Mutations In Gene Research Articles

Biopsy-free diagnosis of primary diffuse large B-cell lymphoma of the central nervous system

Introduction. The use of invasive methods for diagnosing primary diffuse large B-cell lymphoma of the central nervous system (PDLBCL CNS) and primary vitreoretinal lymphoma (PVRL) is often associated with the development of severe neurological deficits and disability of patients. A promising direction in the diagnosis of PDLBCL CNS and PVRL is the use of non-invasive approaches based on molecular genetic methods for the determination of mutations in the MYD88 gene in the cerebrospinal fluid (CSF), vitreous fluid (VF) and free circulating tumor DNA (cfDNA) in blood serum.Aim: to present the potential of non-invasive diagnosis of PDLBCL CNS and PVRL.Main findings. The study included 6 patients (4 — PDLBCL CNS, 2 — PVRL). The average age was 64 (54–75) years. Despite increased cytosis in the CSF in all 4 patients with PDLBCL CNS, the tumor population was determined by flow cytometry in only half of the cases. According to a molecular genetic study, mutations in the MYD88 gene with an allelic load from 2.5% to 15% were detected in the genomic DNA of the CSF of all patients. In 2 patients with PVRL, mutations in the MYD88 gene were detected in VF with an allelic load of 4.2% and 6.6%, which was the only confirmation of the diagnosis. Currently, 5 patients have completed the treatment program and 1 is undergoing therapy. All 5 patients are in complete remission (CR) of the disease with follow-up periods from 1 to 8 months.

Inborn Error of WAS Presenting with SARS-CoV-2-Related Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) has been reported in patients with inborn errors of immunity (IEI), providing insights into disease pathogenesis. Here, we present the first case of MIS-C in a child affected by Wiskott-Aldrich syndrome (WAS) gene mutation, elucidating underlying predisposing factors and the involved inflammatory pathways. Genetic analysis revealed a frameshift truncating variant in the WAS gene, resulting in WAS protein expression between mild and severe forms, despite a clinical phenotype resembling X-linked thrombocytopenia (XLT). IL-1β secretion by LPS-stimulated peripheral blood mononuclear cells from patient during MIS-C was lower compared to healthy subjects but increased during follow-up. Conversely, the percentage of ASC (apoptosis-associated speck-like protein containing a CARD) specks in the patient's circulating monocytes during the acute phase was higher than in healthy subjects. The type I interferon (IFN) signature during MIS-C was normal, in contrast to the raised IFN signature measured far from the acute event. This case supports the association of IEI with MIS-C, potentially linked to delayed immune responses to SARS-CoV-2. The XLT phenotype underlies a subclinical immunodysregulation involving the NLRP3 inflammasome and the type-I IFN response.

Osteogenesis Imperfecta: Case Report and Literature Review

Osteogenesis imperfecta or brittle bone disease is a disorder of congenital bone fragility caused by genetic mutations in the procollagen type 1 code (COL1A1 and COL1A2). The global prevalence of children born with osteogenesis imperfecta is estimated to be 1 in 100,000 children worldwide. The diagnosis of osteogenesis imperfecta is based on history taking, physical examination, and supporting examination. Management of patients with osteogenesis imperfecta requires multidisciplinary treatment. There are several treatments that can be done, including conservative management, surgical management, and medical rehabilitation. This case report will discuss an 8-year-old girl who was treated at the West Nusa Tenggara Provincial Hospital with a diagnosis of closed fracture of the sinistra femur, osteogenesis imperfecta, and Congenital Talipes Equinovarus (CTEV).

Enhancing Mycotoxin Detection in Table Olives: The Role of Enzyme-Linked Immunosorbent Assay and Method Optimization

The issue of mycotoxin contamination in food is particularly important for consumer safety. The consumption of these fungi can cause genetic mutations or lead to the formation of cancer cells. For years, foods have been studied for this problem, but for table olives, there is currently a significant lack of scientific literature. This study aimed to point out the possible application of the Enzyme-Linked Immunosorbent Assay (ELISA) method for the detection of mycotoxins in table olives, assessing the presence of aflatoxins and ochratoxin A. Different tests were carried out on commercial and inoculated olives to assess the efficacy of the method by comparison with the conventional one, based on Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS). Initial unsatisfactory results led us to refine the extraction method via the substitution of the chemical extractant 70% methanol with dichloromethane; this procedure allowed for improving the recovery of inoculated mycotoxins. The results show the effectiveness of the ELISA test, especially in mycotoxin recovery, after the modification of the extraction method. The recoveries increased for aflatoxins from 55% to over 70% and for ochratoxin A from 40% to over 55%. When samples were analyzed with commercial kits, the results showed some false positives, especially for the first sample of ochratoxin A, but the reliability of the ELISA method test was largely verified in the rest of the samples. The validity, the reduced cost, and the speed of application of the ELISA test make this method suitable as a control program for mycotoxin detection in table olives, which can be supported by LC-MS/MS analysis in case of positive results.

Early Neonatal Epilepsy Caused by Homozygous Mutation in the SLC13A5 Gene: A Case Report From India

Early Neonatal Epilepsy Caused by Homozygous Mutation in the SLC13A5 Gene: A Case Report From India

Dravet Syndrome – Clinical and Developmental Characteristics: A Case Report

Introduction: Dravet syndrome is a rare, genetically determined epilepsy and epileptic encephalopathy primarily caused by a loss-of-function mutation in the SCN1A gene, also associated with autism spectrum disorder. Follow-ing birth, patients have typical neurodevelopment, but the regression of cognitive, motor and speech abilities become noticeable after the onset of seizures.Aims and case report: We report on the case of a five-year-old girl with characteristic clinical features of Dravet syndrome, detailing her basic clinical and developmental characteristics, disease course and treatment. The first seizure occured at four months of age, coinciding with increased body temperature, and by the end of first year she developed recurrent seizures. While the baseline electroencephagram was nomal, follow-up examinations revealed continuous high-amplitude and sharp, multifocal spike waves. During her second year of life, significant delay in psychomotor development became apparent. The Bayley-III scale was used to assess psychomotor de-velopment in cognition area, comprehension and quality of speech, as well as fine and gross motor skills. The results indicated that her cognitive abilities corresponded to those of an eight-month-old child, while her motor skills were at the level of an 18-month-old. Notable gait impairment was observed, with a wide-based crouch gait. The patient was also diagnosed with an autism spectrum disorder.Conclusion: Although rare, Dravet syndrome is an important differential diagnosis in children presenting with early-onset epilepsy and progressive developmental delays. It is essential to evaluate patients for common comor-bidities, such as autism, gait disorders, and intellectual disability, important determinants of patients’ quality of life.

A four-hit mechanism is sufficient for meningioma development.

Meningiomas are central nervous system tumors whose incidence increases with age. Benign meningioma pathogenesis involves germline or somatic mutation of target genes, such as NF2, leading to clonal expansion. We used an established cancer epidemiology model to investigate the number of rate-limiting steps sufficient for benign meningioma development. Incidence data was obtained from the Surveillance, Epidemiology and End Results Program (SEER) for nonmalignant meningioma from 2004 to 2020. Age-adjusted incidence rates per 100,000 person-years were divided into 5-year bands. This was repeated for vestibular schwannomas as a negative control. The Armitage-Doll methodology was applied. Mathematical solutions correcting for volatile tumor microenvironments were applied to fit higher-order models using polynomial regression when appropriate. A 75:25 training:test split was utilized for validation. 222,509 cases of benign meningiomas were identified. We noted strong linear relationships between log-transformed incidence and age across the cohort and multiple subpopulations: male, white, black, Hispanic, Asian/Pacific Islander, and American Indian subpopulations all demonstrated R2 = 0.99. Slopes were between 3.1 and 3.4, suggesting a four-step process for benign meningioma development. Female patients exhibited nonlinear deviations, but the corrected model demonstrated R2 = 0.99 with a four-hit pathway. This model performed robustly on test data with R2 = 0.99. Vestibular schwannomas demonstrated a slope of 2.1 with R2 = 0.99, suggesting a separate three-step process. Four mutations are uniquely required for the development of benign meningiomas. Correcting for volatile tumor microenvironments reliably accounted for nonlinear deviations in behavior. Further studies are warranted to elucidate genomic findings suggestive of key mutations in this pathway.

Molecular and hematological spectrum of α-thalassemia in Saudi patients

α-thalassemia (α-thal) is a genetic disorder characterized by a decreased synthesis of α-globin chains. A deletion mutation most often causes it in one or more α-globin chains. No comprehensive characterization studies have been conducted on α-thal patients in the Saudi population. Therefore, this research aims to identify the spectrum of genetic mutations responsible for α-thal in our region. Individuals with microcytic, hypochromic red blood cells and normal hemoglobin (Hb) A2 were enrolled. Sixty samples of individuals suspected of α-thal were selected for further genetic analysis. Multiplex ligation-dependent probe amplification assay was used to detect deletion mutations in α-globin genes. Among all samples tested, the -α3.7 deletion mutation was detected in 57 (95%) cases, whereas no mutation was detected in the remaining 3 (5%). In addition, 9 (15%) individuals were heterozygous for -α3.7, while -α3.7 homozygosity was found in 85% of the analyzed cases. The hematological characteristics of α3.7 subjects were significantly lower than the control group in the mean of Hb, hematocrit, mean corpuscular volume, mean corpuscular Hb, and mean corpuscular Hb concentration (p<0.001). These results highlight the importance of α-thal diagnosis and investigation in Saudi Arabia’s pre-marital screening program for microcytic hypochromic individuals. Thus, it contributes to reducing the spread of genetic diseases.

PIEZO1 attenuates Marfan syndrome aneurysm development through TGF-β signaling pathway inhibition via TGFBR2.

Marfan syndrome (MFS) is a hereditary disorder primarily caused by mutations in the FBN1 gene. Its critical cardiovascular manifestation is thoracic aortic aneurysm (TAA), which poses life-threatening risks. Owing to the lack of effective pharmacological therapies, surgical intervention continues to be the current definitive treatment. In this study, the role of Piezo-type mechanosensitive ion channel component 1 (Piezo1) in MFS was investigated and the activation of PIEZO1 was identified as a potential treatment for MFS. PIEZO1 expression was detected in MFS mice (Fbn1C1041G/+) and patients. Piezo1 conditional knockout mice in vascular smooth muscle cells of MFS mice (MFS × CKO) was generated, and bioinformatics analysis and experiments in vitro and in vivo were performed to investigate the role of Piezo1 in MFS. PIEZO1 expression decreased in the aortas of MFS mice; MFS × CKO mice showed aggravated TAA, inflammation, extracellular matrix remodelling, and TGF-β pathway activation compared to MFS mice. Mechanistically, PIEZO1 knockout exacerbated the activation of the TGF-β signalling pathway by inhibiting the endocytosis and autophagy of TGF-β receptor 2 mediated by Rab GTPase 3C. Additionally, the pharmacological activation PIEZO1 through Yoda1 prevented TGF-β signalling pathway activation and reversed TAA in MFS mice. Piezo1 deficiency aggravates MFS aneurysms by promoting TGF-β signalling pathway activation via TGF-β receptor 2 endocytosis and a decrease in autophagy. These data suggest that PIEZO1 may be a potential therapeutic target for MFS treatment.

A novel homozygous mutation in the NLRP2 gene causes early embryonic arrest.

Successful reproduction in humans requires maturation and fertilization of gametes as well as early embryonic development. Any deviation from these processes leads to infertility. Early embryonic arrest (EEA) is common in female infertility and is primarily attributed to genetic factors. Mutations in the NLRP2 gene have been identified as the causative factors for EEA. In the present study, a novel mutation identified in NLRP2 underscored the novel homozygous variant and phenotypes that might contribute to its inclusion in the genetic counseling of infertile patients. We recruited a proband from a consanguineous family with a diagnosis of EEA. Peripheral blood samples were collected from the proband and family members for whole-exome sequencing to identify the genes and inheritance patterns associated with infertility; the results were substantiated by Sanger sequencing. All genetic variants and protein structures were analyzed based on computational predictions. Wild-type and mutant plasmids were constructed and transfected into HeLa cells. Subsequent in vitro analyses elucidated the functional impact of the variant. A novel homozygous mutation in NLRP2 was identified in the proband. The patient harbored a frameshift deletion mutation (c.195delC: p.Tyr66Thrfs*32) in the pyrin structural domain. This genetic alteration resulted in the down-regulation of NLRP2 mRNA expression, truncation of the protein structure, and altered protein localization in cells. The current findings broaden the spectra of NLRP2 variants, especially concerning EEA. Also, potential diagnostic markers for patients experiencing recurrent IVF/ICSI failure were identified, and a solid foundation was laid for genetic counseling for female infertility.

A Rare Case of an Infant With TFE3 Mutation Presenting With Direct Hyperbilirubinemia and Hepatomegaly.

Translocations within the TFE gene resulting in oncogenic fusion proteins have been associated with multiple neoplasms. De novo mutations in the X-linked gene TFE3 in exons 3 and 4 are considered to contribute to lysosomal storage disorder-like features. However, the histologic findings within the livers of patients with TFE3 mutations are not well characterized. The authors report a case of a 12 day old term male who was admitted to the pediatric intensive care unit and went on to develop worsening direct hyperbilirubinemia and hepatomegaly. Due to the constellation of clinical findings, whole genome sequencing was performed and a rare de novo hemizygous mutation was identified in the TFE3 gene (c.560C > T; p.Thr187Met) which was thought to be likely pathogenic. The patient subsequently had 2 liver biopsies performed, both with similar histologic findings. The liver was found to have a giant cell hepatitis pattern of injury with severe cholestasis and extensive pseudorosette formation. Additional studies are needed to understand the histologic changes which could be associated with mutations in the TFE3 gene. The impact of a TFE3 mutation on the liver represents an area where further study is necessary to provide prognostic and therapeutic guidance for future patients.

An Update of Phenotypic–Genotypic IMNEPD Cases and a Bioinformatics Analysis of the New PTRH2 Gene Variants

Background/Objectives: Biallelic mutations in the PTRH2 gene are associated with a rare genetic disease known as infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD). In this study, we describe a new case carrying a previously identified mutation, provide an updated analysis of the relative frequencies of the clinical features across all published cases (including the three latest studies), and perform a bioinformatics analysis of the newly identified PTRH2 protein variants from a structural perspective. Methods: Clinical examination of the patients was carried out, and genetic testing was performed using a genome sequencing strategy. A bioinformatics analysis was carried out for the newly reported mutations using PYMOL that was utilized to view the structure and analyze the mutations. Additionally, the ThermoMPNN webserver was employed to check the effect of point mutations on the overall stability of the protein. Results: Our findings indicate that motor delay, neuropathy, intellectual disability, distal weakness, hearing impairment, and ataxia are the most common symptoms, while the other clinical features fall into two frequency categories: moderately common ones and the least common ones. The bioinformatics analysis revealed that the Q85 residue is highly conserved, suggesting that mutations at this position could disrupt key signaling pathways or cellular functions. Indeed, the Q85R mutation was shown to significantly impair the stability and functionality of the protein. Conclusions: The clinical presentation of IMNEPD remains highly variable in terms of both severity and progression. Mutations at the Q85 residue have been identified in nearly 50% of reported cases, highlighting this position as a potential mutational hotspot in the PTRH2 protein.

Congenital diarrhoea due to microvillus inclusion disease: a case report

Inherited diarrheal diseases are group of disorders which cause significant morbidity with dependence on parental nutrition. Microvillus inclusion disease (MVID) is a secretory enteropathy with autosomal recessive inheritance associated with bi allelic mutations in MYO5B gene or more rarely STX3 gene. We report a newborn, which developed diarrhea since birth and whose exome sequencing showed the rare mutation of biallelic STX3. There is a possibility of familial involvement (two other siblings having similar symptoms involved, but not evaluated and died) in this particular case. All three had antenatal polyhydramnios and dilated bowel loops. This case report describes an infant diagnosed with congenital diarrhea due to a genetic mutation, explores the diagnostic process, and outlines the management strategies and outcomes. The report emphasizes the importance of early diagnosis and tailored treatment in improving patient outcomes.

Prevalence and Antibiotic Susceptibility of Nontyphoidal Salmonella from Food-Animal Products in Bejaia, Algeria.

Nontyphoidal Salmonella presents a significant threat to animal and human health as a food-borne infectious agent. This study focused on the characterization of Salmonella isolates obtained from fresh animal products consumed in Bejaia, Algeria. In total, 495 beef products, 600 chicken products, and 355 dairy products were collected from retail outlets and slaughterhouses in the region. Out of the 1450 samples collected, a Salmonella positivity rate of 3.5% (51/1450) was observed. Traditional Algerian sausages exhibited the highest prevalence of Salmonella contamination (14.1%, 36/256). Fifteen different serotypes were identified, with S. Kentucky (n = 12), S. Anatum (n = 11), and S. Bredeney (n = 9) being the most prevalent. Antimicrobial susceptibility testing showed resistance to diverse antibiotics, particularly against tetracycline, ampicillin, nalidixic acid, and sulphonamides. Whole-genome sequencing conducted on 21 isolates enabled the comparison of phylogenetic links between isolates. We notably identified clones circulating across the region in different locations and food types, suggesting contamination at the early stages of the food chain (in the herd or slaughterhouses) that disseminated to numerous butcher shops in various cities. We also identified acquired antibiotic resistance genes and point mutations in the quinolone resistance-determining region genes, contributing to the observed resistance patterns. This study describes the genomic and phylogenetic characteristics of several Salmonella clones found in diverse food samples in Algeria. It suggests potential transmission dynamics that could better understand Salmonella's contamination routes.

Molecular analysis of mucopolysaccharidosis type VI in Iranian patients; the influence of founder effect and consanguinity.

Mucopolysaccharidosis type VI (MPS VI), also known as Manteaux-Lamy syndrome, is an autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme arylsulfatase B(ARSB). This syndrome is progressive and affects many tissues and organs, leading to inflammation and scarring. The classic clinical features of Maroteaux-Lamy syndrome are significant impairment of the osteoarticular system with dysostosis multiplex, short stature and motor dysfunction. The rate at which symptoms appear and worsen can vary between affected individuals. Mutations in the ARSB gene are responsible for MPS VI. We investigated the clinical presentation and molecular basis of patients with MPS VI for the first time in Yazd province, Iran. Of the 52 people who took part in this project, there were 13 probands. Whole exome sequencing (WES) was performed in 2 of them and the nominated mutation in the ARSB (c.430G > A) was verified by Sanger sequencing in the remaining patients. All patients had parental consanguinity, except for one family in which the parents were unrelated. All patients were of Fars ethnicity and had characteristic phenotypes such as severe short stature, cardiac involvement, coarse facial features, and corneal opacities. Sequence analysis of the ARSB gene revealed a pathogenic homozygous missense mutation c.430G > A (p. Gly144Arg) in all patients. This type of mutation influenced the phenotypes of the severe patients. These results expand the genetic databases of Iranian patients with MPS VI and would be very helpful for the high-risk families to accelerate the detection of carriers and to perform prenatal testing for the disorder in this population in a cost-effective manner. There is a possibility that other unknown mutations are responsible for the disease. The decision to screen for and detect carriers of this disease at a national level is awaited. The results of the present study could be an asset for married families in part of the city of Meybod. The results offer a way for early detection of patients and carriers of the disease.

Feasibility study of the psychosocial effects of an online mindfulness intervention in children and adolescents with achondroplasia and their parents

IntroductionAchondroplasia is a common skeletal dysplasia caused by a mutation in the FGFR3 gene, leading to disproportionate short stature and various clinical features. Despite the absence of definitive pharmacological treatments, mindfulness-based interventions may offer psychosocial benefits for affected individuals and their families. ObjectivesThis study aimed to assess the feasibility and psychosocial effects of an online mindfulness intervention for children and adolescents with achondroplasia and their parents. MethodologyThe intervention was an eight-week, synchronous online program with 15 participants: seven children and adolescents with achondroplasia and eight parents. Quantitative outcomes were assessed pre-and post-intervention using the State-Trait Anxiety Inventory for Children (STAIC) and the Multifactorial Self-Assessment Child Adaptation Test (TAMAI) for the younger participants, along with the Brief Symptom Inventory (BSI-18) for parents. Participant satisfaction was assessed using a customized survey, and qualitative data were collected through semi-structured interviews. ResultsThe study demonstrated high feasibility, with 84.61% adherence and 93.75% participant satisfaction. Average anxiety levels decreased in children and adolescents (30.60 ± 5.12 to 26.80 ± 6.94, p = .285) and parents (3.67 ± 3.98 to 1.00 ± .89, p = .066). Emotional regulation was the most reported improvement category by children and adolescents (31.4%), while general well-being was the most noted by parents (29.63%). ConclusionsThe results support the feasibility of this online mindfulness intervention for individuals with achondroplasia and their parents, indicating benefits for psychosocial well-being. Future studies should address these limitations by expanding sample sizes, exploring hybrid intervention models, and ensuring data anonymity. Integrating mindfulness into comprehensive psychosocial care strategies could enhance the quality of life for these populations.

A case report of mild hereditary hemochromatosis caused by HAMP gene mutation

The incidence of juvenile Hereditary Hemochromatosis caused by HAMP gene mutation is low, which is rarely reported in China. This patient took abnormal liver function as the first symptom, and was finally diagnosed by genetic testing and hepatic histopathology, and treated by venous bloodletting.

Relationship between ripretinib concentration and the prognosis of advanced gastrointestinal stromal tumors in China: a multicenter study

Objective: To investigate the prognosis and safety of ripretinib in the treatment of patients with advanced gastrointestinal mesenchymal stromal tumors (GISTs) and to analyze the relationship between blood concentrations of this drug and prognosis. Methods: In this retrospective study, we investigated the effects of ripretinib in patients with advanced GISTs. The inclusion criteria comprised: (1) daily oral administration of ripretinib scheduled; and (2) uninterrupted treatment for at least 1month, with a stable and relatively fixed daily dosage maintained for a minimum of 2 weeks. Exclusion criteria comprised concurrent use of other tyrosine kinase inhibitors and presence of significant organ dysfunction. We retrospectively identified 79 patients with advanced GISTs who had received ripretinib across seven medical centers, namely Jiangsu Provincial Hospital, Jiangsu Cancer Hospital, Nanjing Drum Tower Hospital Affiliated to Nanjing University, Sir Run Run Shaw Hospital of Zhejiang University, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and the General Hospital of the People's Liberation Army, from 1 June 2021 to 31 March 2024. The cohort included 48 men and 31 women, 19 of whom had received ripretinib as second-line, 13 as third-line, and 47 as fourth-line therapy. Two peripheral venous blood samples were obtained from each participant and high-performance liquid chromatography-tandem mass spectrometry used to determine peak (Cmax) and trough (Cmin) concentrations of ripretinib. Machine learning methodologies, specifically the K-nearest neighbor algorithm combined with the Gridsearch CV strategy, were employed to establish the threshold for Cmin. We analyzed adverse reactions, treatment efficacy, median progression-free survival (mPFS), and the relationship between drug blood concentration and selected clinical parameters. Results: In the entire cohort, the Cmin and Cmax of ripretinib were 467 ± 360 μg/L and 986 ± 493 μg/L, respectively. Notably, female patients and individuals in the high-dose group exhibited significantly higher values for both Cmin and Cmax (both P<0.05). However, variations in drug concentrations associated with the line of ripretinib therapy, treatment efficacy, disease progression, and presence of selected specific genetic mutations were not significantly associated with values of Cmin and Cmax (P>0.05). Among the 79 patients with advanced GISTs receiving ripretinib, reported adverse reactions included alopecia (53, 67.09%), hand-foot syndrome (24, 30.38%), fatigue (22, 27.85%), and myalgia (21, 26.58%). Two patients (2.53%) had grade III complications, both classified as hand-foot syndrome. The correlation between Cmax and adverse reactions was not statistically significant (P > 0.05). By the time of the latest follow-up, five deaths (6.3%) had occurred within the cohort. The mPFS for the group was 16.3 months, with a mPFS of 14.4 months for those receiving standard dosage and 7.0 months for those receiving escalating dosage. Among the 65 patients treated with standard doses of ripretinib, those with Cmin exceeding a threshold of 450 μg/L exhibited a significantly longer mPFS (18.0 months vs.13.7 months; P < 0.05). Conclusion: In China, patients with advanced GISTs exhibit a notable tolerance to ripretinib, with no evidence for a correlation between adverse reactions and Cmax for the drug. Additionally, a Cmin exceeding 450 μg/L may be associated with an extended mPFS.

Rapid and supersensitive allele detection of Plasmodium falciparum chloroquine resistance via a Pyrococcus furiosus argonaute-triggered dual-signal biosensing platform

BackgroundMalaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute (PfAgo) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study.MethodsBy meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt. The recombinant plasmids pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt-CVIET plasmid mixture was added to the pUC57/Pfcrt-CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA-PfAgo platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing.ResultsThe entire process achieves the key mutation Pfcrt-CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 104 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples.ConclusionsThis study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site.Graphical abstract

Lung Cancer Associated with Cystic Airspaces: Current Insights into Diagnosis, Pathophysiology, and Treatment Strategies

Lung cancer associated with cystic airspaces (LCCA) is a rare subtype of non-small-cell lung cancer (NSCLC), accounting for 1–4% of cases. LCCA is characterized by the presence of cystic airspaces within or at the periphery of the tumor on imaging. LCCA poses significant clinical challenges due to its high risk of misdiagnosis or missed diagnosis, often leading to a worse prognosis compared to other forms of lung cancer. While previous studies have identified correlations between the pathological features and imaging characteristics of LCCA, research on its associated driver gene mutations and responses to chemotherapy and immunotherapy remains limited. Furthermore, the development of an appropriate T-staging system is necessary to improve prognostic outcomes. This review provides an overview of the current research on the definition, imaging classification, pathological and molecular mechanisms, and prognosis of LCCA, aiming to provide a reference for clinical decision-making.