Prevalence Of Mutations Research Articles

Insecticide resistance and its intensity in urban Anopheles arabiensis in Kisumu City, Western Kenya: Implications for malaria control in urban areas.

The rise of insecticide resistance poses a growing challenge to the effectiveness of vector control tools, particularly in rural areas. However, the urban setting has received comparatively less focus despite its significance in attracting rural to urban migration. Unplanned urbanization, often overlooked, exacerbates insecticide resistance as Anopheles mosquitoes adapt to the polluted environments of rapidly expanding cities. This study aimed to assess the insecticide susceptibility status of malaria vectors and identify potential underlying mechanisms across three distinct ecological settings characterized by differing levels of urbanization in Kisumu County, Kenya. The study was conducted in 2022-2023 in Kisumu County, western Kenya. Field-derived An. gambiae (s.l.) larvae collected from a long stretch of urban-to-rural continuum were phenotyped as either resistant or susceptible to six different insecticides using the World Health Organization (WHO) susceptibility test. Polymerase chain reaction (PCR) techniques were used to identify the species of the An. gambiae complex and screened for mutations at voltage-gated sodium channels (Vgsc-1014F, Vgsc-1014S, Vgsc-1575Y) and acetylcholinesterase (Ace1) target site mutation 119S. Metabolic enzyme activities (non-specific β-esterases and monooxygenases) were evaluated in mosquitoes not exposed to insecticides using microplate assays. Additionally, during larval sampling, a retrospective questionnaire survey was conducted to determine pesticide usage by the local inhabitants. Anopheles arabiensis dominated in urban (96.2%) and peri-urban (96.8%) areas, while An. gambiae (s.s.) was abundant in rural settings (82.7%). Urban mosquito populations showed high resistance intensity to deltamethrin (Mortality rate: 85.2% at 10x) and suspected resistance to Pirimiphos-methyl and bendiocarb while peri-urban and rural populations exhibited moderate resistance intensity to deltamethrin (mortality rate >98% at 10x). Preexposure of mosquitoes to a synergist piperonyl butoxide (PBO) significantly increased mortality rates: from 40.7% to 88.5% in urban, 51.9% to 90.3% in peri-urban, and 55.4% to 87.6% in rural populations for deltamethrin, and from 41.4% to 78.8% in urban, 43.7% to 90.7% in peri-urban, and 35% to 84.2% in rural populations for permethrin. In contrast, 100% mortality to chlorfenapyr and clothianidin was observed in all the populations tested. The prevalence of L1014F mutation was notably higher in urban An. arabiensis (0.22) unlike the peri-urban (0.11) and rural (0.14) populations while the L1014S mutation was more prevalent in rural An. gambiae (0.93). Additionally, urban An. arabiensis exhibited elevated levels of mixed function oxidases (0.8/mg protein) and non-specific esterases (2.12/mg protein) compared to peri-urban (0.57/mg protein and 1.5/mg protein, respectively) and rural populations (0.6/mg protein and 1.8/mg protein, respectively). Pyrethroids, apart from their use in public health through LLINs, were being highly used for agricultural purposes across all ecological settings (urban 38%, peri-urban 36% and rural 37%) followed by amidine group, with organophosphates, neonicotinoids and carbamates being of secondary importance. These findings show high resistance of An. arabiensis to insecticides commonly used for vector control, linked with increased levels of detoxification enzymes. The observed intensity of resistance underscores the pressing issue of insecticide resistance in urban areas, potentially compromising the effectiveness of vector control measures, especially pyrethroid-treated LLINs. Given the species' unique behavior and ecology compared to An. gambiae, tailored vector control strategies are needed to address this concern in urban settings.

Inactivation of Pseudovirus Expressing the D614G Spike Protein Mutation using Nitric Oxide-Plasma Activated Water.

Variants of concern (VOCs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) exhibit high infectivity due to mutations, particularly in the spike protein, that facilitate enhanced binding of virus to human angiotensin-converting enzyme 2 (hACE2). The D614G mutation, situated in S1-domain, promotes the open conformation of spike protein, augmenting its interaction with hACE2. Activated water neutralizes pathogens by damaging biological molecules; however, its effect on mutated SARS-CoV-2 or VOCs requires further exploration. Here, the efficacy of nitric oxide (NOx)-plasma activated water (PAW) in inhibiting infections by SARS-CoV-2 pseudovirus expressing D614G-mutated spike protein is investigated, which serves as a model for mutated SARS-CoV-2. Results demonstrated high prevalence of D614G mutation in SARS-CoV-2 and its VOCs. NOx-PAW is non-toxic to cells at high concentration, inhibiting infection by 71%. Moreover, NOx-PAW induced structural changes in S1-domain of spike protein, reducing its binding affinity and lowering clathrin-mediated endocytosis-related gene expression. Additionally, in silico analysis revealed NOx species in NOx-PAW played key role in impairing S1-domain function of the mutated SARS-CoV-2 pseudovirus by interacting directly with it. Collectively, these findings reveal the potent inactivation ability of PAW against mutated SARS-CoV-2 and suggest its potential application in combating emerging variants of SARS-CoV-2 and other viral threats.

Noonan Syndrome: Relation of Genotype to Cardiovascular Phenotype—A Multi-Center Retrospective Study

Background: Noonan syndrome (NS) is a congenital genetic disorder with a prevalence of 1 in 1000 to 2500 live births, and is characterized by distinctive facial features, short stature, chest deformities, and congenital heart disease. This study aims to evaluate the prevalence of specific genetic mutations and their impact on cardiovascular and other outcomes in NS. Methods: We conducted a retrospective clinical study of 25 pediatric patients diagnosed with NS at two institutions: The Mother and Child Health Care Institute of Serbia and the Clinic for Children Diseases, University Clinical Center of the Republic of Srpska. Patients underwent whole-exome sequencing (WES) to identify genetic mutations. Clinical data, including cardiovascular manifestations, psychomotor development, and stature, were analyzed in relation to mutation types. Results: The cohort comprised 60% male and 40% female patients, with a median age at diagnosis of 7.2 years. Cardiovascular abnormalities were present in 88% of patients. Mutations in PTPN11 were most commonly associated with pulmonary valve stenosis (PVS), while RAF1 mutations were prevalent in patients with hypertrophic cardiomyopathy (HCM). No significant association was found between cardiac disease and delayed psychomotor development (p = 0.755), even though the likelihood ratio showed significance in that regard (p = 0.018). Short stature was observed in 48% of patients but was not significantly correlated with genetic type of disease, presence of cardiac disease, or developmental delay. Conclusions: The study confirms the high prevalence of cardiovascular manifestations in NS and highlights genotype–phenotype correlations. While cardiac abnormalities are common, their impact on psychomotor development and stature is less clear. Further research is needed to explore genetic interactions influencing these outcomes and refine clinical management strategies.

Prevalence of BRCA1 and BRCA2 mutations in ovarian cancer patients from Yunnan Province in southwest China.

Carriers with germline breast cancer 1/2 gene mutations (BRCAm) are likely to develop ovarian cancer (OC). Therefore, identifying these mutations may enable individualized therapy for OC and preventive measures to reduce OC risk in BRCAm carrier families. Thus, we investigated the prevalence of BRCAm in OC patients from Yunnan Province in Southwest China. In total, 674 unselected OC patients were enrolled and tested for BRCAm via next-generation sequencing. Data on clinicopathological characteristics and personal/family history of cancer were collected. The prevalence rates of pathogenic/likely pathogenic BRCAm were 26.6% overall, 20.8% among BRCA1m carriers, 5.5% among BRCA2m carriers, and 0.3% among carriers of both BRCA1m and BRCA2m. The most common pathogenic mutation in the BRCA1 gene was c.5114T>C (n = 9). The number of BRCAm carriers was significantly greater among patients with serous cancer, a personal tumor history, a family history of hereditary breast and ovarian cancer (HBOC)-related tumors, and bilateral tumors. The most common pathogenic mutation in this cohort was c.5114T>C (n = 9) in BRCA1. The prevalence and spectrum of BRCAm in OC patients from Yunnan Province are different from those in other groups. BRCA status testing is advised for all OC patients, particularly those with a family history of HBOC.

Abstract 4138731: Clonal Hematopoiesis of Indeterminant Potential is Associated with Pulmonary Arterial Hypertension

Background: Pulmonary Arterial Hypertension (PAH) is a fatal cardiopulmonary disorder characterized by adverse vascular remodelling of small pulmonary arteries, which increases resistance and demand on the right ventricle (RV), ultimately leading to RV fibrosis and failure. Emerging evidence suggests that inflammation plays a crucial role in PAH pathogenesis. Here, we study Clonal Hematopoiesis of Indeterminate Potential (CHIP), a disorder characterized by somatic mutations in genes such as DNMT3A and TET2 , that promotes an inflammatory phenotype, in the development of PAH. Methods: We performed deep, targeted panel sequencing on 1659 PAH patients from the PAH Biobank and 3644 controls (3401 Vanderbilt University Biorepository patients and 243 participants recruited by the Queen’s Genomics Centre at Ongwanada). Variants in known CHIP genes expressed at a variant allele frequency (VAF) > 2% were classified as CHIP. Genes with a VAF > 25% and <50% were considered CHIP if located in reported CHIP hotspot regions. The prevalence of CHIP mutations was compared between controls and PAH patients. Logistic regression was used for statistical analysis. Results: We identified 242 CHIP variants in the 1659 PAH patients. DNMT3A was the most mutated gene (116/242 = 48%), followed by TET2 (46/242=19%). After adjusting for age and sex, we identified a significant association between all CHIP variants combined and PAH (OR: 23.35 [7.67, 71.03]; p<0.0001). Further, DNMT3A CHIP (OR: 25.44 [5.37, 120.40]; p<0.0001), non- DNMT3A CHIP (OR: 26.80 [5.56, 129.23]; p<0.0001) and TET2 CHIP (OR: 13.69 [1.29, 145.30]; p=0.03) were all associated with PAH. A higher proportion of PAH patients under the age of 70 were found to have CHIP variants compared to controls and this was significant for individuals between 60-69 years of age (p=0.037). While there was a 3.8:1 female-to-male ratio of PAH patients, there was a 5.8:1 female-to-male preponderance of DNMT3A variants (p=0.039) (4.3:1 for all CHIP; p=ns), suggesting DNMT3A variants are more common in female patients. The presence of CHIP variants had no significant effect on PAH patient 10-year survival. Conclusion: This research reveals a significant association between CHIP variants, specifically in DNMT3A and TET2, and PAH. The prevalence of CHIP is higher in PAH patients aged 40 to 70 compared to controls and is more common in female patients. These findings suggest that CHIP is a significant risk factor for the development of PAH.

PATH-61. GENOMIC SIGNATURE IN NON-SMALL CELL LUNG CANCER PATIENTS METASTATIC TO BRAIN-A SINGLE INSTITUTION EXPERIENCE

Abstract BACKGROUND Lung cancer is leading cause of cancer-related death in US, with 10-30 % of NSCLC patients developing brain metastasis, which challenges curative treatment and life expectancy. Somatic mutations in KRAS and EGFR genes have been commonly implicated with brain metastasis. Other studies have shown higher amplification frequencies in MYC, YAP1, and MMP13 and deletions in CDKN2A/B. METHODS We conducted a retrospective study on observational cohort of NSCLC patients from January 2022 to July 2023, using IRB-approved data in patients with confirmed brain metastasis (CBS). Beyond demographic information, data on genomic signatures was obtained from maximum parallel/next-generation sequencing on tumor biopsy samples. Comparative analysis was conducted to predict commonalities in brain metastasis gene signatures. RESULTS The study involved 143 newly diagnosed lung cancer patients, with 29 having CBS. Genomic data was available in 24 patients. Most common abnormality was noted in TP53 gene in about 47% of patients, followed by LRP1B (12%). Mutations in KRAS, EGFR, NF1, MET, SMARCA4, KEAP1, and STK11 genes were seen in two patients each. Interestingly, 3 of 4 patients with LRP1B had brain metastasis. Actionable mutations/rearrangements were significantly lower compared to general prevalence of these mutations (KRAS gene 8% and EGFR gene 5%). CONCLUSIONS Lung cancer with brain metastasis has an abysmal prognosis due to poor CNS penetration of chemotherapeutic agents. Surgical resection and RT remain cornerstones of treatment, but certain TKI has shown some potential. Our study uncovered a notably higher prevalence of TP53 mutation among patients with brain metastasis. While patient cohort was small, those harboring an LRP1B mutation exhibited significantly higher incidence of brain metastasis. Contrary to prevalent epidemiological data, our patient population demonstrated a lower frequency of targetable mutations (specifically in EGFR and KRAS genes). We hypothesize that this observation is due to a markedly higher smoking prevalence in our region.

BIOM-07. SURVIVAL-ASSOCIATED GENETIC VARIATIONS IN TAIWANESE GLIOBLASTOMA MULTIFORME PATIENTS - A RETROSPECTIVE NGS STUDY

Abstract This retrospective study, conducted at Taichung Veterans General Hospital in Taiwan, employed Next-Generation Sequencing (NGS) technology to analyze genetic variations associated with survival in patients with glioblastoma multiforme (GBM). The study encompassed 30 newly diagnosed GBM patients, divided into two groups based on survival duration: long-term (over two years) and short-term (under two years). The findings are outlined below: Key Mutations: Significant mutations included CHEK2, IDH1, and TP53, along with variations in the TERT promoter and TP53 RNA splicing sites. Correlation Between Mutations and Survival: Contrary to Western data, TP53 mutations were more prevalent in the long-term survival group, suggesting that regional genetic differences may influence GBM prognosis. Therapeutic Effectiveness: The application of bevacizumab was significantly associated with improved survival rates, indicating its potential efficacy in treatment. The study highlights the critical role of personalized medicine in the treatment of GBM in Taiwan and points to the necessity of further research into the genetic characteristics and treatment responses of GBM patients in Taiwan and potentially other non-Western regions. These insights underline the importance of continuing to explore effective personalized treatment strategies and enhancing our understanding of genetic determinants in diverse populations. Moreover, the prevalence of certain mutations in Taiwanese patients, which differ from those commonly observed in Western populations, prompts a reevaluation of global treatment frameworks and suggests the potential for region-specific therapeutic protocols. The implication of these findings extends beyond immediate clinical applications, providing a foundation for future clinical trials and research into genetic markers that could dictate treatment choices. In conclusion, this study not only underscores the importance of individualized treatment plans based on genetic profiling but also highlights the variations in genetic mutations across different populations, which could have profound implications for the management of GBM worldwide. Further investigation into these disparities and their implications on treatment efficacy is essential for advancing GBM treatment and improving patient outcomes globally.

Mutations in human cytymegalovirus (Orthoherpesviridae: Herpesvirales: Cytomegalovirus: Cytomegalovirus humanbeta 5) UL97 gene lead to increase in viremia duration and poor antiviral response in recipients of allogeneic hematopoietic stem cells.

Human cytomegalovirus (Orthoherpesviridae: Herpesvirales: Cytomegalovirus: Cytomegalovirus humanbeta 5) (HCMV) is one of the most commonly detected viruses in recipients of allogeneic hematopoietic stem cell (allo-HSCT) transplants. However, the emergence of resistance to antiviral drugs such as ganciclovir (GCV) poses a challenge in managing these patients. This study aims to investigate the prevalence and impact of mutations in the HCMV UL97 gene associated with resistance to GCV on the course of infection among allo-HSCT patients. The study examined the association between UL97 mutations and the clinical course of HCMV infection in allo-HSCT patients. Genetic sequencing was performed to identify mutations, and their impact on viral replication and resistance to GCV was assessed. Six mutations were identified (D490A, T502A, C592G, C592F, E596G, C603W). C592G, C592F, E596G, and C603W are associated with resistance to antiviral drugs, while D490A and T502A described for the first time. When comparing patients with wild-type and those carrying the mutant variant, several parameters of peripheral blood were significantly lower in the former group. The median time to peak viral load following allo-HSCT, duration of viremia, and rate of virological response to high-dose therapy also differed significantly between the two groups. It was shown that approximately one third (4 out of 14) of allogeneic stem cell transplant recipients had mutations associated with resistance to GCV. Patients carrying the mutant variant of HCMV had longer viremia and took longer to achieve a negative virological test result after starting high-dose therapy. Performing genotyping may help make more evidence-based therapeutic decisions.

Redefining pancreatic cancer management with tumor-agnostic precision medicine.

Precision oncology and tumor-agnostic drug development provide hope for enhancing outcomes among patients with pancreatic cancer. Tumor-agnostic therapies have emerged across various tumor types, driven by insights into shared biomarkers. In the case of pancreatic cancer, the prevalence of the KRAS gene mutation is noteworthy. However, there exist other actionable alterations, such as BRCA1/2 mutations and fusion genes (BRAF, FGFR2, RET, NTRK, NRG1, and ALK), which present potential targets for therapy. Notably, tumor-agnostic drugs have demonstrated efficacy in specific subsets of pancreatic cancer patients who harbor these genetic alterations. Despite the rarity of NTRK fusions in pancreatic cancer, larotrectinib and entrectinib have exhibited effectiveness in NTRK fusion-positive pancreatic cancers. Additionally, repotrectinib, a next-generation NTRK inhibitor, has shown promising activity in NTRK positive pancreatic cancer patients who have developed acquired resistance to previous NTRK inhibitors. Immune checkpoint inhibitors, such as pembrolizumab and dostarlimab, have proven to be effective in dMMR/MSI-H pancreatic cancers. Moreover, targeted therapies for BRAF V600, RET fusions, and HER2/neu overexpression have displayed promising results in specific subsets of pancreatic cancer patients. Emerging targets like NRG fusions, FGFR2 fusions, TP53 mutations, and KRAS G12C mutations present potential avenues for targeted therapy. Tumor-agnostic therapies have the potential to revolutionize pancreatic cancer treatment by focusing on specific genetic alterations. It is crucial to continue implementing comprehensive screening strategies that encompass the ability to detect all these tumor-agnostic biomarkers. This will be essential in identifying pancreatic cancer patients who may benefit from these therapies.

Temporal genomics in Southern Zambia shows rising prevalence of Plasmodium falciparum mutations linked to delayed clearance after artemisinin-lumefantrine treatment

The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013 and 2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample from 2016 carrying K13 622I, no other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations were observed. However, in the more recent years (2016–2017) five novel K13-propeller-domain mutations, C532S, A578S, Q613E, D680N and G718S were identified at low prevalence. Moreover, 13% (CI, 9.6–17.2) of isolates had the AP2MU 160N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015 and 2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 NFD haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia.

Emerging threat of artemisinin partial resistance markers (pfk13 mutations) in Plasmodium falciparum parasite populations in multiple geographical locations in high transmission regions of Uganda

BackgroundArtemisinin-based combination therapy (ACT) is currently recommended for treatment of uncomplicated malaria. However, the emergence and spread of partial artemisinin resistance threatens their effectiveness for malaria treatment in sub-Saharan Africa where the burden of malaria is highest. Early detection and reporting of validated molecular markers (pfk13 mutations) in Plasmodium falciparum is useful for tracking the emergence and spread of partial artemisinin resistance to inform containment efforts.MethodsGenomic surveillance was conducted at 50 surveillance sites across four regions of Uganda in Karamoja, Lango, Acholi and West Nile from June 2021 to August 2023. Symptomatic malaria suspected patients were recruited and screened for presence of parasites. In addition, dried blood spots (DBS) were collected for parasite genomic analysis with PCR and sequencing. Out of 563 available dried blood spots (DBS), a random subset of 240 P. falciparum mono-infections, confirmed by a multiplex PCR were selected and used for detecting the pfk13 mutations by Sanger sequencing using Big Dye Terminator method. Regional variations in the proportions of pfk13 mutations were assessed using the chi square or Fisher’s exact tests while Kruskal–Wallis test was used to compare absolute parasite DNA levels between wild type and mutant parasites.ResultsOverall, 238/240 samples (99.2%) contained sufficient DNA and were successfully sequenced. Three mutations were identified within the sequenced samples; pfk13 C469Y in 32/238 (13.5%) samples, pfk13 A675V in 14/238 (5.9%) and pfk13 S522C in (1/238 (0.42%) samples across the four surveyed regions. The prevalence of pfk13 C469Y mutation was significantly higher in Karamoja region (23.3%) compared to other regions, P = 0.007. The majority of parasite isolates circulating in West Nile are of wild type (98.3), P = 0.002. Relative parasite DNA quantity did not differ in samples carrying the wild type, C469Y and A675V alleles (Kruskal–Wallis test, P = 0.6373).ConclusionDetection of validated molecular markers of artemisinin partial resistance in multiple geographical locations in this setting provides additional evidence of emerging threat of artemisinin partial resistance in Uganda. In view of these findings, periodic genomic surveillance is recommended to detect and monitor levels of pfk13 mutations in other regions in parallel with TES to assess potential implication on delayed parasite clearance and associated treatment failure in this setting. Future studies should consider identification of potential drivers of artemisinin partial resistance in the different malaria transmission settings in Uganda.

An Ala/Glu difference in E1 of Cx26 and Cx30 contributes to their differential anionic permeabilities.

Two closely related connexins, Cx26 and Cx30, share widespread expression in the cochlear cellular networks. Gap junction channels formed by these connexins have been shown to have different permeability profiles, with Cx30 showing a strongly reduced preference for anionic tracers. The pore-forming segment of the first extracellular loop, E1, identified by computational studies of the Cx26 crystal structure to form a parahelix and a narrowed region of the pore, differs at a single residue at position 49. Cx26 contains an Ala and Cx30, a charged Glu at this position, and cysteine scanning in hemichannels identified this position to be pore-lining. To assess whether the Ala/Glu difference affects permeability, we modeled and quantified Lucifer Yellow transfer between HeLa cell pairs expressing WT Cx26 and Cx30 and variants that reciprocally substituted Glu and Ala at position 49. Cx26(A49E) and Cx30(E49A) substitutions essentially reversed the Lucifer Yellow permeability profile when accounting for junctional conductance. Moreover, by using a calcein efflux assay in single cells, we observed a similar reduced anionic preference in undocked Cx30 hemichannels and a reversal with reciprocal Ala/Glu substitutions. Thus, our data indicate that Cx26 and Cx30 gap junction channels and undocked hemichannels retain similar permeability characteristics and that a single residue difference in their E1 domains can largely account for their differential permeabilities to anionic tracers. The higher anionic permeability of Cx26 compared with Cx30 suggests that these connexins may serve distinct signaling functions in the cochlea, perhaps reflected in the vastly higher prevalence of Cx26 mutations in human deafness.

High-Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis.

Candida parapsilosis is a pathogenic yeast that has reduced susceptibility to echinocandins and ranks as the second or third leading cause of candidaemia, depending on the geographical region. This yeast often causes nosocomial infections, which are frequently detected as outbreaks. In recent years, resistance to azoles in C. parapsilosis has increased globally, primarily due to the accumulation of mutations in the ERG11 gene. In this study, we have developed an assay based on real-time PCR and high-resolution melting (HRM) curve analysis to detect two of the most prevalent mutations at ERG11 that confer resistance to fluconazole (Y132F and G458S). We designed allele-specific oligonucleotides that selectively bind to either the wild type or mutated sequences and optimised the conditions to ensure amplification of the specific allele, followed by detection via high-resolution melting (HRM) analysis. The designed oligonucleotides to detect the Erg11Y132F and Erg11G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real-time PCR combining oligonucleotides for the wild-type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach. This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis.

A Gene Expression Signature that Predicts Gastric Cancer Sensitivity to PARP Inhibitor Therapy.

Biomarkers indicating sensitivity to poly ADP-ribose polymerase (PARP) inhibitors have not yet been identified in gastric cancer. PARP inhibitors target homologous recombination deficiency (HRD); however, homologous recombination (HR) induces complex changes in gene expression, which makes it difficult to identify reliable biomarkers. In this study, we identified a multi-gene expression signature as a marker of PARP inhibitor sensitivity in gastric cancer. Seven gastric cancer cell lines were evaluated for susceptibility to PARP inhibitors using a growth inhibition assay. Gene expression profiling (GEP) was used to identify differentially expressed genes between PARP inhibitor-sensitive and -resistant cell lines. The resulting gene set was subjected to cluster analysis using tumor samples from 250 patients who underwent gastrectomy for primary gastroesophageal junction and gastric adenocarcinoma. HRD was defined as a truncating mutation in one or more of 22 HR-related genes and HRD scores were calculated using whole-exome sequencing data. In the growth inhibition assays, the HGC27 and HSC39 cell lines were sensitive to the PARP inhibitors, olaparib, and rucaparib, and were significantly correlated with the GEP results. Seven (2.8%) patients harbored truncating mutations in HR-related genes. A gene expression signature based on the top 100 high and low differentially expressed genes between sensitive and resistant cell lines revealed a patient cluster with a high prevalence of HR-related gene mutations and high HRD scores. The 100-gene expression signature identified in this study may serve as a valuable predictive biomarker for PARP inhibitor sensitivity in gastric cancer.

P56 Azathioprine-induced alopecia totalis and knuckle hyperpigmentation in a child with uveitis: test before you leap!

Abstract Introduction Azathioprine is a common disease-modifying anti-rheumatic drug used in non-infectious uveitis. Although unusual, it has been associated with myelosuppression in genetically predisposed individuals who exhibit mutations in thiopurine S-methyltransferase (TPMT) and nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) genes. Alopecia secondary to azathioprine treatment is rare, and is an early marker of azathioprine-induced myelosuppression. It usually precedes the onset of myelosuppression, but can also occur simultaneously with pancytopenia. Knuckle pad hyperpigmentation is strongly associated with megaloblastic anemia. We report a patient of panuveitis with both alopecia totalis and knuckle pad hyperpigmentation secondary to azathioprine-induced myelosuppression. Case description A 13-year-old girl presented with acute-onset pain, redness and blurred of both eyes. Ophthalmological evaluation revealed bilateral granulomatous panuveitis. Visual acuity was 6/12 in both eyes, and anterior chamber had 2+ cells. Extensive evaluation for infective and autoimmune causes did not yield any aetiology. Following laboratory tests, a diagnosis of non-infectious panuveitis was made, and oral corticosteroids (2 mg/kg followed by taper) were started. As steroid sparing agents, subcutaneous methotrexate (15 mg/m2/week) and azathioprine (2 mg/kg) were also started after a normal baseline hemogram [Hb: 132 g/L, total leucocyte count (TLC) 12 x 109/L and platelet count: 310 x 109/L]. Two weeks later, she presented with complete loss of hair (alopecia totalis) and knuckle pad hyperpigmentation. Hemogram revealed pancytopenia (Hb: 86 g/L, TLC: 1.7 x 109/L and platelet count: 25 x 109/L). Considering drug-induced myelosuppression, azathioprine was stopped. Subcutaneous injections of Granulocyte colony-stimulating factor (G-CSF) (300 day) were given for two weeks. Mycophenolate mofetil (1200 mg/m2/day) was added. Quantitative polymerase-chain reaction genotyping assay showed a homozygous variant in NUDT15 gene (c.415 C>T). Normalization of hemogram was seen after two weeks (Hb: 120 g/L, TLC: 10.44 x 109/L and platelet count: 199 x 109/L). Reversal of hair loss and knuckle hyperpigmentation was nearly complete at five months and six weeks of follow-up, respectively. Panuveitis remained resolved on methotrexate and mycophenolate, with 6/6 vision and no recurrence at 16-months of follow up. Discussion In India, the prevalence of TPMT mutations (heterozygous 1.2-10%, and homozygous <1%), and NUDT15 mutations (heterozygous 11.6%, and homozygous 1.4%) is low, and the tests are expensive. In routine clinical practice, monitoring of blood counts is recommended in patients on azathioprine (two weekly initially, and then four weekly). Both TPMT and NUDT15 enzymes influence metabolism of azathioprine. Their polymorphisms result in accumulation of active 6-mercaptopurine, which gets converted to 6-thioguanine nucleotide metabolites, producing cytotoxic effects. As a result of disruption in mitotic activity of actively growing hair (anagen), more catagen follicles are seen than anagen, leading to hair loss. While massive hair loss is well recognized secondary to azathioprine, there is no report of knuckle pad hyperpigmentation as a direct adverse reaction of azathioprine. Both knuckle pad hyperpigmentation and azathioprine toxicity are associated with megaloblastic anemia. Knuckle pad hyperpigmentation may be explained by vitamin B12deficiency, with decreased intracellular glutathione. As a result, melanin deposition increases due to loss of tyrosinase inhibition. In addition, melanocyte pigmentary incontinence may also contribute to knuckle pad hyperpigmentation in such cases. Key learning points • While alopecia is often massive, knuckle pad hyperpigmentation may often be overlooked as a clinical sign in patients with azathioprine-induced megaloblastic anemia. Our case highlights the significance of two simple clinical cutaneous signs (alopecia totalis and knuckle pad hyperpigmentation) as clinical indicators of impending, and potentially fatal complication of pancytopenia secondary to azathioprine. In developed countries, genetic analysis is often performed for identifying variants in TPMT and NUDT15 genes before initiation of azathioprine. However, in resource-constrained settings, regular and close monitoring of hemogram, and prompt identification of clinical clues such as alopecia and knuckle pad hyperpigmentation may be a practical method to anticipate azathioprine-induced toxicity including myelosuppression.

High Prevalence of MYD88 and CD79B Mutations in Primary Sinonasal Diffuse Large B-Cell Lymphoma: Identification of an MCD-like Subtype.

Primary sinonasal diffuse large B-cell lymphoma (PSDLBCL) is a rare aggressive lymphoma. Recently, genetic classification using Next Generation Sequencing (NGS) demonstrated that PSDLBCL largely consists of the MCD genotype, which has a poor prognosis mainly driven by MYD88 L265P and CD79B gene abnormalities. This study investigated the prevalence and clinicopathological significance of MYD88 L265P and CD79B Y196 mutations using droplet digital PCR in 55 patients with PSDLBCL, as well as the translocation of BCL2/BCL6/c-Myc with FISH. We found mutations in MYD88 L265P (29/55, 52.7%) and CD79B Y196 (20/55, 36.4%). The MCD-like subtype, defined by the mutation of MYD88 and/or CD79B, was found in 32 out of 55 cases (58.2%). This subtype largely consists of non-GCB type (31/32, 96.9%; P<0.01) and double-expressor cases (20/32, 62.5%; P=0.01) compared with the MYD88/CD79B co-wild type, with BCL6 translocation in a small subset (2/32, 6.3%) and no translocations of BCL2 (0/32) or c-Myc (0/32). The MCD-like subtype tended to relapse in specific sites such as the central nervous system, testis, and/or skin compared with the co-wild type (P=0.03), showing poorer outcomes in overall survival (P=0.02) and progression-free survival (P=0.01). In conclusion, our study highlights a high prevalence of MYD88 and CD79B mutations in PSDLBCL, identifying an aggressive MCD-like subtype with a distinct relapse pattern. This molecular subclassification can be helpful for both prognostic prediction and therapeutic strategy in patients with PSDLBCL.

Evolutionary Dynamics and Pathogenicity Analysis of Feline Panleukopenia Virus in Xinjiang, China

Feline panleukopenia virus (FPV), a globally pervasive and highly pathogenic pathogen, has garnered significant attention recently due to the cross-species transmission of its variants. Despite the vast body of research conducted on FPV, studies exploring its evolutionary history, dynamics, and the factors driving its evolution remain scarce. The pathogenicity of strains with the prevalent mutations (A91S and I101T) in the VP2 protein has also not been fully elucidated. This study conducted a comparative analysis of FPV VP2 sequences sourced from Xinjiang province in China, other provinces in China, and other countries. It was confirmed that the evolutionary rate of FPV approached that of RNA viruses, at approximately 1.13 × 10−4 substitutions/site/year. The study reconstructed molecular models of the VP2 protein with the A91S and I101T mutations and used viral strains carrying these mutations to perform the animal regression experiment. It was confirmed that isolates with the A91S and I101T mutations could cause typical leukopenia and acute enteritis symptoms, suggesting that the mutant strains still possess certain pathogenicity. This is the first study to report on the evolutionary dynamics of FPV in Xinjiang, China, and it emphasized the importance of continuously monitoring FPV evolutionary dynamics.

Evaluation of the relationship between thrombophilia gene mutations and demographic data, imaging findings and stroke subtypes in young patients followed up with a diagnosis of stroke

Aims: A stroke is defined as a focal or generalized neurological deficit that develops suddenly, lasts for more than 24 hours, or ends in death within this period, with a cause other than a vascular cause. Stroke is among the leading causes of morbidity and mortality in the general population. In our study, we examined the prevalence of all prothrombotic gene mutations observed in patients with and without predisposing risk factors for young patients with stroke and discussed their roles in stroke formation in the context of existing literature. Methods: In the present study, we conducted a retrospective review of the medical records of 300 patients aged 18 to 49 years who were hospitalized in the neurology clinic of our hospital between June 2023 and June 2024 and diagnosed with acute arterial ischemic stroke based on anamnesis, neurological examination, and radiological imaging (CT and MRI) results. The study cohort comprised 47 patients (23 males, 24 females) aged between 18 and 49 years with a diagnosis of ischemic stroke. In our study, we examined ischemic stroke-related gene polymorphisms in patients with early-onset ischemic stroke (before the age of 50 years). This study aims to investigate the prevalence of Factor V-Leiden, prothrombin-G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C, SERPIN 1, and Factor II polymorphisms in young patients with ischemic stroke. Results: A total of 47 patients, comprising 23 males and 24 females, were included in the study. The mean age of the patients was 40.34 ± 6.37 years. According to the TOAST classification system, 17 patients (36.2%) were diagnosed with large artery atherothrombosis, one patient (2.1%) had a cardioembolic infarction, and 29 patients (61.7%) had a small artery occlusion. In our study, when evaluating the thrombophilia mutation subtypes in young ischemic stroke patients, 23.4% (n=11) of the patients were found to be heterozygous for the FVL mutation, while 76.6% (n=36) had no mutation. Concerning the SERPIN 1 mutation, 40.4% (n=19) of the patients were heterozygous, 23.4% (n=11) were homozygous, and 36.2% (n=17) exhibited no mutation. The distribution of patients according to MTHFR C677T mutation revealed that 57.4% (n=27) were heterozygous, 19.1% (n=9) were homozygous, and 23.4% (n=11) had no mutation. Considering the distribution of patients according to the MTHFR A1298C gene mutation, a total of 38.3% (n=18) of the patients were heterozygous, 14.9% (n=7) were homozygous, and 46.9% (n=22) had no mutation. Upon analysis of the distribution of patients according to factor XIII mutation, it was identified that 19.1% (n=9) were heterozygous, 2.1% (n=1) were homozygous, and 78.7% (n=37) of the patients exhibited no mutation. Upon analysis of the distribution of patients according to prothrombin (Factor II) mutation, it was identified that 8.5% (n=4) were heterozygous, while 91.5% (n=43) exhibited no mutation. Conclusion: Although stroke is less prevalent in young adults, the underlying etiology is highly varied. Further research, including the investigation of genetic and prothrombotic mutations, is vital for the prevention of recurrent strokes in young adults.

Novel rapid molecular diagnosis methods for comprehensive genetic analysis of 21-hydroxylase deficiency

BackgroundMolecular analysis of the CYP21A2 gene is highly important for understanding the aetiology of 21-hydroxylase deficiency (21-OHD). The aim of this study was to use a novel approach named CNVplex, together with the SNaPshot assay and direct sequencing, to identify CYP21A2 mutations efficiently and comprehensively. Targeted CYP21A2 mutation analysis was performed in 113 patients and 226 parents. Large rearrangements of CYP21A2 were characterized by CNVplex; twenty prevalent mutations, including nine common micro-conversions and eleven high-frequency mutations reported in the literature, were detected by SNaPshot; and rare mutations were investigated by direct sequencing.ResultsAmong the 113 21-OHD patients, 95.6% of the affected alleles were detected accurately by SNaPshot and CNVplex. Prevalent mutations were detected in 69.5% of the alleles; 62.4% of alleles contained pseudogene-derived micro-conversions, 1.8% contained nonpseudogene-derived mutations, and 5.3% contained complex variations resulting from multiple recombinations between CYP21A2 and CYP21A1P. Large rearrangements were identified in 27.0% of the alleles, including five types (CH-1, CH-3, CH-4, CH-5 and CH-8) of chimeric CYP21A1P/CYP21A2 genes. Two novel CYP21A2 haplotypes and four de novo CYP21A2 mutations were characterized. A rare haplotype with a c.955 C > T mutation in the duplicated CYP21A2 gene was found in 0.9% of the probands and 33.3% of the parents. In addition, four parents were also diagnosed with 21-OHD.ConclusionCNVplex and SNaPshot appear to be highly efficient and reliable techniques for use in a molecular diagnosis laboratory, and combined with direct sequencing based on locus-specific PCR, they might constitute a definitive way to detect almost all common and rare 21-OHD-related alleles.

Somatic DICER1-Mutant Benign Thyroid Nodules in Adults: A Group of Follicular Nodular Disease With Continuous Growth.

Germline DICER1 mutations cause familial multinodular goiter (MNG). However, the prevalence of somatic DICER1 mutations in non-MNG benign thyroid nodules and their characteristics remain unknown. Adult-onset thyroid nodules with a pathological diagnosis were genotyped via targeted sequencing. DICER1-mutant nodules were assessed clinically and pathologically. Organoids were established to investigate follicular formation and growth. Transcriptomic analysis was conducted to evaluate transcriptional features, which were validated by immunofluorescence. Among 931 adult-onset thyroid nodules, we identified 13 benign thyroid nodules with DICER1 hotspot mutations. The majority harbored a somatic DICER1 hotspot mutation with a somatic DICER1 truncating variant. Clinically, 38.5% of the DICER1-mutant nodules exhibited substantial growth. DICER1-mutant nodules with durations longer than 2 years were substantially enlarged (P=.0448). Pathologically, all DICER1-mutant nodules were defined as thyroid follicular nodular disease (TFND). The TFND nodules with DICER1 mutations grew faster than those with wild-type DICER1. Organoid culture of a DICER1-mutant nodule revealed increased active follicular formation. Compared with the normal thyroid tissues, the DICER1-mutant nodules had comparable thyroid differentiation scores, significantly higher ERK scores (P=.0141) and lower epithelial‒mesenchymal transition scores (P=.0001). Moreover, the expression of genes related to follicular polarity, such as CDH16, SLC5A5, TSHR and TPO, was downregulated in the DICER1-mutant nodules. Somatic DICER1 two-hit mutations represent a notable percentage in adult TFND patients, and DICER1-mutant benign thyroid nodules were characterized by continuous growth.