[Clinical Value of Detecting ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia Based on High-Throughput Sequencing Technology].

OBJECTIVE: To compare the efficacy and clinical value of high-throughput sequencing (HTS) and Sanger sequencing in detecting ABL kinase domain mutations in patients with chronic myeloid leukemia (CML). METHODS: A total of 198 samples of 147 CML patients from July 2017 to March 2021 in Henan Cancer Hospital were collected and underwent high-throughput sequencing and Sanger sequencing to detect the mutations in ABL kinase domain, and the relevant clinical data were collected for comparative analysis. RESULTS: The proportion of total mutations and ≥2 mutations detected by high-throughput sequencing were significantly higher than those detected by Sanger sequencing (P =0.01; P =0.046). ≥2 mutations were detected in 22 cases, of which 5 cases (22.7%) had compound mutations. High-throughput sequencing can detect low level mutations that cannot be detected by Sanger sequencing. In 198 samples, 25 (12.6%) were low level mutations, 33 (16.7%) were high level mutations and 10 (5.1%) were mixed high and low level mutations. In the analysis of related clinical factors, the total mutation rate and the low level mutation rate in the optimal period, failure period and warning period were gradually increased (total mutation rate, P =0.016; low level mutation rate, P =0.005). The mutation rate of the samples with additional chromosomal abnormalities was also significantly increased (P =0.009). The mutation rate of patients who received first- and second-line treatment was significantly lower than that of patients who received third- or higher-line treatment (P =0.006). Analysis based on variant allele frequency (VAF) of the mutation site was helpful to visually evaluate the clonal evolution status of TKI-resistance CML cells. CONCLUSION: High-throughput sequencing is more sensitive and accurate than Sanger sequencing in mutation detection, which is helpful to accurately and visually evaluate TKI treatment response and optimize treatment strategy for CML.

Regulatory Effect and Mechanism of Erythroblastic Island Macrophages on Anemia in Patients with Newly Diagnosed Multiple Myeloma.

Objective: To examine the clinical characteristics and anemia-related factors in patients with newly diagnosed multiple myeloma (NDMM), as well as the effect and mechanism of erythroblastic islands (EBIs) and EBI macrophages in NDMM patients with anemia. Methods: We collected and analyzed clinical data to find anemia-related factors. Using flow cytometry, the numbers and ratios of erythroblasts and EBI macrophages were determined. RNA sequencing (RNA-seq) was used to determine the differences of EBI macrophages in NDMM patients with or without anemia. Results: Based on the clinical characteristics of NDMM patients with anemia, MCV, abnormal levels of albumin, osteolytic lesions, and Durie-Salmon (DS) stage are risk factors for anemia. Patients with anemia have fewer erythroblasts, erythroblastic islands (EBIs), and EBI macrophages in their bone marrow than patients without anemia. RNA-seq analysis of EBI macrophages from the bone marrow of patients with and without anemia revealed that macrophages from patients with anemia are impaired and tend to promote the production of interleukin-6, which has been demonstrated to be an essential survival factor of myeloma cells and protects them from apoptosis. Conclusion: In NDMM patients with anemia, EBI macrophages are impaired, which causes anemia in those patients. Our finding highlights the significance of EBI macrophages in anemia in NDMM patients and provides a new strategy for recovery from anemia in these patients.

Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome.

Copy number aberrations (CNA) are the core determinants for diagnosis, risk stratification and prognosis in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In this study, a shallow whole-genome sequencing-based assay, LeukoPrint, was utilized to depict genomic CNA profiles from the bone marrow of 137 newly diagnosed AML/MDS patients. It demonstrated 98.1% concordance of CNA profiles with cytogenetics and/or fluorescence in situ hybridization (FISH). It is advantageous in detecting CNAs of short segments (1 Mb) and from samples with low leukemic cell content, more accurate for describing complex karyotypes and less confounded by subjective bias. LeukoPrint improved the overall diagnostic yield by redefining the risk categories for 16 patients by presenting new information. In summary, LeukoPrint provided an automated, convenient, and cost-effective approach to describe genomic CNA profiles. It brought greater diagnostic yield and risk stratification information by incorporating into the routine cytogenetics based on the CNA-related criteria of standard ELN/IPSS-R guidelines.

[Strategy Optimization and Clinical Analyze of Multiple Nucleotide Polymorphism Analysis in the Chimerism Detection after Allogeneic Hematopoietic Stem Cell Transplantation].

AbstractObjective: To investigate the sample selection, result correction and clinical application value of multi nucleotide polymorphism chimerism detection method based on Next-generation sequencing. METHODS: The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method. RESULTS: The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance. CONCLUSION: MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

Residue of thiram in food, suppresses immune system stress signals and disturbs sphingolipid metabolism in chickens.

Thiram, a well-known sulfur containing organic compound is frequently and extensively used in agriculture because of high biological activity to control different pests. In certain cases, due to long persistence in the environment pesticides and other environmental contaminants induce undesirable toxic impacts to public health and environment. To ascertain the potential mechanisms of toxicity of thiram on different immune organs of broilers, a total of 100 one-day-old chicks were obtained and randomly divided into two groups including thiram group (50 mg/kg) and untreated control group. Thymus and spleen tissues were collected at the age of 14 days from the experimental birds. At necropsy level, thymus was congested, enlarged and hyperemic while spleen had no obvious lesions. The results on mechanisms (apoptosis and autophagy) of immunotoxicity showed significantly increased expression of bax, caspase3, cytc, ATG5, beclin1 and p62 in spleen of treated mice. Results indicated significantly decreased expression of m-TOR and bcl2 to activate apoptosis and autophagy. The expressions of bax, p53 and m-TOR were up-regulated in the thymus while the expressions of ATG5 and Beclin1 were down-regulated to mediate cell apoptosis and inhibit autophagy. The results on different metabolome investigation showed that the sphingolipid metabolism in the thymus of chicks exposed to thiram was disrupted resulting in up-regulation of metabolites related to cell membrane components such as SM, galactosylceramide and lactosylceramide. The results of our experimental research suggest that thiram can interfere with the sphingolipid metabolism in thymus and angiogenesis, inhibit the proliferation of vascular endothelial cells to induce potential toxic effects in chicken.

The potential risks of herbicide butachlor to immunotoxicity via induction of autophagy and apoptosis in the spleen.

Butachlor being an important member of chloroacetanilide herbicides, is frequently used in agriculture to control unwanted weeds. Exposure to butachlor can induce cancer, human lymphocyte aberration, and immunotoxic effects in animals. The current experimental trial was executed to determine the potential risks of herbicide butachlor to immunotoxicity and its mechanism of adverse effects on the spleen. For this purpose, mice were exposed to 8 mg/kg butachlor for 28 days, and the toxicity of butachlor on the spleen of mice was evaluated. We found that butachlor exposure led to an increase in serum ALB, GLU, TC, TG, and TP and changes in the morphological structure of the spleen of mice. More importantly, results showed that butachlor significantly increased the expression level of ATG-5, decreased the protein expression of LC3B and M-TOR, and significantly decreased the mRNA content of M-TOR and p62. Results revealed that the mRNA contents of APAF-1, CYTC, and CASP-9 related genes were significantly decreased after butachlor treatment. Subsequently, the mRNA levels of inflammatory cytokines (IL-1ß, TNF-α, IL-10) were reduced in the spleen of treated mice. This study suggested that butachlor induce spleen toxicity and activate the immune response of spleen tissue by targeting the CYTC/BCL2/M-TOR pathway and caspase cascading activation of spleen autophagy and apoptosis pathways which may ultimately lead to immune system disorders.

The potential risks of chronic fluoride exposure on nephrotoxic via altering glucolipid metabolism and activating autophagy and apoptosis in ducks.

Fluoride is one of the most widely distributed elements in nature, while some fluorine-containing compounds are toxic to several vertebrates at certain levels. The current study was performed to evaluate the nephrotoxic effects of fluoride exposure in ducks. The results showed that the renal index was decreased in NaF group, and fluoride exposure significantly decreased the levels of serum Albumin, Glucose, Total cholesterol, Urea, protein and Triglycerides, confirming that NaF exhibited adverse effects on the kidney. The overall structure of renal cells showed damage with the signs of nuclelytic, vacuolar degeneration, atrophy, renal cystic cavity widening after fluoride induction. Renal vascular growth was impaired as the expression of VEGF and HIF-1α decreased (p > 0.05). More importantly, autophagy and apoptosis levels of CYT C, LC3, p62, Beclin, M-TOR, Bax and Caspase-3 were increased (p < 0.05) in the NaF treated group. Interestingly, our results showed that Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC) activated the M-TOR autophagy pathway. Meanwhile, the PE acted on Atg5/ LC3 autophagy factor, followed by the auto-phagosome generation and activation of cell autophagy. These results indicate that NaF exposure to duck induced nephron-toxicity by activating autophagy, apoptosis and glucolipid metabolism pathways, which suggest that fluorine exposure poses a risk of poisoning.

Transcriptome analysis of the response provided by Lasiopodomys mandarinus to severe hypoxia includes enhancing DNA repair and damage prevention.

BACKGROUND: Severe hypoxia induces a series of stress responses in mammals; however, subterranean rodents have evolved several adaptation mechanisms of energy metabolisms and O2 utilization for hypoxia. Mammalian brains show extreme aerobic metabolism. Following hypoxia exposure, mammals usually experience irreversible brain damage and can even develop serious diseases, such as hypoxic ischemic encephalopathy and brain edema. To investigate mechanisms underlying the responses of subterranean rodents to severe hypoxia, we performed a cross-species brain transcriptomic analysis using RNA sequencing and identified differentially expressed genes (DEGs) between the subterranean rodent Lasiopodomys mandarinus and its closely related aboveground species L. brandtii under severe hypoxia (5.0% O2, 6 h) and normoxia (20.9% O2, 6 h). RESULTS: We obtained 361 million clean reads, including 69,611 unigenes in L. mandarinus and 69,360 in L. brandtii. We identified 359 and 515 DEGs by comparing the hypoxic and normoxia groups of L. mandarinus and L. brandtii, respectively. Gene Ontology (GO) analysis showed that upregulated DEGs in both species displayed similar terms in response to severe hypoxia; the main difference is that GO terms of L. brandtii were enriched in the immune system. However, in the downregulated DEGs, GO terms of L. mandarinus were enriched in cell proliferation and protein transport and those of L. brandtii were enriched in nuclease and hydrolase activities, particularly in terms of developmental functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that upregulated DEGs in L. mandarinus were associated with DNA repair and damage prevention as well as angiogenesis and metastasis inhibition, whereas downregulated DEGs were associated with neuronal synaptic transmission and tumor-associated metabolic pathways. In L. brandtii, upregulated KEGG pathways were enriched in the immune, endocrine, and cardiovascular systems and particularly in cancer-related pathways, whereas downregulated DEGs were associated with environmental information processing and misregulation in cancers. CONCLUSIONS: L. mandarinus has evolved hypoxia adaptation by enhancing DNA repair, damage prevention, and augmenting sensing, whereas L. brandtii showed a higher risk of tumorigenesis and promoted innate immunity toward severe hypoxia. These results reveal the hypoxic mechanisms of L. mandarinus to severe hypoxia, which may provide research clues for hypoxic diseases.

Identification of a novel nonsense mutation in SH2D1A in a patient with X-linked lymphoproliferative syndrome type 1: a case report.

BACKGROUND: X-linked lymphoproliferative syndrome type 1 (XLP1) is an X-linked recessive genetic disorder with a strong resemblance to hemophagocytic lymphohistiocytosis (HLH). Causative mutations for XLP1 have been identified in SH2D1A, located on chromosome Xq25. CASE PRESENTATION: We report a case of an 18-month-old male with a novel nonsense mutation in SH2D1A. The patient presented the typical phenotype of HLH, including splenomegaly and hemophagocytosis in the bone marrow. Thus, he was initially diagnosed with HLH based on HLH-2004 guidelines. High-throughput amplicon sequencing was performed to detect mutations in the most commonly reported causative genes of HLH, i.e., PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP. A likely pathogenic nonsense mutation was detected in SH2D1A (NM_002351.4:c.300T>A). The mutation was inherited from the patient's mother, and an X-linked recessive mode of inheritance was confirmed by a two-generation pedigree analysis based on Sanger sequencing results. CONCLUSIONS: The nonsense mutation in SH2D1A (NM_002351.4:c.300T>A) was reported for the first time in a case of XLP1 and was considered to be likely pathogenic based on the truncation of the mRNA sequence. This finding expands the spectrum of known XLP-related mutations in Chinese patients and indicates the utility of amplicon sequencing for XLP and HLH diagnosis.

Inhibition of miR­34a prevents endothelial cell apoptosis by directly targeting HDAC1 in the setting of atherosclerosis.

Despite recent medical advances, atherosclerosis is a global burden accounting for numerous mortalities and hospital admissions. MicroRNAs (miRNAs/miRs) regulate cardiovascular biology and disease, but the role of microRNA­34a in atherosclerosis remains unclear. In the present study, it was demonstrated that miR­34a was highly expressed in atherosclerotic lesions and oxidized low­density lipoprotein (Ox­LDL)­treated human aortic endothelial cells (HAECs) (atherosclerotic cell model) using reverse transcription­quantitative polymerase chain reaction. The expression of histone deacetylase (HDAC) 1 was reduced in atherosclerotic lesions and Ox­LDL treated HAECs. TargetScan predicted that HDAC1 is the potential target of miR­34a and the double­luciferase reporter assay confirmed that HDAC1 was directly targeted by miR­34a. Furthermore, miR­34a inhibitor significantly enhanced the cell viability of HAECs and the cell apoptosis was suppressed. In addition, the expression of apoptotic­related proteins was detected by western blotting. The results showed that miR­34a inhibitor significantly upregulated B­cell lymphoma 2, procaspase­3, procaspase­9 and proto­oncogene c­Myc protein expression, and downregulated the expression of p21. In contrast, co­transfection of HDAC1­small interfering RNA and miR­34a inhibitor eliminated the effects of miR­34a on HAECs. This indicated that miR­34a inhibitor promoted cell viability and prevented cell apoptosis of HAECs through regulating HDAC1. In conclusion, it was demonstrated that miR­34a promoted atherosclerotic formation by modulating the proliferation and apoptosis of HAECs, and regulating the expression of apoptosis­related proteins by targeting HDAC1.