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Climate warming and human activities have a major impact on the spatial and temporal characteristics of surface runoff in the Shiyang river basin, and the adaptive management of water resources in the basin is facing serious challenges. With three independent drainage Dajinghe river system, Liuhe river system and Xidahe river system as the research object, the statistical method analyzed the average flow evolution in 1960−2020, using Mann-Kendall test and Pettitt test to determine the runoff sequence mutation point, set the combination of climate and land use change, and used SWAT model to identify the runoff variation in Shiyang river basin, addressing the unequal allocation of water resources. The results showed that: (1) from 1960 to 2020, the annual average runoff decline rate of Xidahe river, Liuhe river and Dajing river system was 0.01m³ ·s−1 , 0.07m³ ·s−1, 0.01m³ ·s−1 respectively. The runoff years for the Liuhe river and Xidahe river were in 1973 and 2002, respectively, while the Dajing river system was in its natural state. (2) The SWAT model had a good adaptability to the Shiyang river basin, and the determination coefficient and the Nash efficiency coefficient were both higher than 0.50. (3) From 1960 to 2020, the contribution of precipitation, average temperature, relative humidity, solar radiation and average wind speed to the annual average runoff were 75%, 53%, 55%, 55%, and 52%, respectively. The contribution of land use change to the annual average runoff was 17%. The coupled contribution of six influence factors to annual average runoff reduction was 67%, indicating that climate change had great influence on runoff in Shiyang river basin. The research results could provide a reference for the adaptive management of watershed water resources. [ABSTRACT FROM AUTHOR]

Summary: Despite the clinical and molecular heterogeneity of follicular lymphoma (FL), there remains a lack of biomarker‐directed therapeutic approaches in routine clinical practice, with the notable exception of the EZH2 inhibitor tazemetostat in EZH2‐mutant FL. Here we examined whether gene mutation status predicts response to clinical mTOR inhibitors (mTORi) in FL, by performing targeted mutational profiling of biopsies from 21 relapsed/refractory FL patients treated with mTORi everolimus or temsirolimus within clinical trials. We observed an enrichment of mutations within the catalytic histone acetyltransferase (HAT) domain of CREBBP in mTORi‐responders, and describe distinct transcriptional characteristics and co‐occurring mutations of FL harbouring these mutations; reinforcing the growing appreciation of CREBBPHAT mutation as a key biological determinant and its promise as a therapeutic biomarker in FL. [ABSTRACT FROM AUTHOR]

Objectives We sought to characterize the immunophenotype of acute myeloid leukemia (AML) with CBFB rearrangement and correlate the results with cytogenetic and molecular data. Methods Sixty-one cases of AML with CBFB rearrangement were evaluated. Results The sample population consisted of 33 men and 28 women, with a median age of 49 years. Flow cytometry immunophenotypic analysis showed that myeloblasts were positive for CD34 and CD117 in all cases, and myeloperoxidase was positive in 52 of 55 (95%) cases. The most common abnormalities included decreased CD38 in 90%, increased CD13 in 85%, increased CD123 in 84%, and decreased HLA-DR in 84% of cases. Monocytes were increased, with a mature immunophenotype, and accounted for 23.7% of total cells. Among 60 cases with available karyotype, inv(16)(p13.1q22) was most common in 50 (83%) cases, followed by t(16;16) (p13.1;q22) in 6 (10%). Type A CBFB::MYH11 transcript was most common, detected in 84% of cases. Mutational analysis showed mutations of NRAS in 37%, FLT3 in 25%, and KIT in 24% of cases. Comparing cases with type A vs non–type A transcripts, blasts in type A cases more frequently exhibited CD64 positivity and increased CD13 levels while showing a lower frequency of CD7 and CD56 expression. Trisomy 22 and mutations in KIT, NF1, and TET2 were identified only in cases with type A transcript. Conclusions Myeloblasts of AML with CBFB rearrangement are positive for CD34, CD117, and myeloperoxidase. These neoplasms most frequently carry inv(16)(p13.1q22) and type A fusion transcript. NRAS mutation was the most common mutation. Some immunophenotypic and genetic correlations occurred with different types of transcripts. [ABSTRACT FROM AUTHOR]

Canine distemper virus (CDV) is a highly contagious and often fatal disease affecting wild and domesticated carnivores. The virus is a single-stranded RNA virus from the genus Morbillivirus and the family Paramyxoviridae. While domestic dogs are the most common hosts, the virus poses a significant threat to endangered wildlife due to its broad host range. This study aimed to characterize the CDV Haemagglutinin (H) gene in golden jackals and explore the molecular evolution of the virus in an underrepresented host. A total of 88 brain samples from hunted golden jackals were tested for the presence of CDV viral nucleic acid, and the H gene of positive samples was amplified and sequenced using the Sanger method. Phylogenetic analysis, conducted using maximum likelihood methods, revealed that all Serbian sequences clustered within the Arctic lineage. Notably, the analysis identified a tyrosine (Y) at position 549 of the H protein, a mutation commonly associated with wildlife hosts, instead of the histidine (H) typically found in domestic strains. Additionally, a mutation at position 310 was observed, which could potentially affect the protein's function and virus-host interactions. These findings provide valuable insights into the genetic diversity and evolutionary dynamics of CDV in golden jackals, with broader implications for understanding the virus's adaptability to different hosts. Further research is needed to investigate the functional impact of these mutations, particularly their role in vaccine efficacy and disease transmission across wildlife and domestic species. [ABSTRACT FROM AUTHOR]

Congenital bilateral absence of vas deferens (CBAVD) is a urological syndrome of Wolffian ducts and is responsible for male infertility and obstructive azoospermia. This study is designed to explore the integrity of exon 10 of CFTR and its role in male infertility in a cohort of CBVAD patients in Pakistan. Genomic DNA was extracted from 17 male patients with CBAVD having clinical symptoms, and 10 healthy controls via phenol-chloroform method. Exon 10 of the CFTR gene was amplified, using PCR with specific primers and DNA screening was done by Sanger sequencing. Sequencing results were analyzed using freeware Serial Cloner, SnapGene, BioEdit and FinchTV. Furthermore, bioinformatics tools were used to analyze the mutations and their impact on the protein function and stability. We have identified 4 mutations on exon 10 of CFTR in 6 out of 17 patients. Two of the mutations were missense variants V456A, K464E, and the other two were silent mutations G437G, S431S. The identified variant V456A was present in 4 of the studied patients. Whereas, the presence of K464E in our patients further weighs on the crucial importance for its strategic location to influence the gene function at post-transcriptional and protein level. Furthermore, Polyphen-2 and SIFT analyze the mutations as harmful and deleterious. The recurrence of V456A and tactically conserved locality of K464E are evidence of their potential role in CBAVD patients and in male infertility. The data can contribute in developing genetic testing and treatment of CBAVD. [ABSTRACT FROM AUTHOR]

The Qinba Mountain range is a typical climate-sensitive and ecologically fragile region. Monitoring of vegetation dynamics is crucial for ecological protection and achieving sustainable development goals. Various mutation-detection methods, along with slope analysis, hot-spot analysis, and residual analysis, were used to examine changes in the Normalized Difference Vegetation Index (NDVI) during the growing and non-growing seasons over 41 years and to distinguish the relative effects of the drivers. This revealed four key findings. (1) NDVI increased at 0.02 decade−1, with mutation points in 2006 for growing-season NDVI and 2007 for non-growing-season NDVI. (2) The trend in NDVI changed markedly at the mutation point. After the mutation point, NDVI was impacted more by human activity than by climate change. The hot and cold spots of the NDVI trend rate change in location and range in the growing season; in the non-growing season, it shows an obvious north–south distribution. (3) The spatial patterns in the effects of the drivers changed at this point. In the growing season, before this point, climate change and human activity collectively enhanced NDVI in ca. 81.3% of the region; after the mutation point, this value declined to 59.9% of the area, and human activity became the dominant driver in the area formerly dominated by both factors in combination. In the non-growing season, after the mutation point, the areas where both factors promoted vegetation growth decreased by 12.6% and those where climate change alone promoted it decreased by 11.1%, whereas the area affected only by human activity increased by 11.6%. (4) Before this point, human activity contributed >60% to the change in NDVI in the western Qinling region, with climate change contributing >60% in the other areas. After this point, human activity exerted a stronger influence than climate change, contributing >60% to enhancing vegetation growth and >80% reducing it. These findings provide a scientific basis for protecting the Qinba Mountain ecosystem and are essential for achieving sustainable development goals. [ABSTRACT FROM AUTHOR]

Yunxia Zhu,1 Deng Zhang,1 Liang Wu,1 Xiaoliang Ouyang,2 Shengcai Zhu,1 Xiuping Wang,1 Zhen Xiao,3 Yanping Tan,4 Chunming Li1 1Department of Dermatology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Plastic Surgery, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 3Department of Dermatology, Taiyuan Central Hospital, Taiyuan, Shanxi, People’s Republic of China; 4Department of Dermatology, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi, People’s Republic of ChinaCorrespondence: Chunming Li, Department of Dermatology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, People’s Republic of China, Tel/Fax +86 79186278821, Email chunminglincu@163.comPurpose: Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant inherited pigmentary dermatosis. The gene responsible for DSH has been identified as adenosine deaminase acting on RNA1 (ADAR1). This study aimed to identify the causative variants in the ADAR1 gene in three Chinese families with DSH.Patients and Methods: Data and blood samples were collected from three Chinese families with DSH. Whole-exome and Sanger sequencing were performed to detect pathogenic gene mutation in the patients. Bioinformatics tools were used to predict the pathogenicity of the variants.Results: Four heterozygous ADAR1 variants were identified, including two novel missense variants c.2369G>C (Arg790Pro), and 503C>T (Pro168Leu), and two previously reported variants: c.3232C>T(R1078C), and c.1472C>G (p.S491X). The novel c.503C>T variant was predicted as “deleterious” (score =− 2.704) by PROVEAN, and “probably damaging” (score = 1) by PolyPhen2. The other novel variant c.2369G>C was also predicted as “deleterious” (score =− 4.167) by PROVEAN, “probably damaging” (score = 1) by PolyPhen2, and “disease-causing” (p = 0.999) by Mutation Taster.Conclusion: Two novel ADAR1 variants were found in Chinese patients with DSH. This research has expanded the ADAR1 gene database for DSH, enhancing our comprehension of the underlying mechanisms.Keywords: dyschromatosis symmetrica hereditaria, adenosine deaminase acting on RNA1, mutation analysis, Chinese

Abstract Canine distemper virus (CDV) is a highly contagious and often fatal disease affecting wild and domesticated carnivores. The virus is a single-stranded RNA virus from the genus Morbillivirus and the family Paramyxoviridae. While domestic dogs are the most common hosts, the virus poses a significant threat to endangered wildlife due to its broad host range. This study aimed to characterize the CDV Haemagglutinin (H) gene in golden jackals and explore the molecular evolution of the virus in an underrepresented host. A total of 88 brain samples from hunted golden jackals were tested for the presence of CDV viral nucleic acid, and the H gene of positive samples was amplified and sequenced using the Sanger method. Phylogenetic analysis, conducted using maximum likelihood methods, revealed that all Serbian sequences clustered within the Arctic lineage. Notably, the analysis identified a tyrosine (Y) at position 549 of the H protein, a mutation commonly associated with wildlife hosts, instead of the histidine (H) typically found in domestic strains. Additionally, a mutation at position 310 was observed, which could potentially affect the protein’s function and virus-host interactions. These findings provide valuable insights into the genetic diversity and evolutionary dynamics of CDV in golden jackals, with broader implications for understanding the virus’s adaptability to different hosts. Further research is needed to investigate the functional impact of these mutations, particularly their role in vaccine efficacy and disease transmission across wildlife and domestic species.

The COVID-19 pandemic in Ukraine, from March 2020 to June 2022, witnessed distinct waves, each characterized by an increase in cases and fatalities. Next-generation sequencing has been used to understand the impact of viral variants on the pandemic situation in Ukraine. We analyzed SARS-CoV-2 genome sequencing data to identify viral variants circulating during each wave. By integrating epidemiological information, we established associations between viral variants and disease spread. The adoption of next-generation sequencing for SARS-CoV-2 surveillance in Ukraine, despite limited resources, yielded adequate and trustworthy results, reflecting the pandemic situation. After the Russian military invasion of Ukraine in February 2022, a large number of refugees crossed the border with neighboring countries. Mutation analysis on sequencing data from Ukraine and Poland was used to estimate the exchange of SARS-CoV-2 variants between the countries during this period. Omicron subvariants detected in both countries were similar. The analysis of SARS-CoV-2 sequences from Poland and Ukraine revealed shared nucleotide mutations that can be used to identify the directions of spreading.

Background: In cases of chronic pancreatitis, a set of behavioral, genetic, and environmental factors are involved. The SPINK1 and CTFR genes are important genetic factors, but they might have different patterns in different regions. There are only few studies conducted to investigate the prevalence of these mutations, especially in Iran. In this study, we determined the prevalence of mutations in SPINK1 and CFTR in patients with chronic pancreatitis in an Iranian referral hospital. Materials and Methods: A total of 56 patients diagnosed with chronic pancreatitis (based on the criteria of the American Pancreatic Association) were included in this cross-sectional study. Using specific primers for the CFTR exon 11 and SPINK1 exon 3, two conventioan PCRs were performed, and products were examined by nucleotide sequencing. The raw data were edited with bioinformatics software against the reference sequence. Mutated samples were added to the patient data file and analyzed with the rest of the variables in SPSS software. Results: Among the 56 patients, the average age±SD of the patients was 48.1±14.9 years, 20 were women (35.7%), 44 (78.6%) were ever- smokers and 25 people (44.6%) had a history of long-term alcohol consumption. According to the results of mutation analysis, seven people (12.5%) had CFTR gene mutation (F508del and Gly469Asp), three people (5.4%) had SPINK1 gene mutation (S34N), and one person (1.8%) had both CFTR and SPINK1 gene mutation. The average age and other demographical data of mutated people were not statistically different from other patients. CFTR mutation was observed more in men (85.7%), smokers (85.7%), with gallstones (57.1%), and in people with SPINK1 mutation, it was seen more in ever-smoking patients, diabetics, and those with gallstones, and family history of acute pancreatitis (2/3; 66.7%). Conclusions: We observed S34N and F508del mutations in the SPINK1 and CFTR genes are associated with chronic pancreatitis, which is in agreement with previous studies. The younger age, smoking, gallstone, and diabetes are probable risk factors for chronic pancreatitis. The limited sample size, lack of a control group, and small number of examined exons may affect our findings and significant results. [ABSTRACT FROM AUTHOR]

Background: Cell-free DNA (cfDNA) is free DNA found in circulating blood that originates from apoptosis or necrosis, and elevated cfDNA concentrations have been reported in cancers and other diseases. Methods: In this study, the concentrations and fragment distributions of plasma cfDNA were preliminary investigated in elderly (n = 1) and paediatric (n = 1) patients with acute promyelocytic leukaemia (APL) treated with arsenic trioxide (ATO). Results: A slight increase in cfDNA concentrations was observed in the APL patients compared with healthy controls. The change in plasma cfDNA concentrations corresponded to the change in plasma arsenic concentrations during ATO treatment. The fragment distribution pattern did not differ before and during treatment. Three ladder fragments were observed in part of the cfDNA in the second consolidation therapy in an elderly APL patient and the first consolidation therapy of a paediatric APL patient, while two fragments were observed in all other treatment periods. Moreover, APL-related gene mutations were successfully genotyped from plasma cfDNA by using polymerase chain reaction-based methods and these results are consistent with those from leukocytes. Conclusion: This study is the first to report the concentrations and fragment patterns of cfDNA from APL patients treated with ATO. The results suggested that plasma cfDNA concentration in APL patients increased with ATO treatment and that cfDNA is released mainly via neutrophil extracellular traps (and/or necrosis) in addition to apoptosis. To confirm whether cfDNA concentrations and fragment patterns can be used as a biomarker for APL treated with ATO, further accumulative data are needed. [ABSTRACT FROM AUTHOR]

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The COVID-19 pandemic in Ukraine, from March 2020 to June 2022, witnessed distinct waves, each characterized by an increase in cases and fatalities. Next-generation sequencing has been used to understand the impact of viral variants on the pandemic situation in Ukraine. We analyzed SARS-CoV-2 genome sequencing data to identify viral variants circulating during each wave. By integrating epidemiological information, we established associations between viral variants and disease spread. The adoption of next-generation sequencing for SARS-CoV-2 surveillance in Ukraine, despite limited resources, yielded adequate and trustworthy results, reflecting the pandemic situation. After the Russian military invasion of Ukraine in February 2022, a large number of refugees crossed the border with neighboring countries. Mutation analysis on sequencing data from Ukraine and Poland was used to estimate the exchange of SARS-CoV-2 variants between the countries during this period. Omicron subvariants detected in both countries were similar. The analysis of SARS-CoV-2 sequences from Poland and Ukraine revealed shared nucleotide mutations that can be used to identify the directions of spreading. [ABSTRACT FROM AUTHOR]

Protein kinases are crucial cellular enzymes that facilitate the transfer of phosphates from adenosine triphosphate (ATP) to their substrates, thereby regulating numerous cellular activities. Dysfunctional kinase activity often leads to oncogenic conditions. Chosen by using structural similarity to 5UG9, we selected 79 crystal structures from the PDB and based on the position of the phenylalanine side chain in the DFG motif, we classified these 79 crystal structures into 5 group clusters. Our approach applies our kinematic flexibility analysis (KFA) to explore the flexibility of kinases in various activity states and examine the impact of the activation loop on kinase structure. KFA enables the rapid decomposition of macromolecules into different flexibility regions, allowing comprehensive analysis of conformational structures. The results reveal that the activation loop of kinases acts as a "lock" that stabilizes the active conformation of kinases by rigidifying the adjacent α‐helices. Furthermore, we investigate specific kinase mutations, such as the L858R mutation commonly associated with non‐small cell lung cancer, which induces increased flexibility in active‐state kinases. In addition, through analyzing the hydrogen bond pattern, we examine the substructure of kinases in different states. Notably, active‐state kinases exhibit a higher occurrence of α‐helices compared to inactive‐state kinases. This study contributes to the understanding of biomolecular conformation at a level relevant to drug development. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) causes severe systemic and tissue-invasive disease in immunocompromised patients, particularly solid organ and hematopoietic stem cell transplant recipients. While antiviral drugs offer promising efficacy, clinical management is complicated by the high frequency of drug resistance-associated mutations. The most commonly encountered mutations occur in the genes encoding for the drug targets: UL54 (DNA polymerase), UL56 (terminase complex), and UL97 (phosphotransferase), conferring resistance to ganciclovir/cidofovir/foscarnet, letermovir, and ganciclovir/maribavir, respectively. Currently, standard practice for detecting drug resistance is sequencing-based genotypic analysis by commercial reference laboratories with strictly prescribed sample requirements and reporting parameters that can often restrict testing in a highly vulnerable population. In order to circumvent these limitations, we developed a dual-step next-generation sequencing (NGS)-based clinical assay that utilizes full-length gene amplification by long-range PCR followed by shotgun sequencing for mutation analysis. This laboratory-developed test (LDT) achieved satisfactory performance with 96.4% accuracy, 100% precision, and an analytical sensitivity of 300IU/mL with 20% allele frequency. Highlighted by two clinical cases, our NGS LDT was able to provide critical results from patient specimens with viral loads

ABSTRACT: Gyrovirus galga1 (GyVg1), formerly known as AGV2, was initially identified in chickens in southern Brazil. The prevalence of GyVg1 from 2021 to 2024 in 28 out of the 63 poultry farms located in Jiangsu, Anhui, Henan, Hunan, Shandong, and Hubei provinces in eastern and central China was detected via PCR. The complete genomes of the 28 strains were sequenced and exhibited a full length of 2,376 bp. Similarity analysis of these strains did not suggest definite correlation with evolutionary branching and geographical distribution. Compared with the reference GyVg1 strains, HN2202 shared the highest similarity of 99.71% with HLJ1511 (chicken-originated) from northeastern China in 2015 to 2016. Recombination analysis revealed that AH2102 was a potential recombinant of peafowl-originated HN2019-PF1 and chicken-originated HLJ1506-2, whereas HN2304 was a recombinant of peafowl-originated HN2019-PF1 and the Hungarian ferret strain G13. Mutation site analysis of the capsid protein revealed that highly mutated regions occurred between sites 288 to 316 and 383 to 419. These results indicate that GyVg1 may have undergone an interspecies transmission, which involved complex mutations and recombination. This study may provide a reference for subsequent investigations targeting the molecular epidemiology and viral evolution of GyVg1.

Congenital bilateral absence of vas deferens (CBAVD) is a urological syndrome of Wolffian ducts and is responsible for male infertility and obstructive azoospermia. This study is designed to explore the integrity of exon 10 of CFTR and its role in male infertility in a cohort of CBVAD patients in Pakistan. Genomic DNA was extracted from 17 male patients with CBAVD having clinical symptoms, and 10 healthy controls via phenol-chloroform method. Exon 10 of the CFTR gene was amplified, using PCR with specific primers and DNA screening was done by Sanger sequencing. Sequencing results were analyzed using freeware Serial Cloner, SnapGene, BioEdit and FinchTV. Furthermore, bioinformatics tools were used to analyze the mutations and their impact on the protein function and stability. We have identified 4 mutations on exon 10 of CFTR in 6 out of 17 patients. Two of the mutations were missense variants V456A, K464E, and the other two were silent mutations G437G, S431S. The identified variant V456A was present in 4 of the studied patients. Whereas, the presence of K464E in our patients further weighs on the crucial importance for its strategic location to influence the gene function at post-transcriptional and protein level. Furthermore, Polyphen-2 and SIFT analyze the mutations as harmful and deleterious. The recurrence of V456A and tactically conserved locality of K464E are evidence of their potential role in CBAVD patients and in male infertility. The data can contribute in developing genetic testing and treatment of CBAVD.

Objectives: In this study, stool samples were evaluated for tumor mutation analysis via a targeted next generation sequencing (NGS) approach in a small patient cohort suffering from localized rectal cancer. Introduction: Colorectal cancer (CRC) causes the second highest cancer-related death rate worldwide. Thus, improvements in disease assessment and monitoring that may facilitate treatment allocation and allow organ-sparing "watch-and-wait" treatment strategies are highly relevant for a significant number of CRC patients. Methods: Stool-based results were compared with mutation profiles derived from liquid biopsies and the gold standard procedure of tumor biopsy from the same patients. A workflow was established that enables the detection of de-novo tumor mutations in stool samples of CRC patients via ultra-sensitive cell-free tumor DNA target enrichment. Results: Notably, only a 19% overall concordance was found in mutational profiles across the compared sample specimens of stool, tumor, and liquid biopsies. Conclusion: Based on these results, the analysis of stool and liquid biopsy samples can provide important additional information on tumor heterogeneity and potentially on the assessment of minimal residual disease and clonal tumor evolution. [ABSTRACT FROM AUTHOR]

The evolutionary conserved Notch signaling pathway functions as a mediator of direct cell–cell communication between neighboring cells during development. Notch plays a crucial role in various fundamental biological processes in a wide range of tissues. Accordingly, the aberrant signaling of this pathway underlies multiple genetic pathologies such as developmental syndromes, congenital disorders, neurodegenerative diseases, and cancer. Over the last two decades, significant data have shown that the Notch signaling pathway displays a significant function in the mature brains of vertebrates and invertebrates beyond neuronal development and specification during embryonic development. Neuronal connection, synaptic plasticity, learning, and memory appear to be regulated by this pathway. Specific mutations in human Notch family proteins have been linked to several neurodegenerative diseases including Alzheimer's disease, CADASIL, and ischemic injury. Neurodegenerative diseases are incurable disorders of the central nervous system that cause the progressive degeneration and/or death of brain nerve cells, affecting both mental function and movement (ataxia). There is currently a lot of study being conducted to better understand the molecular mechanisms by which Notch plays an essential role in the mature brain. In this study, an in silico analysis of polymorphisms and mutations in human Notch family members that lead to neurodegenerative diseases was performed in order to investigate the correlations among Notch family proteins and neurodegenerative diseases. Particular emphasis was placed on the study of mutations in the Notch3 protein and the structure analysis of the mutant Notch3 protein that leads to the manifestation of the CADASIL syndrome in order to spot possible conserved mutations and interpret the effect of these mutations in the Notch3 protein structure. Conserved mutations of cysteine residues may be candidate pharmacological targets for the potential therapy of CADASIL syndrome. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis developing resistance to anti-tubercular therapy leads to pioneering innovative treatment protocols featuring carefully curated combinations of potent medications. The aim of this study was to analyze the frequency and pattern of fluoroquinolone (FQ) resistant mutations among the first-line drug-resistant specimens. By employing an elective sampling strategy, we deliberately focused our investigation on a targeted subset of multidrug resistant (MDR) and FQ resistant (FQR) TB cases. Method: Sputum samples from first-line drug-resistant patients were subjected to a GenoType MTBDRsl VER 2.0 assay to determine FQR. To investigate the effect of bedaquiline (BDQ) on these mutation-conferring genes (Rv0678, pepQ, and atpE), sanger sequencing was carried out on the MDR + FQR subset that was obtained. We have also conducted a computational analysis of the gene products of Rv0678 and pepQ with BDQ using molecular docking methods. Results: Eighty-three samples were observed to have FQR, and the majority of these specimens were associated with naïve and treatment failure cases. Among them, 64 isolates exhibited mutations in gyrA gene, while 19 isolates showed mutations in gyrB gene. The predominant occurrence of genetic variations, particularly in D94G and A90V, was observed within the specimens displaying resistance in gyrA gene, and the same has been confirmed by PCR-based detection. And the sequence analysis of BDQ genes among MDR + FQR subset indicated that the majority of the samples did not exhibit mutations in pepQ and atpE genes. However, only 2 samples were found to have the genetic variant G337A, which involves replacing the amino acid glutamic acid (E) at codon position 113 of Rv0678 gene with lysine (K). Conclusions: Most of the mutations were harbored in rpoB + katG + gyrA gene pattern. These findings pave the way for the development of databases and rapid diagnostic probes.

Abstract Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Estimating software testability can crucially assist software managers to optimize test budgets and software quality. In this paper, we propose a new approach that radically differs from the traditional approach of pursuing testability measurements based on software metrics, e.g., the size of the code or the complexity of the designs. Our approach exploits automatic test generation and mutation analysis to quantify the evidence about the relative hardness of developing effective test cases. In the paper, we elaborate on the intuitions and the methodological choices that underlie our proposal for estimating testability, introduce a technique and a prototype that allows for concretely estimating testability accordingly, and discuss our findings out of a set of experiments in which we compare the performance of our estimations both against and in combination with traditional software metrics. The results show that our testability estimates capture a complementary dimension of testability that can be synergistically combined with approaches based on software metrics to improve the accuracy of predictions.

Abstract Background Patients with pancreatic ductal adenocarcinoma (PDCA) carrying impaired mismatch repair mechanisms seem to have an outcome advantage under treatment with conventional chemotherapy, whereas the role for the tumor mutation burden on prognosis is controversial. In this study, we evaluated the prognostic role of the mutated genes involved in genome damage repair in a real-life series of PDAC patients in a hospital-based manner from the main Institution deputed to surgically treat such a disease in North Sardinia. Methods A cohort of fifty-five consecutive PDAC patients with potentially resectable/border line resectable PDAC (stage IIB-III) or oligometastatic disease (stage IV) and tumor tissue availability underwent next-generation sequencing (NGS)-based analysis using a panel containing driver oncogenes and tumor suppressor genes as well as genes controlling DNA repair mechanisms. Results Genes involved in the both genome damage repair (DR) and DNA mismatch repair (MMR) were found mutated in 17 (31%) and 15 (27%) cases, respectively. One fourth of PDAC cases (14/55; 25.5%) carried tumors presenting a combination of mutations in repair genes (DR and MMR) and the highest mutation load rates (MLR-H). After correction for confounders (surgery, adjuvant therapy, stage T, and metastasis), multivariate Cox regression analysis indicated that mutations in DR genes (HR = 3.0126, 95% CI 1.0707 to 8.4764, p = 0.0367) and the MLR (HR = 1.0018, 95%CI 1.0005 to 1.0032, p = 0.009) were significantly related to worse survival. Conclusions The combination of mutated repair genes and MLR-H, which is associated with a worse survival in our series of PDAC patients treated with conventional chemotherapy protocols, might become a predictive biomarker of response to immunotherapy in addition to its prognostic role in predicting survival.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues. [ABSTRACT FROM AUTHOR]

Background: Patients with pancreatic ductal adenocarcinoma (PDCA) carrying impaired mismatch repair mechanisms seem to have an outcome advantage under treatment with conventional chemotherapy, whereas the role for the tumor mutation burden on prognosis is controversial. In this study, we evaluated the prognostic role of the mutated genes involved in genome damage repair in a real-life series of PDAC patients in a hospital-based manner from the main Institution deputed to surgically treat such a disease in North Sardinia. Methods: A cohort of fifty-five consecutive PDAC patients with potentially resectable/border line resectable PDAC (stage IIB-III) or oligometastatic disease (stage IV) and tumor tissue availability underwent next-generation sequencing (NGS)-based analysis using a panel containing driver oncogenes and tumor suppressor genes as well as genes controlling DNA repair mechanisms. Results: Genes involved in the both genome damage repair (DR) and DNA mismatch repair (MMR) were found mutated in 17 (31%) and 15 (27%) cases, respectively. One fourth of PDAC cases (14/55; 25.5%) carried tumors presenting a combination of mutations in repair genes (DR and MMR) and the highest mutation load rates (MLR-H). After correction for confounders (surgery, adjuvant therapy, stage T, and metastasis), multivariate Cox regression analysis indicated that mutations in DR genes (HR = 3.0126, 95% CI 1.0707 to 8.4764, p = 0.0367) and the MLR (HR = 1.0018, 95%CI 1.0005 to 1.0032, p = 0.009) were significantly related to worse survival. Conclusions: The combination of mutated repair genes and MLR-H, which is associated with a worse survival in our series of PDAC patients treated with conventional chemotherapy protocols, might become a predictive biomarker of response to immunotherapy in addition to its prognostic role in predicting survival. [ABSTRACT FROM AUTHOR]

Background: Accurate genetic diagnosis of end‐stage renal disease patients with a family history of renal dysfunction is very essential. It not only helps in proper prognosis, but becomes crucial in designating donor for live related renal transplant. We here present a case of family with deleterious mutations in INF2 and ROBO2 and its importance of genetic testing before preparing for kidney transplantation. Case Presentation: We report the case of a 29‐year‐female with end‐stage renal disease and rapidly progressive renal failure. Mutational analysis revealed an Autosomal Dominant inheritance pattern and mutation in exon 4 of the INF2 gene (p. Thr215Ser) and exon 26 of the ROBO2 gene (p. Arg1371Cys). Her mother was diagnosed for CKD stage 4 with creatinine level of 4.3 mg/dL. Genetic variants (INF2 and ROBO2) identified in proband were tested in her sisters and mother. Her elder sister was positive for both heterozygous variants (INF2 and ROBO2). Her mother was positive for mutation in INF2 gene, and her donor elder sister did not showed mutation in INF2 gene and had mutation in ROBO2 gene without any clinical symptoms. Conclusion: This case report emphasize that familial genetic screening has allowed us in allocating the donor selection in family where family member had history of genetic defect of Chronic Kidney Disease. Information of the causative renal disorder is extremely valuable for risk‐assessment and planning of kidney transplantation. [ABSTRACT FROM AUTHOR]

Introduction: Hermansky-Pudlak syndrome (HPS) is a rare autosomal-recessive disease characterized by ocular albinism (OA) or oculocutaneous albinism (OCA), platelet dysfunction, and other symptoms. This study aimed to analyze the molecular defect in two Chinese families with suspected OA, as well as to investigate the profile of HPS6 variants and their genotype-phenotype correlations. Methods: Seven members from two families were recruited and underwent clinical ophthalmologic examinations. The genomic DNA was extracted from peripheral blood leukocytes. Whole-exome sequencing was performed on the proband of family JX. The single coding exon of HPS6 was directly Sanger sequenced based on PCR amplification in all available family members. An additional 46 probands from families or sporadic cases with the pathogenic variants of HPS6 reported in the literature were reviewed. Results: We identified two different compound heterozygous truncating variants of HPS6 in probands with suspected OA from two independent families. The proband of family JX had c.1674dup and c.503-504del variants, and the other proband from family CZ had a nonsense variant of c.1114C>T and a frameshift variant of c.1556del. Among them, c.1674dup and c.1556del variants in HPS6 have not been reported previously. Therefore, our patients were diagnosed as HPS6 disease by molecular diagnostics. In the retrospective cohort of HPS6 patients, we delineated the profile of HPS6 variants and revealed a significant overlap between CpG islands and the variants of HPS6, suggesting a potential link between DNA methylation and HPS6 variants. We also observed a spatial aggregation of the variants in 3D structure of HPS6 protein, implying the possible functional significance of these structural regions. In addition, we did not find any significant genotype-phenotype correlation of HPS6, and neither did we observe a correlation between the truncation length of the HPS6 protein and the phenotype of HPS6 disease. Conclusion: Our research expands the spectrum of HPS6 variants, providing a comprehensive delineation of their profile and systematically investigating genotype-phenotype correlations in HPS6. These findings could offer potentially valuable clues for investigating the molecular mechanism underlying HPS6 pathogenesis, as well as aiding the clinical diagnosis of HPS6 patients and improving disease prognosis. [ABSTRACT FROM AUTHOR]

Clonal hematopoiesis of indeterminate potential (CHIP) has fascinated the medical community for some time. Discovered about a decade ago, this phenomenon links age-related alterations in hematopoiesis not only to the later development of hematological malignancies but also to an increased risk of early-onset cardiovascular disease and some other disorders. CHIP is detected in the blood and is characterized by clonally expanded somatic mutations in cancer-associated genes, predisposing to the development of hematologic neoplasms such as MDS and AML. CHIP-associated mutations often involve DNA damage repair genes and are frequently observed following prior cytotoxic cancer therapy. Genetic predisposition seems to be a contributing factor. It came as a surprise that CHIP significantly elevates the risk of myocardial infarction and stroke, and also contributes to heart failure and pulmonary hypertension. Meanwhile, evidence of mutant clonal macrophages in vessel walls and organ parenchyma helps to explain the pathophysiology. Besides aging, there are some risk factors promoting the appearance of CHIP, such as smoking, chronic inflammation, chronic sleep deprivation, and high birth weight. This article describes fundamental aspects of CHIP and explains its association with hematologic malignancies, cardiovascular disorders, and other medical conditions, while also exploring potential progress in the clinical management of affected individuals. While it is important to diagnose conditions that can lead to adverse, but potentially preventable, effects, it is equally important not to stress patients by confronting them with disconcerting findings that cannot be remedied. Individuals with diagnosed or suspected CHIP should receive counseling in a specialized outpatient clinic, where professionals from relevant medical specialties may help them to avoid the development of CHIP-related health problems. Unfortunately, useful treatments and clinical guidelines for managing CHIP are still largely lacking. However, there are some promising approaches regarding the management of cardiovascular disease risk. In the future, strategies aimed at restoration of gene function or inhibition of inflammatory mediators may become an option. [ABSTRACT FROM AUTHOR]

BackgroundExportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood.MethodsWe performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor).ResultsWe report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL.ConclusionsThese findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.

Abstract Background Mal de Meleda is an autosomal recessive palmoplantar keratoderma, with SLURP1 identified as the pathogenic gene responsible. Although over 20 mutations in SLURP1 have been reported, only the mutation c.256G > A (p.G87R) has been detected in Chinese patients. Here, we report a novel heterozygous SLURP1 mutation in a Chinese family. Methods We assessed the clinical manifestations of two Chinese patients with Mal de Meleda and collected specimens from the patients and other family members for whole-exome and Sanger sequencing. We used algorithms (MutationTaster, SIFT, PolyPhen-2, PROVEAN, PANTHER, FATHMM, mCSM, SDM and DUET) to predict the pathogenetic potential of the mutation detected. We also employed AlphaFold2 and PyMOL for protein structure analysis. Results Both patients displayed the typical manifestation of palmoplantar keratoderma. In Proband 1, we detected a novel compound heterozygous mutation (c.243C > A and c.256G > A) in exon 3 of SLURP1. Proband 2 was an adult female born to a consanguineous family and carried a homozygous mutation (c.211C > T). Algorithms indicated both mutations to be probably disease causing. We used AlphaFold2 to predict the protein structure of these mutations and found that they cause instability, as shown by PyMOL. Conclusions Our study identified a novel compound heterozygous mutation (c.243C > A and c.256G > A) in a Chinese patient with Mal de Meleda that has the potential to cause instability in protein structure. Moreover, this study expands on the existing knowledge of SLURP1 mutations and contributes to knowledge of Mal de Meleda.

Background: This study compares the characteristics, referral and treatment patterns and overall survival (OS) of gastrointestinal stromal tumor (GIST) patients treated in reference and non-reference centers in the Netherlands. Patients and methods: This retrospective cohort study on patients diagnosed between 2016 and 2019, utilises data from the Netherlands Cancer Registry and the Dutch Nationwide Pathology Database. Patients were categorized into two groups: patients diagnosed in or referred to reference centers and patients diagnosed in non-reference centers without referral. Results: This study included 1,550 GIST patients with a median age of 67.0 in reference and 68.0 years in non-reference centers. Eighty-seven per cent of patients were diagnosed in non-reference centers, of which 36.5% (493/1,352) were referred to a reference center. Referral rates were higher for high-risk (62.2% [74/119]) and metastatic patients (67.2% [90/134]). Mutation analysis was performed in 96.9% and 87.6% of these cases in reference and in non-reference centers (p < 0.01), respectively. Systemic therapy was given in reference centers versus non-reference in 89.5% versus 82.0% (p < 0.01) of high-risk and in 94.1% versus 65.9% (p < 0.01) of metastatic patients, respectively. The proportion of positive resection margins and tumor rupture did not differ between reference and non-reference centers. Median OS was not reached. Conclusion: A substantial amount of metastatic GIST patients in non-reference centers did not receive systemic treatment. This might be due to valid reasons. However, optimisation of the referral strategy of GIST patients in the Netherlands could benefit patients. Further research is needed to explore reasons for not starting systemic treatment in metastatic GIST patients.

Introduction: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor therapy is effective as a first-line treatment of advanced non-small-cell lung cancer (NSCLC). This research study investigated the distribution of EGFR mutations in patients diagnosed with NSCLC to assist in selecting patients who could benefit from tyrosine kinase inhibitor therapy. Materials and Methods: This cross-sectional study was conducted between July 2017 and November 2022. A real-time multiplex ​Polymerase Chain Reaction (PCR) assay supplied by Roche Diagnostics was used to examine DNA obtained from 682 tumor biopsies collected from NSCLC patients. DNA amplification was performed in a Cobas z 480 instrument for mutation analysis. The PCR assay was designed using specific primers and probes to detect 43 different mutations targeting exons 18-21. Results: Among the 682 samples, 466 (68.3%) were males, and 216 were females. The male-to-female ratio was 2.1. Twenty per cent of the male and 37% of the female samples were positive for EGFR mutations. The most common mutations were the in-frame deletion of exon 19, followed by L858R in exon 21, exon 20 insertion and S769I, exon 18 G719X. In addition, three mutations, namely del exon 19, T790M and exon 20 insertions were also detected in a patient, suggesting an actively progressive disease. Conclusions: This study showed that EGFR mutations are more common in Pakistani female patients than males. Secondly, in-frame deletion of exon 19 and exon 21 mutation L858R are prevalent in most of the NSCLC patients. The prevalence of common and rare EGFR mutations in Pakistani patients provides an opportunity for a subset of patients' chance of therapy.

Clonal hematopoiesis of indeterminate potential (CHIP) has fascinated the medical community for some time. Discovered about a decade ago, this phenomenon links age-related alterations in hematopoiesis not only to the later development of hematological malignancies but also to an increased risk of early-onset cardiovascular disease and some other disorders. CHIP is detected in the blood and is characterized by clonally expanded somatic mutations in cancer-associated genes, predisposing to the development of hematologic neoplasms such as MDS and AML. CHIP-associated mutations often involve DNA damage repair genes and are frequently observed following prior cytotoxic cancer therapy. Genetic predisposition seems to be a contributing factor. It came as a surprise that CHIP significantly elevates the risk of myocardial infarction and stroke, and also contributes to heart failure and pulmonary hypertension. Meanwhile, evidence of mutant clonal macrophages in vessel walls and organ parenchyma helps to explain the pathophysiology. Besides aging, there are some risk factors promoting the appearance of CHIP, such as smoking, chronic inflammation, chronic sleep deprivation, and high birth weight. This article describes fundamental aspects of CHIP and explains its association with hematologic malignancies, cardiovascular disorders, and other medical conditions, while also exploring potential progress in the clinical management of affected individuals. While it is important to diagnose conditions that can lead to adverse, but potentially preventable, effects, it is equally important not to stress patients by confronting them with disconcerting findings that cannot be remedied. Individuals with diagnosed or suspected CHIP should receive counseling in a specialized outpatient clinic, where professionals from relevant medical specialties may help them to avoid the development of CHIP-related health problems. Unfortunately, useful treatments and clinical guidelines for managing CHIP are still largely lacking. However, there are some promising approaches regarding the management of cardiovascular disease risk. In the future, strategies aimed at restoration of gene function or inhibition of inflammatory mediators may become an option.

Background: Early detection and diagnosis are important crucial to prevent life-threatening acute attacks in patients with acute intermittent porphyria (AIP). We aim to provide comprehensive data on the clinical and hydroxymethylbilane synthase (HMBS) gene variant characteristics and genotype-phenotype association of Chinese patients with AIP in order to improve clinicians’ knowledge of AIP and reduce misdiagnosis and mistaken treatment.Methods: We searched the literature on Chinese patients with AIP in PubMed, Web of Science, Wiley Online Library, ScienceDirect and Chinese literature databases up to August 2023 in our analysis to explore the clinical and HMBS gene variant characteristics of Chinese patients with AIP.Results: A total of 41 original articles associated with Chinese AIP patients were included for analysis: 97 variants were detected in 160 unrelated families, including 35 missense, 29 frameshift, 24 splicing and 9 nonsense variants, with c.517C>T being the most common variant. Clinical data were reported in 77 of 160 patients: Most of them were female (67/77) and the age was 28.8 ± 9.9 years. The most common symptom was abdominal pain (73/77, 94.8%), followed by central nervous system symptoms (45/77, 58.4%). 13.0% (10/77) of patients experienced psychiatric symptoms. Hyponatremia was the most common electrolyte abnormality (42/77). 31 patients received carbohydrate loading therapy, and 30 of them were improved. 6 patients were treated with carbohydrate loading combined with hemin therapy and 5 eventually improved. All variants causing premature stop codons, frameshifts or enzyme activity center may experience more severe clinical phenotypes such as seizures, respiratory paralysis, intracranial hemorrhage disorder or respiratory failure.Conclusion: The most common presenting symptom in Chinese AIP patients was abdominal pain, followed by central nervous system symptoms. The HMBS gene analysis in Chinese AIP patients revealed that the heterogeneity is strong and the most common variant was missense mutation, with c.517C>T being the most common variant. The genotype-phenotype association helps guide clinical diagnosis and treatment. However, the treatment for AIP in China is limited and monolithic, and more attention needs to be paid to the treatment.

Paget's disease of bone (PDB) is a common, late-onset bone disorder, characterized by focal increases of bone turnover that can result in bone lesions. Heterozygous pathogenic variants in the Sequestosome 1 (SQSTM1) gene are found to be the main genetic cause of PDB. More recently, PFN1 and ZNF687 have been identified as causal genes in patients with a severe, early-onset, polyostotic form of PDB, and an increased likelihood to develop giant cell tumors. In our study, we screened the coding regions of PFN1 and ZNF687 in a Belgian PDB cohort (n = 188). In the PFN1 gene, no variants could be identified, supporting the observation that variants in this gene are extremely rare in PDB. However, we identified 3 non-synonymous coding variants in ZNF687. Interestingly, two of these rare variants (p.Pro937His and p.Arg939Cys) were clustering in the nuclear localization signal of the encoded ZNF687 protein, also harboring the p.Pro937Arg variant, a previously reported disease-causing variant. In conclusion, our findings support the involvement of genetic variation in ZNF687 in the pathogenesis of classical PDB, thereby expanding its mutational spectrum. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) is an extremely widespread pathogen characterized by recurrent infections. HSV-1 most commonly causes painful blisters or sores around the mouth or on the genitals, but it can also cause keratitis or, rarely, encephalitis. First-line and second-line antiviral drugs used to treat HSV infections, acyclovir and related compounds, as well as foscarnet and cidofovir, selectively inhibit herpesvirus DNA polymerase (DNA-pol). It has been previously found that (S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (compound 1) exhibits selective anti-herpesvirus activity against HSV-1 in cell culture, including acyclovir-resistant mutants, so we consider it as a lead compound. In this work, the selection of HSV-1 clones resistant to the lead compound was carried out. High-throughput sequencing of resistant clones and reference HSV-1/L2 parent strain was performed to identify the genetic determinants of the virus's resistance to the lead compound. We identified a candidate mutation presumably associated with resistance to the virus, namely the T321I mutation in the UL15 gene encoding the large terminase subunit. Molecular modeling was used to evaluate the affinity and dynamics of the lead compound binding to the putative terminase binding site. The results obtained suggest that the lead compound, by binding to pUL15, affects the terminase complex. pUL15, which is directly involved in the processing and packaging of viral DNA, is one of the crucial components of the HSV terminase complex. The loss of its functional activity leads to disruption of the formation of mature virions, so it represents a promising drug target. The discovery of anti-herpesvirus agents that affect biotargets other than DNA polymerase will expand our possibilities of targeting HSV infections, including those resistant to baseline drugs. [ABSTRACT FROM AUTHOR]

Mutations in the 43 kDa transactive-response (TAR)-DNA-binding protein (TARDBP) are associated with 2–5% of familial Amyotrophic Lateral Sclerosis (ALS) cases. TAR DNA-Binding Protein 43 (TDP-43) is an RNA/DNA-binding protein involved in several cellular mechanisms (e.g., transcription, pre-mRNA processing, and splicing). Many ALS-linked TARDBP mutations have been described in the literature, but few phenotypic data on monogenic TARDBP-mutated ALS are available. In this paper, (1) we describe the clinical features of ALS patients carrying mutations in the TARDBP gene evaluated at the Tertiary ALS Center at Maggiore della Carità University Hospital, Novara, Italy, from 2010 to 2020 and (2) present the results of our review of the literature on this topic, analyzing data obtained for 267 patients and highlighting their main clinical and demographic features. [ABSTRACT FROM AUTHOR]

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The Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS‐CoV‐2) is the virus responsible for the COVID‐19 pandemic. COVID‐19 continues to cause millions of deaths globally in part due to immune‐evading mutations. SARS‐CoV‐2 main protease (Mpro) is an important enzyme for viral replication and potentially an effective drug target. Mutations affect the dynamics of enzymes and thereby their activity and ability to bind ligands. Here, we use kinematic flexibility analysis (KFA) to identify how mutations and ligand binding changes the conformational flexibility of Mpro. KFA decomposes macromolecules into regions of different flexibility near‐instantly from a static structure, allowing conformational dynamics analysis at scale. Altogether, we analyzed 47 mutation sites across 69 Mpro–ligand complexes resulting in more than 3300 different structures which includes 69 mutated structures with all 47 sites mutated simultaneously and 3243 single residue mutated structures. We found that mutations generally increased the conformational flexibility of the protein. Understanding the impact of mutations on the flexibility of Mpro is essential for identifying potential drug targets in the treatment of SARS‐CoV‐2. Further studies in this area can offer valuable insights into the mechanisms of molecular recognition. [ABSTRACT FROM AUTHOR]

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss‐of‐function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss‐of‐function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N‐terminal mutations in other disease‐associated genes should generally be taken into consideration in the variant interpretation process. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing, with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuing to evolve and accumulate mutations. While various bioinformatics tools have been developed for SARS-CoV-2, a well-curated mutation-tracking database integrated with in silico evaluation for molecular diagnostic assays is currently unavailable. To address this, we introduce CovidShiny, a web tool that integrates mutation profiling, in silico evaluation, and data download capabilities for genomic sequence-based SARS-CoV-2 assays and data download. It offers a feasible framework for surveilling the mutation of SARS-CoV-2 and evaluating the coverage of the molecular diagnostic assay for SARS-CoV-2. With CovidShiny, we examined the dynamic mutation pattern of SARS-CoV-2 and evaluated the coverage of commonly used assays on a large scale. Based on our in silico analysis, we stress the importance of using multiple target molecular diagnostic assays for SARS-CoV-2 to avoid potential false-negative results caused by viral mutations. Overall, CovidShiny is a valuable tool for SARS-CoV-2 mutation surveillance and in silico assay design and evaluation. [ABSTRACT FROM AUTHOR]

The heterogeneity of Myelodysplastic Neoplasm (MDS) extends beyond mutational diversity to include significant ethnic variability, a factor that has been underexplored. While the development of the IPSS‐M prognostic tool has advanced our understanding of MDS, its reliance on data primarily from European cohorts limits its applicability to non‐European populations. Duployez et al.'s review highlighted the importance of molecular markers in MDS for personalized treatment and disease monitoring yet did not address the impact of genetic ancestry. This commentary critiques the IPSS‐M's limited sample of 110 Brazilian patients, questioning its adequacy in reflecting the influence of patient ancestry on prognostic accuracy. Given the potential for differing mutation profiles and prognostic implications across diverse ethnic groups, robust genomic ancestry studies are urgently needed. These studies should stratify MDS patients by ethnic background to investigate mutation incidence and impacts, thereby validating IPSS‐M and potentially identifying new prognostic markers. Incorporating ethnic diversity into prognostic models is essential for ensuring they are truly universal and inclusive, thereby improving personalized treatment and care for all MDS patients. Commentary on: Duployez and Preudhomme. Monitoring molecular changes in the management of myelodysplastic syndromes. Br J Haematol 2024; 205:772‐779. [ABSTRACT FROM AUTHOR]

Background Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

Background Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy. Conclusion This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.

Purpose: The genetic characteristics and mismatch repair (MMR) status of the primary tumor and corresponding metastases in colorectal cancer (CRC) are generally considered to be highly concordant. This implies that either the primary or metastatic tumor can be used for testing gene mutation and MMR status. However, whether this is also true for CRC and their ovarian metastases is currently unknown. Ovarian metastases generally show a poorer response to systemic therapy compared to other metastatic sites. Differences in biomarker status between primary CRC and ovarian metastases could possibly explain this difference in therapy response. Methods: The study cohort was selected from CRC patients treated in two Dutch hospitals. Eligible patients with CRC and ovarian metastasis who were surgically treated between 2011 and 2018 were included. CRC and corresponding ovarian metastatic tissues were paired. Gene mutation status was established using next-generation sequencing, while the MMR status was established using either immunohistochemistry or microsatellite instability analysis. Results: Matched samples of CRC and ovarian metastasis from 26 patients were available for analysis. A biomarker concordance of 100% was detected. Conclusion: Complete biomarker concordance was found between MMR proficient CRC and their matching ovarian metastasis. Biomarker testing of MMR proficient CRC tissue appears to be sufficient, and additional testing of metastatic ovarian tissue is not necessary. Differences in therapy response between ovarian metastases and other metastases from CRC are thus unlikely to be caused by differences in the genetic status. [ABSTRACT FROM AUTHOR]

A measure of the "goodness" or efficiency of the test suite is used to determine the proficiency of a test suite. The appropriateness of the test suite is determined through mutation analysis. Several Finite State Machine (FSM) mutants are produced in mutation analysis by injecting errors against hypotheses. These mutants serve as test subjects for the test suite (TS). The effectiveness of the test suite is proportional to the number of eliminated mutants. The most effective test suite is the one that removes the most significant number of mutants at the optimal time. It is difficult to determine the fault detection ratio of the system. Because it is difficult to identify the system's potential flaws precisely. In mutation testing, the Fault Detection Ratio (FDR) metric is currently used to express the adequacy of a test suite. However, there are some issues with this metric. If both test suites have the same defect detection rate, the smaller of the two tests is preferred. The test case (TC) is affected by the same issue. The smaller two test cases with identical performance are assumed to have superior performance. Another difficulty involves time. The performance of numerous vehicles claiming to have a perfect mutant capture time is problematic. Our study developed three metrics to address these issues: FDR/|TS|, FDR/|TC|, and FDR/|Time| generation tools were examined and tested using the developed metrics. Thanks to the metrics we have developed, the research contributes to eliminating the problems related to performance measurement by integrating the missing parameters into the system. [ABSTRACT FROM AUTHOR]

Background: Mal de Meleda is an autosomal recessive palmoplantar keratoderma, with SLURP1 identified as the pathogenic gene responsible. Although over 20 mutations in SLURP1 have been reported, only the mutation c.256G > A (p.G87R) has been detected in Chinese patients. Here, we report a novel heterozygous SLURP1 mutation in a Chinese family. Methods: We assessed the clinical manifestations of two Chinese patients with Mal de Meleda and collected specimens from the patients and other family members for whole-exome and Sanger sequencing. We used algorithms (MutationTaster, SIFT, PolyPhen-2, PROVEAN, PANTHER, FATHMM, mCSM, SDM and DUET) to predict the pathogenetic potential of the mutation detected. We also employed AlphaFold2 and PyMOL for protein structure analysis. Results: Both patients displayed the typical manifestation of palmoplantar keratoderma. In Proband 1, we detected a novel compound heterozygous mutation (c.243C > A and c.256G > A) in exon 3 of SLURP1. Proband 2 was an adult female born to a consanguineous family and carried a homozygous mutation (c.211C > T). Algorithms indicated both mutations to be probably disease causing. We used AlphaFold2 to predict the protein structure of these mutations and found that they cause instability, as shown by PyMOL. Conclusions: Our study identified a novel compound heterozygous mutation (c.243C > A and c.256G > A) in a Chinese patient with Mal de Meleda that has the potential to cause instability in protein structure. Moreover, this study expands on the existing knowledge of SLURP1 mutations and contributes to knowledge of Mal de Meleda. [ABSTRACT FROM AUTHOR]

Executable Domain-Specific Languages (xDSLs) allow the definition and the execution of behavioral models. Some behavioral models are reactive, meaning that during their execution, they accept external events and react by exposing events to the external environment. Since complex interaction may occur between the reactive model and the external environment, they should be tested as early as possible to ensure the correctness of their behavior. In this paper, we propose a set of generic testing facilities for reactive xDSLs using the standardized Test Description Language (TDL). Given a reactive xDSL, we generate a TDL library enabling the domain experts to write and run event-driven TDL test cases for conforming reactive models. To further support the domain expert, the approach integrates interactive debugging to help in localizing defects, and mutation analysis to measure the quality of test cases. We evaluate the level of genericity of the approach by successfully writing, executing, and analyzing 247 event-driven TDL test cases for 70 models conforming to two different reactive xDSLs. [ABSTRACT FROM AUTHOR]