable tool for initial diagnostic approach in children with B-ALL, even in smaller institutions in low- and middle-income countries.Key words: B-acute lymphoblastic leukemia, IKZF1 deletions, IKZF1 ^plus , MLPA, pediatric, copy number variations (CNVs)     

In vivo study of propolis supplementation effects on antioxidative status and red blood cells

In vivo study has been conducted on 47 healthy women and men in order to investigate whether daily intake of powdered propolis extract during 30 days has any influence on the following blood parameters: activity of superoxide dismutase, glutathione peroxidase and catalase, concentration of plasma malondialdehyde, total cholesterol, low- and high-density lipoprotein cholesterol, triglycerides, glucose, uric acid, ferritin and transferrin, together with routine red blood cell parameters.

The effect of daily propolis intake seems to be time and gender related. For the men test group after the initial 15 days of propolis treatment, 23.2% (p = 0.005) decrease in concentration of malondialdehyde was observed. After 30 days of treatment, statistically significant (p = 0.010) 20.9% increase in superoxide dismutase activity and change in some of the red blood cell parameters were detected. For the women test group, the propolis treatment did not induce a change in any of the measured parameters.     

Data mining and visualization for decision support and modeling of public health-care resources

This paper proposes an innovative use of data mining and visualization techniques for decision support in planning and regional-level management of Slovenian public health-care. Data mining and statistical techniques were used to analyze databases collected by a regional Public Heath Institute. We also studied organizational aspects of public health resources in the selected Celje region with the objective to identify the areas that are atypical in terms of availability and accessibility of public health services for the population. The most important step was the detection of outliers and the analysis of availability and accessibility deviations. The results are applicable to health-care planning and support in decision making by local and regional health-care authorities. In addition to the practical results, which are directly useful for decision making in planning of the regional health-care system, the main methodological contribution of the paper are the developed visualization methods that can be used to facilitate knowledge management and decision making processes.     

False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures

Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.Detecting driver mutations in cancer genomes is of increasing importance for patient care, both for prognostic significance and for allowing better utilization of targeted therapies. Determining the mutational status of specific genes, such as KRAS, BRAF, and EGFR in lung adenocarcinoma and KRAS and BRAF in colorectal adenocarcinoma, has become the standard of care in clinical oncology to direct epidermal growth factor receptor (EGFR) inhibitor therapy.^1,2 To achieve this, targeted sequencing of these genes with the use of Sanger sequencing and pyrosequencing is widely available. However, Sanger sequencing is labor intensive and has a relatively poor analytic sensitivity (approximately 20% mutant alleles), requiring specimens with a significant percentage of tumor nuclei (>40%) to detect heterozygous mutations.^3,4 Pyrosequencing, although less labor intensive with a better limit of detection (analytic sensitivity approximately 5%), is typically limited to short regions of DNA, requiring the clustering of mutations (eg, KRAS codons 12 and 13). With each of these approaches, a single amplicon from a single patient is analyzed in a single well or capillary.Massively parallel, next-generation sequencing (NGS) platforms, such as the Ion Torrent Personal Genome Machine (PGM) and the Illumina MiSeq, provide limits of detection superior to pyrosequencing combined with even broader genomic coverage.^5–7 Although NGS platforms have the capability to perform cancer whole genome/exome sequencing, targeted sequencing of panels of amplicons with actionable and hotspot mutations is currently more practical in a clinical laboratory setting.^8–11 One panel, the Ion Torrent AmpliSeq Cancer Hotspot Panel, uses multiplex-PCR to cover >200 amplicons in 50 genes known to be involved in carcinogenesis. We recently transitioned to this platform to determine KRAS, BRAF, and EGFR mutation status in formalin-fixed, paraffin-embedded (FFPE) lung and colon cancer specimens.A major issue with FFPE specimens is significant variability in both the quality and quantity of DNA that can be isolated. Sources of this variability include the amount of tumor in the biopsy, time from biopsy/resection to fixation, and time in formalin before processing.^12,13 As a result, we often are left with relatively low DNA concentrations, which may require use of less DNA than the recommended 10- to 30-ng amount for the AmpliSeq panel.Here, we report that off-target amplification is common in multiplex-PCR–based NGS, yielding 19 mispriming events in 208 amplicons (9%) in our study. We define the signature features to identify mispriming events and show that false-positive mutations can be avoided by using multiple bioinformatic analysis tools in the pipeline. We also show that these events are more common with lower input DNA amounts. We demonstrate that the phenomenon is due to multiplex PCR and is not seen when primers are used in monoplex reactions. Finally, because the vast majority of primers do not show significant mispriming, we hypothesize that NGS may be the ultimate multiplex PCR primer design tool by allowing for sensitive detection of off-target amplification and consequent iterative primer design.     

Haplotype Counting by Next-Generation Sequencing for Ultrasensitive Human DNA Detection

Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Myeloablative conditioning and allogeneic stem cell transplantation have historically been limited to the treatment of lethal hematological malignancies in children or young adults. More recently, with the advent of highly immunosuppressive, nonmyeloablative regimens, the clinical use of allogeneic stem cell transplantation has expanded to include older, less fit patients with hematological malignancies and patients with nonmalignant disorders, such as sickle-cell disease.^{1, 2, 3, 4} Nonmyeloablative conditioning regimens offer the additional safeguard of recovery of autologous hematopoiesis in the event of graft rejection and may be a safer option in patients at risk for immune-mediated rejection of the donor graft.Chimerism testing at set intervals is an effective method for detecting graft rejection or recurrence of the original hematopoietic neoplasm after allogeneic hematopoietic stem cell transplantation (HSCT) [with either bone marrow (BM) or peripheral blood stem cells]. Decades ago, BM engraftment (BME) monitoring was performed using Southern blot analysis and minisatellite or variable number of tandem repeats loci.⁵ Today, short tandem repeat (STR) or microsatellite loci are most commonly used for this purpose.^{6, 7, 8} STRs are composed of 10 to 60 tandemly repeated units, in which each unit is 1 to 6 bases in length. They are widely distributed throughout the human genome and highly variable between individuals; therefore, they allow for excellent differentiation between individuals, including patient and donor, even if they are closely related. Most laboratories use multiplex PCR-based kits, originally developed for forensics analysis using Combined DNA Index System loci.^{7, 9, 10} STR analysis most commonly involves PCR amplification using fluorescently labeled primers, followed by amplicon separation by capillary electrophoresis.Other polymorphic DNAs that could be used to monitor BME include single-nucleotide polymorphisms (SNPs).^{11, 12, 13} SNPs are theoretically superior to STR-based analyses because analysis of STR loci by capillary electrophoresis is relatively insensitive [limit of detection (LD), 1% to 5%] and microsatellite alleles of varying length amplify with different efficiencies, thus making them inherently biased. STR amplification can also be difficult in the setting of highly degraded DNA. However, SNPs are less attractive as targets because of their inherently lower informativity (eg, only two possible bases for a bi-allelic SNP versus ≥10 alleles for some microsatellites), requiring many more SNPs to be tested to identify those that distinguish donor from recipient. For example, we previously estimated that one would need to screen >20 to 30 individual SNPs to confidently identify one SNP where the donor is homozygous for one allele and an unrelated recipient is homozygous for the other allele.¹¹ Fewer would need to be included if heterozygotes were included, but more would have to be analyzed for related individuals.Recently emerging next-generation sequencing (NGS) technologies, along with their decreasing costs, are now feasible for clinical testing. However, all NGS technologies currently have high error rates, in the range of 0.04% to 1% at each base,¹⁴ which precludes their use for ultrasensitive detection of one SNP. One solution to this problem is sequencing blocks of closely spaced SNPs (ie, haplotypes). Haplotypes are regions of the genome, where polymorphic areas are sufficiently close that they are inherited together, including either genes (eg, HLA-A and HLA-B) within a locus or multiple SNPs within a region of DNA.Herein, we first used the HLA-A locus as proof-of-principle to demonstrate that this approach permits high sensitivity, precision, and accuracy. We then studied BM samples from a cohort of patients who engrafted after HSCT and tested as all donors by STRs, and found that low-level patient DNA is commonly present. To identify additional loci that could be used for this purpose, we comprehensively analyzed the human genome and identified other regions with highly informative haplotypes. We discuss additional situations where routine haplotyping patient samples could improve patient safety.     

Optimization of gas chromatographic method for the enantioseparation of arylpropionic non-steroidal anti-inflammatory drug methyl esters

The gas chromatography (GC) method for enantioseparation of well-known non-steroidal anti-inflammatory drugs ibuprofen, fenoprofen and ketoprofen methyl esters mixture was developed. Best enantioseparation was performed on capillary column with heptakis-(2,3-di-O-methyl-6-O-t-butyldimethyl-silyl)-beta-cyclodextrin stationary phase and hydrogen used as a carrier gas. Initial temperature, program rate and carrier pressure were optimized to obtain best resolution between enantiomers.