Mutations In Loci Research Articles

A novel C19ORF12 mutation in two MPAN sisters treated with deferiprone

BackgroundMitochondrial membrane protein-associated neurodegeneration (MPAN) is a rare and devastating disease caused by pathogenic mutations in C19orf12 gene. MPAN is characterized by pathological iron accumulation in the brain and fewer than 100 cases of MPAN have been described. Although the diagnosis of MPAN has achieved a great breakthrough with the application of the whole exome gene sequencing technology, the therapeutic effect of iron chelation therapy in MPAN remains controversial.Case presentationWe reported that two sisters from the same family diagnosed with MPAN had dramatically different responses to deferiprone (DFP) treatment. The diagnosis of MPAN were established based on typical clinical manifestations, physical examination, brain magnetic resonance imaging (MRI), cerebrospinal fluid analysis (CSF) and gene sequencing results. The clinical presentations of the two sisters with MPAN due to novel gene locus mutations were similar to those previously reported. There is no other difference in basic information except that the proband had a later onset age and fertility history. Both the proband and his second sister were treated with deferiprone (DFP), but they had dramatically different responses to the treatment. The proband’s condition deteriorated sharply after treatment with DFP including psychiatric symptoms and movement disorders. However, the second sister of the proband became relatively stable after receiving the DFP treatment. After four years of follow-up, the patient still denies any new symptoms of neurological deficits.ConclusionThe findings of this study enriched the MPAN gene database and indicated that DFP might ameliorate symptom progression in patients without severe autonomic neuropsychiatric impairment at the early stage of the disease.

Mouse model of PRSS1 p.R122H-related hereditary pancreatitis highlights context-dependent effect of autolysis-site mutation

Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.

Knockdown resistance mutations distribution and characteristics of Aedes albopictus field populations within eleven dengue local epidemic provinces in China.

Aedes albopictus, commonly known as the tiger mosquito, has attracted global attention because its bite can transmit several viruses, such as dengue virus. With the absence of an effective therapy and vaccine, mosquito control is the sole method for dengue fever control. However, Ae. albopictus has developed resistance to most insecticides, especially pyrethroids. Many scholars have conducted thorough research for the target-site of pyrethroids. The main target-site is the voltage-gated sodium channel gene (VGSC) whose mutation causes knockdown resistance (kdr). The spatial distribution of three locus kdr mutations in Ae. albopictus has not been comprehensively analyzed nationwide in China. In addition, the relationship between the frequency of kdr mutations and dengue fever has not yet been explored. A total of 2,241 Ae. albopictus samples from 49 populations from 11 provinces of mainland China were collected in 2020 and analyzed for mutations in the VGSC gene. DNAstar 7.1. Seqman and Mega-X were used to compare the sequences and read the peak map to confirm the genotypes and alleles of each mutation. ArcGIS 10.6 software was used to make interpolation and extract meteorological data of collection sites and to conduct spatial autocorrelation analysis. R 4.1.2 software was used to conduct a chi-square test for kdr mutations and dengue area and to analyze the correlation between meteorological factors and kdr mutations. The overall frequencies of mutant alleles at 1016G, 1532T, and 1534S/C/L were 13.19%, 4.89%, and 46.90%, respectively. Mutations at the three loci were found at 89.80% (44/49), 44.90% (22/49), and 97.96% (48/49) of the field populations. At each of the loci V1016 and I1532, only one allele was detected, which was GGA(G) and ACC(T), respectively. Five mutant alleles were found at codon 1534: TCC/S (33.49%), TGC/C (11.96%), TTG/L (0.60%), CTC/L (0.49%), and TTA/L (0.58%). In total, 31 triple-locus genotype combinations were found, and the single locus mutation was the most common. We also found firstly triple-locus mutant individuals, whose genotypes were V/G+I/T+F/S and V/G+I/T+S/S. The 1016 and 1532 mutation rates were significantly negatively related to the annual average temperature (AAT), but the 1534 mutation rate was significantly positively related to AAT. The 1532 mutation rate was significantly positively related to the 1016 mutation rate but negatively related to the 1534 mutation rate. A relationship was observed between the 1534 codon mutation rate and dengue epidemic areas in this study. Furthermore, spatial autocorrelation analysis results showed that the mutation rates of different codons in different geographical areas had spatial aggregation and positive spatial correlation. This study showed that the multiple kdr mutations at codon 1016, 1532 and 1534 of Ae. albopictus were found in most areas of China. Two novel triple-locus genotype combinations, V/G+I/T+F/S and V/G+I/T+S/S, were detected in this study. In addition, the relationship between mosquito resistance and dengue fever outbreak should be further explored, especially considering the insecticide-usage history in different areas. The characteristic of spatial aggregation of VGSC gene mutation rates reminds us to notice the gene exchange and similarity of insecticide usage in the adjacent areas. The use of pyrethroids should be restricted to delay resistance development. New-type insecticides should be developed to adjust the changes in the resistance spectrum. Our study provides abundant data on the Ae. albopictus kdr gene mutation in China; these findings will be useful for the correlation analysis of molecular mechanism of insecticide resistance.

Fluoroquinolone resistance mutations among Mycobacterium tuberculosis and their interconnection with treatment outcome.

Fluoroquinolone (FQ) antibiotics are among the most potent second-line antitubercular drugs these days. The aim of the study was to analyze the frequency and pattern of genetic mutation in preextensive (pre-XDR) and extensively drug-resistant Mycobacterium tuberculosis using second-line line probe assay (LPA) and to compare drug-resistant mutations with different treatment outcomes. Sputum, lymph node aspirate, and cold accesses from patients with rifampicin-resistant Tuberculosis (TB) were subjected to first-line and second-line LPA (Genotype MTBDRsl by Hain Life Science, Germany) to assess additional drug resistance to fluoroquinolones (levofloxacin and moxifloxacin). Final treatment outcomes as per the National TB Elimination Program were assessed and compared with the mutation profile. One hundred and fifty subjects were observed to have mutations associated with resistance to FQs and constituted the final study population. The most frequent mutation observed among GyrA drug resistance mutation was D94G (Gyr A MUT3C, 44/150, 66%) corresponding to high-level resistance to levofloxacin and moxifloxacin. The same mutation was associated with poor treatment outcome as died or treatment failure (odds ratio 2.50, relative risk 1.67, P = 0.043). The most common hetero-resistance mutation pattern observed in GyrA gene was wild type plus Asp94Gly mutation in 24.6% of isolates. GyrA MUT3C hybridization corresponding to single-point mutation of aspartic acid to glycine at codon 94 constitutes the most common mutation in GyrA gene locus in M. tuberculosis with significant association with treatment outcome as died compared to those with treatment outcome as cured.

Second-line drug resistance markers as proxy indicators of time to sputum culture conversion among second-line drug resistant tuberculosis patients tested in Uganda: A cross-sectional study

Background Increased tuberculosis disease burden arises as a result of low treatment success rates stemming from the emergence of second-line drug resistance. We aimed at determining the usefulness of second-line drug (SLD) resistance markers as proxy indicators of time to sputum culture conversion; a renowned predictor of Tuberculosis treatment outcome, among SLD-resistant tuberculosis (TB) patients tested at the Uganda National TB Reference Laboratory (NTRL). Methods A cross-sectional study was conducted on 72 bacteriologically confirmed SLD resistant TB patients with datasets including culture conversion time and second line probe assay mutation profiles between 01/06/2017 and 31/12/2019. The data were then imported into STATA v15 for descriptive statistical analysis, Univariate cox proportional hazard model analysis and Kaplan-Meier survival curves at a 5% level of significance; p-value ≤0.05. Results Results indicate the median time was achieved at 3 (0–12) months across the studied patients. The rrs G1484T mutation associated with conferring drug resistance to injectable agents was observed to have the shortest median conversion time of 1.5 months, longest by the gryB E540D at 5 months. A single mutation in the gryA gene locus showed higher converted proportions 70.8% (58.9–81.0) than those that had two 8.3% (3.1–17.3) or three 2.7% (0.3–10.0) mutations. Conclusions The studied second-line drug resistance markers had no statistically significant association with the time to sputum culture conversion, although increased drug resistance levels reduced the converted proportions and stressed the need to utilize molecular diagnostics data and other crucial variables to better comprehend proxy indicators of SLD resistant tuberculosis management.

The rate of spontaneous mutations in yeast deficient for MutSβ function.

Mutations in simple sequence repeat loci underlie many inherited disorders in humans, and are increasingly recognized as important determinants of natural phenotypic variation. In eukaryotes, mutations in these sequences are primarily repaired by the MutSβ mismatch repair complex. To better understand the role of this complex in mismatch repair and the determinants of simple sequence repeat mutation predisposition, we performed mutation accumulation in yeast strains with abrogated MutSβ function. We demonstrate that mutations in simple sequence repeat loci in the absence of mismatch repair are primarily deletions. We also show that mutations accumulate at drastically different rates in short (<8 bp) and longer repeat loci. These data lend support to a model in which the mismatch repair complex is responsible for repair primarily in longer simple sequence repeats.

Association between KRAS gene polymorphisms and genetic susceptibility to breast cancer in a Chinese population.

The KRAS gene has a pathophysiological role in the development of many cancers. This study aims to investigate the relationship between KRAS polymorphisms and genetic susceptibility to breast cancer. The rs712, rs12587 and rs9266 gene loci in the KRAS gene of 421 subjects (141 breast cancer patients, 141 benign breast tumours and 139 healthy controls) were analysed by the polymerase chain reaction and SNaPshot sequencing. Transcriptomic information on KRAS and corresponding clinical information was downloaded from the TCGA and GTEx databases. Differences in KRAS expression between breast cancer tissues and control tissues were analysed. We found no significant association between KRAS rs712 and rs12587 locus gene polymorphisms and an increased risk of developing benign breast tumours and breast cancer (p > 0.05). The KRAS rs9266 locus mutation heterozygous model CT and dominant model CT + TT were significantly associated with an increased risk of breast cancer (both p < 0.05). In addition, the TAT haplotype was expressed at an increased frequency, and the GAC haplotype was expressed at a reduced frequency in breast cancer compared with controls (both p < 0.05). We found that KRAS was over expressed in breast cancer tumour tissues compared with the control tissues (p < 0.0001). The KRAS rs9266 gene polymorphism and the TAT haplotype may be associated with an increased risk of breast cancer in Chinese women. The GAC haplotype may be a protective factor against breast cancer.

Polymorphism, Expression, and Structure Analysis of a Key Gene ARNT in Sheep (Ovis aries).

Growth traits are influential factors that significantly affects the development of the sheep industry. A previous TMT proteomic analysis found that a key protein in the HIF signaling pathway, ARNT, may influence embryonic skeletal muscle growth and development in sheep. The purpose of this study was to better understand the association between the polymorphisms of ARNT and growth traits of sheep, and the potential function of ARNT. Real-time qPCR (qRT-PCR) of ARNT was carried out to compare its expression in different developmental stages of the muscle tissues and primary myoblasts in the Hu, Chinese merino, and Gangba sheep. The genetic variance of ARNT was detected using the Illumina Ovine SNP 50 K and 600 K BeadChip in the Hu and Ujimqin sheep populations, respectively. The CDS sequence of the ARNT gene was cloned in the Hu sheep using PCR technology. Finally, bioinformatic analytical methods were applied to characterize the genes and their hypothetical protein products. The qRT-PCR results showed that the ARNT gene was expressed significantly in the Chinese merino embryo after 85 gestation days (D85) (p &lt; 0.05). Additionally, after the sheep were born, the expression of ARNT was significant at the weaning stage of the Hu sheep (p &lt; 0.01). However, there was no difference in the Gangba sheep.In addition, six SNP loci were screened using 50 K and 600 K BeadChip. We found a significant association between rs413597480 A &gt; G and the Hu sheep weight at weaning and backfat thickness in the 5-month-old sheep (p &lt; 0.05), and four SNP loci (rs162298018 G &gt; C, rs159644025 G &gt; A, rs421351865 G &gt; A, and rs401758103 A &gt; G) were also associated with growth traits in the Ujimqin sheep (p &lt; 0.05). Interestingly, we found that a G &gt; C mutation at 1948 bp in the cloned ARNT CDS sequence of the Hu sheep was the same locus mutation as rs162298018 G &gt; C identified using the 600 K BeadChip, which resulted in a nonconservative missense point mutation, leading to a change from proline to alanine and altering the number of DNA, protein-binding sites, and the α-helix of the ARNT protein. There was a strong linkage disequilibrium between rs162298018 G &gt; C and rs159644025 G &gt; A, and the ARNT protein was conserved among the goat, Hu sheep, and Texel sheep. And, we propose that a putative molecular marker for growth and development in sheep may be the G &gt; C mutation at 1948 bp in the CDS region of the ARNT gene. Our study systematically analyzed the expression, structure, and function of the ARNT gene and its encoded proteins in sheep. This provides a basis for future studies of the regulatory mechanisms of the ARNT gene.

Defective activation and regulation of type I interferon immunity is associated with increasing COVID-19 severity

Host immunity to infection with SARS-CoV-2 is highly variable, dictating diverse clinical outcomes ranging from asymptomatic to severe disease and death. We previously reported reduced type I interferon in severe COVID-19 patients preceded clinical worsening. Further studies identified genetic mutations in loci of the TLR3- or TLR7-dependent interferon-I pathways, or neutralizing interferon-I autoantibodies as risk factors for development of COVID-19 pneumonia. Here we show in patient cohorts with different severities of COVID-19, that baseline plasma interferon α measures differ according to the immunoassay used, timing of sampling, the interferon α subtype measured, and the presence of autoantibodies. We also show a consistently reduced induction of interferon-I proteins in hospitalized COVID-19 patients upon immune stimulation, that is not associated with detectable neutralizing autoantibodies against interferon α or interferon ω. Intracellular proteomic analysis shows increased monocyte numbers in hospitalized COVID-19 patients but impaired interferon-I response after stimulation. We confirm this by ex vivo whole blood stimulation with interferon-I which induces transcriptomic responses associated with inflammation in hospitalized COVID-19 patients, that is not seen in controls or non-hospitalized moderate cases. These results may explain the dichotomy of the poor clinical response to interferon-I based treatments in late stage COVID-19, despite the importance of interferon-I in early acute infection and may guide alternative therapeutic strategies.

TET2 Mutation Is More Valuable in Predicting Thrombosis in Thrombocythemia Patients Compared to Polycythaemia Vera

Background Thrombosis is a common complication of myeloproliferative neoplasm (MPN) and its major cause of disability and death. With the development of next-generation gene sequencing technology, the relationship between non-driver mutations and thrombotic risk factors has also attracted close attention. Methods The clinical data of 125 MPN patients (75 ET and 50 PV) attending Xiyuan Hospital of the China Academy of Chinese Medical Sciences between January 2018 and March 2022 were retrospectively analyzed. A multivariate analysis of risk factors for thrombosis was performed using a Cox proportional hazards model. Results A total of 125 patients with MPN (75 ET and 50 PV) were evaluated, with a median age at diagnosis of 50 (18-78) years, 67 (53.6%) males and 58 (46.4%) females. The mutation rates of driver genes JAK2V617F, CALR, and MPL were 70.4%, 12.8%, and 0.8%, respectively. At least one non-driver mutation was detected in 36% of patients, with the highest rate of mutations in TET2 (12%), followed by ASXL1 (9.6%). Thrombotic events occurred in 35 of 125 patients (28.0%), and the incidence of thrombotic events was 21.3% (16/75) in ET patients and 38.0% (19/50) in PV patients, respectively. For 125 MPN patients, univariate analysis revealed significant associations between thrombosis and age at diagnosis, age >60 years at diagnosis, JAK2V617F mutation, TET2 mutation, WBC, NLR, cardiovascular risk factors and history of remote thrombosis. In the multivariate analysis that included the above 8 factors as covariates, age at diagnosis (HR: 1.03, 95％ CI: 1.00-1.06; P = 0.046), TET2 mutation (HR: 2.84, 95％ CI: 1.26-6.36; P = 0.011), NLR (HR: 1.18, 95％ CI: 1.06-1.32; P = 0.003), and history of remote thrombosis (HR: 5.33, 95％ CI: 1.93-14.70; P = 0.001) remained risk factors for thrombosis in MPN patients. In the subgroup analysis of ET and PV patients, univariate analysis was performed for the parameters, and factors with differences were used as covariates for further multivariate analysis. In ET patients, multivariate analysis showed that TET2 mutation and a history of remote thrombosis were independent risk factors for thrombosis in ET patients, with an HR of 4.1 (95% CI: 1.40-12.01; P = 0.01) for TET2 mutation and 6.89 (95% CI: 1.45-32.68; P = 0.015) for history of remote thrombosis (Table 1). In PV patients, multivariate analysis observed neutrophil-to-lymphocyte ratio (NLR) (HR: 4.77, 95% CI: 1.33-17.16; P = 0.017) and a history of remote thrombosis (HR: 1.67, 95% CI: 1.03-1.32; P = 0.014) as independent risk factors for thrombosis, however, no association of TET2 mutation with thrombosis was observed in PV (Table 1). Among 125 patients, TET2 mutations were detected in 15 patients (9 ET and 6 PV), with a mutation rate of 12%. Among them, 11 patients combined with JAK2V617F mutation (6 ET and 5 PV), 2 ET patients combined with CALR mutation and 1 ET patient combined with ASXL1 mutation. Two loci mutations of TET2 were detected in one patient with PV. The details of the 15 MPN patients with TET2 mutation are shown in Table 2. Our study above have shown that TET2 mutation is an independent risk factor for thrombosis in ET patients, and to understand their clinical characteristics and coagulation status, we compared the clinical characteristics and coagulation function of TET2 mutated and unmutated patients. We found that the age of ET patients with TET2 mutation was significantly higher than that of patients without TET2 mutation (P < 0.05) . In terms of coagulation function, compared with ET patients without TET2 mutation, patients with TET2 mutation were in a relatively hypercoagulable state, as evidenced by a significant increase in D-Dimer (P < 0.01) and a significant decrease in antithrombin Ⅲ (AT-III) (P < 0.05). Conclusion TET2 mutation is an independent risk factor for thrombosis in ET patients, but no such association was observed in PV. Therefore, TET2 mutation is more valuable in predicting thrombosis in ET patients compared to PV. ET patients with TET2 mutation were older and in a relatively hypercoagulable state of coagulation compared to TET2 unmutated patients. However, prospective studies with more patients are needed to validate our results and to fully elucidate the association. Our study reports risk factors for thrombosis in MPN patients in Asia and complements the data on the relationship between non-driver mutations and thrombotic events in Asian MPN patients, providing a reference for future larger studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Downregulation of the Mutated WT1 Alleles in Malignant Hematopoietic Cells Via Nonsense-Mediated mRNA Decay Contradicts Their Prognostic Impact

Background The Wilms Tumor 1 (WT1) gene is highly expressed in almost all types of malignancies including acute myeloid leukemia (AML). It is also mutated in up to 20% of AML blasts in pediatric and adult patients. Both, the increased expression and also the presence of mutations are correlated negatively with chance of survival in different AML patient cohorts. We and others have previously showed devastating survival outcomes in children with AML blasts harboring WT1 mutations in combination FLT3-ITD and NUP98-NSD1 (Niktoreh et al., 2019). As WT1 can act as both, oncogene and tumor suppressor gene, this study was designed to analyze the WT1 genomic locus mutations in acute leukemia blasts. Methods and Results Screening for WT1 sequence alterations. Using the TruSight Myeloid Sequencing PanelTM (Illumina), we initially screened 35 leukemia and lymphoma cell lines for genomic alterations in 54 genes. This panel allowed us to detect even low variant-allele-frequency (VAF) alterations in the mutational hotspots of WT1, exons 7 and 9. In addition to a known WT1 mutation in CTS cells (AML M1), we discovered novel alterations in three other malignant cell lines. KASUMI-6 cells (AML M2) harbored the identical mutation as CTS cells, a 2-bp deletion in WT1 exon 7 leading to frameshift with a premature termination codon (c.1202_1203delGA, p.Arg401IlefsTer3). The U937 histiocytic lymphoma cells carried the known TP53 mutation and also an unknown heterozygous nonsense mutation in WT1 exon 7, changing the arginine 369 into a premature stop codon (c.1105C>T, p.Arg369Ter). Interestingly, MOLM-1 cells (CML in blast crisis) harbored a p.Arg462Gln alteration. Next, we performed Sanger sequencing for the complete coding region of WT1, including the upstream promoter as well as the 5'UTR and 3'UTR regions, all 10 exons and the adjacent 150 bp intronic sequences at the exon-intron boundaries. Sanger sequencing confirmed the mutations discovered via NGS and revealed single bp alterations in the WT1 promoter region of CTS cells and in the 5'UTR of K-562 cells. We also detected a recurring variation in intron 3 of WT1 (c.872+16G>A) in the cell lines K-562, NB-4, M07e and UOC-M1; the functional implications of the alterations are yet to be determined. Functional impact of WT1 mutations. To understand the potential impact of the WT1 sequence alterations, we studied the WT1 mRNA expression in these cells in the presence of cycloheximide (CHX), an inhibitor of the nonsense-mediated mRNA decay (NMD) machinery. As shown in figure 1A, U937 cells were used as negative control cells, as they are known to have no expression of the WT1 mRNA. Despite the presence of mutations in CTS, KASUMI-6 and MOLM-1 cells, all three cell lines expressed elevated levels of WT1, well within the range of 30 other malignant cell lines without any alterations/mutations in the WT1 locus (data not shown). To differentiate the WT1 expression between the mutated and the wildtype allele, we performed a fragment length analysis on a capillary electrophoresis system using the complimentary DNA of CHX-treated cell lines. For these analyses, we employed a multiplex PCR strategy using ABL1 as housekeeping gene and a primer pair binding in exons 4 and 10 of WT1. As delineated in figure 1B, the mutated alleles in CTS and KASUMI-6 cells, which are 2-bps shorter, could be easily separated from the wildtypes (data for KASUMI-6 not shown). Additionally, since our primer pair also amplified the alternatively spliced exon 5 and the alternative splice donor at the end of exon 9, leading to inclusion or exclusion of three amino acids (KTS), we were able to separately analyze the four major WT1 isoforms from both alleles. As shown in figure 1B, the expression levels of the WT1 mutated alleles of CHX-treated cells increased 2- to 3-fold compared to DMSO-treated cells in CTS and 3- to 4-fold in KASUMI-6 (data not shown). Conclusion Careful analyses of the WT1 genomic locus in 35 malignant hematological cell lines revealed previously unknown heterozygous loss-of-function WT1 mutations in KASUMI-6, MOLM-1 and U-937 cells. mRNA expression analysis demonstrated that these mutations are strongly regulated by NMD machinery, leading to (almost) complete downregulation of the mutated WT1 allele. These results strongly question any direct effect of WT1 mutations in the leukemic blasts and therefore cannot explain the role of mutations in WT1 as a prognostic factor in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparative Analysis of Codon Usage Patterns in Chloroplast Genomes of Cherries

Synonymous codon usage bias (SCUB) analysis is an effective method to explore species specificity, evolutionary relationships and mRNA translation, as well as to discover novel genes, which are important for understanding gene function and molecular phylogeny. Cherries (Prunus subg. Cerasus) are flowering plant germplasm resources for edible and ornamental purposes. In this study, we analyzed the codon usage patterns of the 36 chloroplast genomes to provide a scientific basis for elucidating the evolution of subg. Cerasus. The results showed that the average GC content was 0.377, the average GC3 was 0.298, and the average ENC value was 49.69. Neutral-plot analysis, ENC-plot analysis, and PR2-plot analysis all indicated that natural selection was the main factor of codon usage bias in subg. Cerasus, whereas correlation analysis showed that gene expression level and GC1 also affect the codon usage pattern. The codon usage pattern was consistent across 36 species, and 30 high-frequency codons were identified, with preference for A/T endings; there were 23 optimal codons, and only GAU was identified in all individuals; structural differences existed between the clustering tree based on RSCU values and the phylogenetic tree based on CDS, elucidating the importance of locus mutations and no-preference codons in phylogenetic reconstruction. This study describes for the first time the SCUB pattern and characterization of subg. Cerasus chloroplast genomes and provides a new insight to explore the phylogeny of this subgenus.

Intravascular Large B-Cell Lymphoma Genomic Profile Is Characterized by Alterations in Genes Regulating NF-κB and Immune Checkpoints.

Intravascular large B-cell lymphoma (IVLBCL) is an uncommon lymphoma with an aggressive clinical course characterized by selective growth of tumor cells within the vessels. Its pathogenesis is still uncertain and there is little information on the underlying genomic alterations. In this study, we performed a clinicopathologic and next-generation sequencing analysis of 15 cases of IVLBCL using a custom panel for the detection of alterations in 68 recurrently mutated genes in B-cell lymphomagenesis. Six patients had evidence of hemophagocytic syndrome. Four patients presented concomitantly a solid malignancy. Tumor cells outside the vessels were observed in 7 cases, 2 with an overt diffuse large B-cell cell lymphoma. In 4 samples, tumor cells infiltrated lymphatic vessel in addition to blood capillaries. Programmed death-ligand 1 (PD-L1) was positive in tumor cells in 4 of 11 evaluable samples and in macrophages intermingled with tumor cells in 8. PD-L1 copy number gains were identified in a higher proportion of cases expressing PD-L1 than in negative tumors. The most frequently mutated gene was PIM1 (9/15, 60%), followed by MYD88L265P and CD79B (8/15, 53% each). In 6 cases, MYD88L265P and CD79B mutations were detected concomitantly. We also identified recurrent mutations in IRF4 , TMEM30A , BTG2 , and ETV6 loci (4/15, 27% each) and novel driver mutations in NOTCH2 , CCND3 , and GNA13 , and an IRF4 translocation in 1 case each. The mutational profile was similar in patients with and without evidence of hemophagocytic syndrome and in cases with or without dissemination of tumor cells outside the vessels. Our results confirm the relevance of mutations in B-cell receptor/nuclear factor-κB signaling and immune escape pathways in IVLBCL and identify novel driver alterations. The similar mutational profile in tumors with extravascular dissemination suggests that these cases may also be considered in the spectrum of IVLBCL.

A 2-bp frameshift deletion at GhDR, which encodes a B-BOX protein that co-segregates with the dwarf-red phenotype in Gossypium hirsutums L.

Plant architecture and leaf color are important factors influencing cotton fiber yield. In this study, based on genetic analysis, stem paraffin sectioning, and phytohormone treatments, we showed that the dwarf-red (DR) cotton mutant is a gibberellin-sensitive mutant caused by a mutation in a single dominant locus, designated GhDR. Using bulked segregant analysis (BSA) and genotyping by target sequencing (GBTS) approaches, we located the causative mutation to a ~197-kb genetic interval on chromosome A09 containing 25 annotated genes. Based on gene annotation and expression changes between the mutant and normal plants, GH_A09G2280 was considered to be the best candidate gene responsible for the dwarf and red mutant phenotypes. A 2-nucleotide deletion was found in the coding region of GhDR/GH_A09G2280 in the DR mutant, which caused a frameshift and truncation of GhDR. GhDR is a homolog of Arabidopsis AtBBX24, and encodes a B-box zinc finger protein. The frameshift deletion eliminated the C-terminal nuclear localization domain and the VP domain of GhDR, and altered its subcellular localization. A comparative transcriptome analysis demonstrated downregulation of the key genes involved in gibberellin biosynthesis and the signaling transduction network, as well as upregulation of the genes related to gibberellin degradation and the anthocyanin biosynthetic pathway in the DR mutant. The results of this study revealed the potential molecular basis by which plant architecture and anthocyanin accumulation are regulated in cotton.

Genetic Variability Analysis for Yield Attributes and Phenotypic Characterization of TFL1 Homologues for Growth Habit in Pigeonpea [Cajanus cajan (L.) Millspaugh

Background: Pigeonpea [Cajanus cajan (L.) Millspaugh] a versatile food legume, grows mainly in arid and semi-arid regions of the globe. The crop is characterized with distinctive growth habit. An important agronomic trait regulated by mutation in Terminal flower 1 (TFL1) locus and decides the onset of reproductive phase in crop plant. The current experiment designed to identify and characterize the pigeonpea genotypes with variation in TFL1 locus along with other traits, affecting complexity of yield through precise phenotyping, to aid in simplifying the developmental mechanism and puzzle of stagnant productivity in pigeonpea. Methods: For profiling of TFL homologue, phenotyping was done in kharif, 2019 at College Farm, N.M. College of Agriculture, Navsari Agricultural University, Gujarat. Sixty four diverse pigeonpea germplasms were screened for growth habit and yield attributing traits. The individuals where shoot apex terminated into reproductive phase was considered as determinate type (DT) and those where main stem continued vegetative growth throughout plant’s life was recorded as indeterminate type (IDT). Result: The experimental findings clearly identify the variants, arises due to mutation in TFL1 locus. Sufficient variability and flexibility has been observed in pigeonpea to breed short duration, photo-insensitive and determinate plant types with superior seed yield to improve the productivity and area of crop. Genotypes like ICPL 87, GT 100 and BP-16-61 could be incorporated in climate-smart breeding programme to attain substantial and sustainable productivity in pigeonpea.

Role of error catastrophe in transmission ability of virus.

The role played by error catastrophe is explicitly taken into account in a mathematical formulation to analyze COVID-19 data. The idea is to combine the mathematical genetics formalism of the error catastrophe of mutations in virus gene loci with the standard model of epidemics, which lacks the explicit incorporation of the effect of mutation on the spreading of viruses. We apply this formalism to the case of SARS-CoV-2 virus. We assume the universality of the error catastrophe in the process of analyzing the data. This means that some basic parameter to describe the error catastrophe is independent of which group (country or city) we deal with. Concretely, we analyze Omicron variant data from South Africa and then analyze cases from Japan using the same value of the basic parameter derived in the South Africa analysis. The excellent fit between the two sets of data, one from South Africa and the other from Japan, using the common values of genetic parameters, justifies our assumption of the universality of these parameters.

Fine mapping and characterization of RLL6 locus required for anti-silencing of a transgene and DNA demethylation in Arabidopsis thaliana.

DNA methylation patterns in plants are dynamically shaped by the antagonistic actions of DNA methylation and demethylation pathways. Although the DNA methylation pathway has been well studied, the DNA demethylation pathway, however, are not fully understood so far. To gain deeper insights into the mechanisms of DNA demethylation pathway, we conducted a genetic screening for proteins that were involved in preventing epigenetic gene silencing, and then the ones, which were also implicated in DNA demethylation pathway, were used for further studies. Eventually, a mutant with low luciferase luminescence (low LUC luminescence) was recovered, and named reduced LUC luminescence 6–1 (rll6-1). Map-based cloning revealed that rll6-1 mutation was located on chromosome 4, and there were a total of 10 candidate genes residing within such a region. Analyses of genome-wide methylation patterns of rll6-1 mutant showed that mutation of RLL6 locus led to 3,863 hyper-DMRs (DMRs for differentially methylated regions) throughout five Arabidopsis chromosomes, and elevated DNA methylation level of 2 × 35S promoter, which was similar to that found in the ros1 (repressor of silencing 1) mutant. Further analysis demonstrated that there were 1,456 common hyper-DMRs shared by rll6-1 and ros1-7 mutants, suggesting that both proteins acted together in a synergistic manner to remove DNA methylation. Further investigations demonstrated that mutation of RLL6 locus did not affect the expression of the four genes of the DNA glycosylase/lyase family. Thus, our results demonstrate that RLL6 locus-encoded protein not only participates in transcriptional anti-silencing of a transgene, but is also involved in DNA demethylation pathway.

Apricot kernels' extract and amygdalin alter bleomycin-induced Ty1 retrotransposition, mitotic gene conversion in the trp-5 locus and reverse point mutations in ilv1-92 allele in Saccharomyces cerevisiae.

The present study aims to analyze the effect of apricot kernels' extract (AKE) and amygdalin (AMY) on bleomycin-induced genetic alternations. Five endpoints were analyzed: cell survival, Ty1 retrotransposition, mitotic gene conversion in the trp-5 locus, reverse point mutations in ilv1-92 allele, and mitotic crossing-over in the ade2 locus. The present work provides the first experimental evidence that bleomycin induces Ty1 retrotransposition in Saccharomyces cerevisiae. New data is obtained that the degree of DNA protection of AMY and AKE depends on the studied genetic event. AKE has been found to provide significant protection against bleomycin-induced Ty1 retrotransposition due to better-expressed antioxidant potential. On the other side, AMY better-expressed protection against bleomycin-induced mitotic gene conversion and reverse mutations may be attributed to the activation of the repair enzymes.

Differential Gene Expression and Methylation Analysis of Melanoma in TCGA Database to Further Study the Expression Pattern of KYNU in Melanoma

Background: The aim of this study was to analyze and compare melanoma gene expression profiles in TCGA database through the application of different genes to explore the pathogenesis of melanoma. Furthermore, we confirmed the extent of the role of KYNU in melanoma and whether it can be a potential target for the diagnosis and treatment of melanoma. Methods: The gene expression profiles of melanoma samples were downloaded from TCGA database, and matrix files were synthesized to screen differential genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis and GCDA broad institute were used to analyze common gene locus mutations and expression changes in melanoma, as well as methylation. In addition, the expression patterns of KYNU in melanoma were quantified by immunohistochemistry, Western blotting, qRT-PCR, software such as GEO DataSets and the Human Protein Atlas, and meta-analysis of skin diseases. KYNU was overexpressed in keratinocytes (HaCaT and HEKα) and melanoma cells (A375 and H1205-lu). CFDA-SE, Annexin V–PI double staining, and PI single staining were used to investigate the mechanism of KYNU in melanoma and its effects on melanoma proliferation, apoptosis, invasion, and migration. Results: The main signaling pathways involved in melanoma were EGF/EGFR–RAS–BRAF–MEK–ERK–CyclinD1/CDK4, Ras–PI3K–PTEN–PKB/AKT, and p14/p16 (CDKN2A)–MDM2–p53–p21–cyclinD1/CDK4/6–Rb/E2F. Moreover, MITF, KIT, CDH1. NRAS, AKT1, EGFR, TP53, KIT, and CDK4 were elevated in melanoma, whereas PTEN, cAMP, and BCL2 were reduced in melanoma. The copy number of tumor-promoting genes increased, while the copy number of tumor suppressor genes decreased. Changes in the copy number of the above tumor genes enriched in chromosomes were found through SNP gene mutations. The genes whose expression was negatively regulated by DNA methylation in melanoma included KRT18, CDK2, JAK3, BCL2, MITF, MET, CXCL10, EGF, SOX10, SOCS3, and KIT. The mutation rate of KYNU was high according to TCGA database. The KYNU level was decreased in melanoma. Overexpression of KYNU can promote changes in apoptotic BCL-2, metabolic KYN, 3-HAA, invasion and migration MMP9, E-cadherin, and other related proteins in melanoma. Fluorescence staining and flow analysis showed that a slower proliferation rate led to a stronger fluorescence intensity. In melanoma tumor cells with a low expression of KYNU, overexpression of KYNU could promote tumor cell apoptosis. IL-10 induced immunoregulatory changes in melanoma. The expression of MMP9 and AMPK decreased in A375, but the change in BCL-2 was not obvious. The expression of BCL-2 decreased significantly in H1205-lu. A375 showed cell-cycle arrest, indicating that IL-10 could slow down the cell cycle of melanoma. Conclusions: These results provide insights into the pathologic mechanisms of melanoma target genes and KYNU as a biomarker and potential therapeutic factor for melanoma.

Environmental Hypoxia is a Driver of Sonic Hedgehog-dependent Troglomorphic Evolution in Astyanax Cavefish

Astyanax mexicanus cavefish (CF) have evolved numerous troglomorphic traits since their divergence from surface fish (SF) ancestors about 20,000 years ago. A large number of these traits are under the control of Sonic Hedgehog (Shh), a signaling morphogen that is overexpressed along the midline in CF relative to SF embryos. The Shh-dependent traits include eye degeneration and increases in the olfactory lobes, hypothalamus, jaws, and oral taste buds. Eye degeneration and tastebud enhancement are antagonistic tradeoffs governed by the pleiotropic shh gene. CF have also evolved multiple changes in circulatory system development, highlighted by larger hematopoietic domains, more erythrocytes, and reversals in normal heart asymmetry, which are also controlled by Shh signaling. Although Shh signaling is an important factor in CF troglomorphism, the shh gene is not associated with any of the many QTL identified by genetic analysis, suggesting that upstream processes, rather than mutation of the shh locus itself, may be important in Shh regulation. Due to the absence of primary productivity, cave pools harboring Astyanax CF are deficient in dissolved oxygen. Accordingly, the Hypoxia Inducing Factor (HIF) transcription factors and their downstream targets are constitutively active during CF development. To determine if oxygen deficiency is an upstream regulator of Shh signaling, we subjected early SF embryos to 8 or 16 hours of hypoxia in the laboratory and assayed the effects on shh expression, erythrocyte formation, heart asymmetry, and eye development. The results indicated that hypoxia can cause shh overexpression, increase erythropoiesis, reverse heart asymmetry, and induce eye degeneration in SF embryos. We conclude that environmental hypoxia may be responsible for the evolution of troglomorphic traits in Astyanax CF through hyperactivation of the Shh signaling pathway.