Types Of Mutations Research Articles

Myopathy with Lactic Acidosis and Sideroblastic Anemia Caused By a Novel PUS1mutation

Mutations in the mitochondrial aminoacyl tRNA synthetases (ARSs) are associated with various clinical phenotypes, including myopathy, lactic acidosis, and sideroblastic anemia (MLASA), which is a rare autosomal recessive disorder. Due to oxidative phosphorylation disorder and abnormal iron metabolism in the skeletal muscle and erythroid bone marrow lines, patients show progressive muscle weakness, exercise intolerance, lactic acidosis, siderocyte anemia, and growth retardation, etc. Herein, we report a case of MLASA caused by a novel homozygous missense mutation (c.597G>T p.R199S) in the pseudouridine synthase 1 ( PUS1) gene, causing the loss of protein function. The patient's parents had a consanguineous marriage, with a grandmother in common. All mutations were heterozygous without disease. While the patient was homozygous for the mutation and developed symptoms of anemia, no specific treatment exists for this genetic disease. Deferrization therapy can reduce iron overload, which leads to reduced heme synthesis in such patients but cannot treat the disease. Consanguineous marriage often causes autosomal recessive genetic diseases and prohibiting it can reduce the incidence of these diseases. The present patient's condition was caused by a mutation in nuclear PUS1, which manifested as SA, hyperlactatemia, iron overload, and muscle damage. PUS1 mutations can be missense, nonsense, or frameshift. We identified a novel missense mutation type in PUS1 in this patient, located on chromosome 12, c.597G>T (p.R199S), which is a homozygous mutation. This was confirmed by the PUS1 analysis revealing homozygosity for the novel mutation. Both the parents and grandmothers were heterozygous for the mutation, but none of them had a clinical manifestation. Combining the patient's family history, medical history, laboratory examination, and genetic characteristics of the disease, the mutation was identified as a pathogenic variant, and the clinical manifestations and genetic findings of the patient expanded the genotype-phenotype spectrum of MLASA. The possibility of MLASA should be suspected in cases of progressive muscle weakness, growth retardation, exercise intolerance in childhood, SA, and lactic acidosis before or after puberty. With the recognition of MLASA and the advent of NGS, identifying patients with PUS1 mutations characterizing incomplete MLASA syndrome became easier, thus further expanding the phenotypic and genotypic heterogeneities of these mitochondrial disorders. Targeting NGS may also significantly improve the prenatal and preimplantation genetic diagnosis of MLASA, providing better genetic counseling for parents. Genetic diseases lack effective treatment measures, and the curative effect is unsatisfactory. Therefore, prevention is more important. Prevention strategies include avoiding consanguineous marriage, genetic counseling, genetic testing for carriers, and a prenatal diagnosis.

Mutation Clearance of Driver Mutations after Transplantation for Myelofibrosis Predicts Outcome

Myelofibrosis is one of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) characterized by clonal myeloproliferation. Allogeneic hematopoietic stem-cell transplantation is the only potentially curative treatment for patients with myelofibrosis. With that, it can resolve fibrosis and lead to complete loss of the clonal burden. More than 90% of myelofibrosis patients harbor driver mutations in one of the 3 genes JAK2, CALR, and MPL, with JAK2 mutations being associated with worse outcomes. We herein report a study to determine whether the persistence of cells with MPN-associated driver mutations in the early period after allogeneic hematopoietic stem-cell transplantation was associated with outcomes. Patient samples for molecular analysis were prospectively collected from peripheral blood at 5 different time points: at start of conditioning prior to stem cell infusion, at days 30, 100, 180, and 1 year after transplantation. We applied quantitative PCR technology to detect individual driver mutations in JAK2, CALR, and MPL (with a high sensitivity of 0.01%). Simulation was applied to explain variances at different time points. Multivariable modelling was employed to determine independent effects. A total of 312 patients receiving first reduced-intensity conditioning transplantation were included. Distribution of driver mutation genotype at time of transplantation was as follows: 67% with JAK2, 19% with CALR, 4% with MPL, and 8% were triple-negative. Conditioning regimen comprised busulfan and fludarabine. Donor platform for transplantation was: matched related (18%), matched unrelated (58%), haploidentical (1%), and mismatched unrelated (23%). Disease risks at time of transplantation were low (1%), intermediate-1 (22%), intermediate-2 (55%), and high (22%). Median follow-up of the total cohort was 6 years. The median allele burden at time of transplantation was significantly (P<0.001) different between driver mutations: 20% (range, 0.1-100%) for JAK2, 44% (range, 3-54%) for CALR, 60% (range, 43-99%) for MPL. CALR and MPL showed earlier mutation clearance after transplantation compared with JAK2 ( Table). Mutation clearance was achieved at day 30 after transplantation in 46% with JAK2, 67% for CALR, and 30% for MPL (P=0.02). At day 100 after transplantation, 66% of JAK2, 75% of CALR, and 100% of MPL showed complete mutation clearance (P=0.09). Complete mutation clearance at day 30 after transplantation was predictive of post-transplantation relapse at 1 year (P=0.009), showing cumulative incidence of relapse of 6% versus 16%. Similar results were seen for complete mutation clearance at day 100 after transplantation, demonstrating cumulative incidence of relapse of 4% versus 24% (P<0.001). Mutation clearance at day 30 and 100 after transplantation explained 80% and 87% of variance of later time points. The effect of mutation clearance on post-transplant relapse was irrespective of driver mutation status ( Figure). 6-year disease-free survival according to mutation type was 52% for JAK2, 63% for CALR, and 80% for MPL (P=0.045). Mutation clearance at day 30 after transplantation was associated with better disease-free survival, irrespective of driver mutation status ( Figure). Moreover, day-30 complete mutation clearance predicted disease-free survival in JAK2, showing rates of 60% for negative versus 47% in JAK2 positive patients (P=0.05). Importantly, achievement of mutation clearance at day 30 after transplantation could also distinguish different risk groups in patients with onset better prognosis harboring CALR/MPL mutations. The 6-year disease-free survival was 76% for patients with mutation clearance versus 59% for CALR/MPL-positive patients(P=0.02) . Multivariate analysis (adjusting for donor type, diagnosis of primary or secondary myelofibrosis, allele burden at time of transplantation, and age) confirmed that patients who had mutation clearance at day 30 after transplantation had lower risk of relapse, better disease-free and overall survival. In conclusion, this is the first study to show that CALR and MPL are associated with earlier mutation clearance after transplantation compared with JAK2. Mutation clearance at day 30 and 100 after transplantation was the best predictor of relapse and disease-free survival, independent of driver mutation status.

Clinical Characteristics of Shwachman-Diamond Syndrome in China - Result from Childhood Bone Marrow Failure Diseases Register of China Alliance for Blood Diseases(cBMFR-CABD)

OBJECTIVE This study aimed to identify the clinical features of 73 children diagnosed with SDS from 22 centres in China. METHODS Childhood Bone Marrow Failure Diseases Register of China Alliance for Blood Diseases (cBMFR-CABD) was a bidirectional multicenter cohort study that collected data from 22 paediatric blood disease treatment centres from all over China. Patients were enrolled in the sub-centres from 1.1.2011 that were aged < 18 years at diagnosis and met the diagnostic criteria for an inherited bone marrow failure disorder. The clinical data for the 73 SDS patients analyzed in this study were collected between 1.1.2011 and 31.12.2022. RESULTS Among the 73 SDS children, 38 were male and 35 were female. Short stature (height<-2SD) was found in 53.4 % (39/73) patients. Chronic diarrhea was observed in 24.7 % (18/73) patients. In children evaluated for intellectual and physical malformations, 7 patients (17.3 %,6/52) had mental retardation and 14 (20.5 %, 15/73) patients had physical deformities. Genetic testing was available in 63 patients. seventeen (27.0 %) patients carry bi-allelic c.258+2T>C and c.183-184TA>CT SBDS mutations. Eleven patients (17.5%) are homozygous for the c.258+2T>C mutation. Four patients (6.3%) were compound heterozygous for c.258+2T>C and exon 2 deletions. One patient carries bi-allelic mutation of DNAJC21. This patient had a history of repeated infections and pancytopenia.Bone marrow smear shows multilineage dysplasia. Bone marrow results were available for evaluation in 54 patients including 67 % (36/54) BMF, 30 %(16/54) MDS, 4 % (2/54) AML.TP53 mutation testing was available in 15 patinets with eight (53.3%) patients carry TP53 mutation.Karyotype analysis was available in 36 patinets with 5 patients were complex karyotype. Among the 56 patients available for follow-up, 17 patients underwent haematopoietic stem cell transplantation, 5 patients died (one infection,three transplant-related death and one relapse). Among the 39 non-HSCT patients, 3 patients died of AML transformation. Three-year overall survival of all patients were 91.7%(95%CI 99.5 %~ 83.9% ), BMF patients were 100.0%(95%CI 100.0%~100.0% ), MDS/AML patients were 67.7%(95%CI 94.0%~ 41.4% ). CONCLUSION Our results provide the clinical features of the largest SDS cohort in China. A high incidence of short stature was observed. Bi-allelic c.258+2T>C with c. 183-184 TA > CT SBDS mutations were the most common mutation type. HSCT is an effective treatment modality in SDS. However, AML transformation is still a major risk factor for children with SDS.

Application of cfDNA-NGS Test in MRD Monitoring of Acute Leukemia Patients with CAR-T Cell Therapy

Background: The minimal residual disease (MRD) plays an important role in the comprehensive management of CAR-T cell therapy. However, some reasons hinder the application of MRD monitoring in CAR-T cell therapy. Dynamic monitoring of cfDNA in solid tumors and hematological malignancies can effectively evaluate the efficacy of treatment and predict long-term prognosis. However, there is no relevant researches on whether cfDNA can replace bone marrow for MRD monitoring after CAR-T cell therapy for acute leukemia. Methods: 23 patients with acute leukemia who received CAR-T cell therapy from January 2021 to October 2022 in the First Affiliated Hospital of Suzhou University were collected retrospectively, including 14 AML patients and 9 ALL patients. The CAR-T cell targets included CD38, CD7, CLL-1 and CD19/CD22. Bone marrow and peripheral blood samples were collected before CAR-T cell therapy, +28d and every month after CAR-T cell therapy. We used QIAamp Circulating Nucleic Acid Kit to extract cfDNA, and then conducted high-throughput sequencing through Nextseq550 sequencing platform. We compared the test results of cfDNA-NGS and BM-NGS in terms of gene mutation types and variant allele frequency (VAF), and analyzed the efficacy of cfDNA-NGS, BM-NGS and FCM on the prediction of remission and replase of CAR-T cell therapy by the receiver operating characteristic curve (ROC curve). Results: In this study, a total of 123 peripheral blood samples were collected and cfDNA was extracted from all samples. The results of 121 cfDNA-NGS and BM-NGS test were consistent, with a consistency rate of 98.4% (121/123), and the consistency rate with bone marrow FCM detection was 85.4% (105/123). cfDNA-NGS test was a good indicator for predicting the efficacy and replase after CAR-T cell therapy: compared with BM-NGS and FCM, cfDNA-NGS test before CAR-T cell therapy had the highest prediction efficiency for CR (AUC=0.767, 95%CI: 0.576-0.958, P=0.039). Survival analysis showed no difference in 1-year OS and LFS between cfDNA-NGS (+), BM-NGS (+) , FCM (+) patients at +28 days after CAR-T cell therapy (OS: 27.3% vs 33.3% vs 30%, P=0.949; LFS: 27.3% vs 33.3% vs 30%, P=0.970). At +28 days after CAR T treatment, there was no difference in 1-year OS and LFS between patients with cfDNA-NGS (-), BM-NGS (-), FCM (-) (OS: 91.7% vs 76.2% vs 83.3%, P=0.671; LFS: 73.3% vs 76.2% vs 82.1%, P=0.726). Then we assessed the long-term prognosis by cfDNA-NGS combined with traditional MRD detection methods after CAR-T cell therapy: Survival analysis showed that 1-year OS and LFS of patients with cfDNA (-), BM-MRD (-) and cfDNA1 (-), BM-MRD (+) were significantly better than those with cfDNA (+), BM-MRD (-) and cfDNA1 (+), BM-MRD (+) (OS: 90.9% vs 100% vs 50% vs 22.2%, P=0.008: LFS：68.2% vs 100% vs 50% vs 22.2%, P=0.017). Conclusions: The cfDNA-NGS test was a good indicator for predicting the efficacy and replase after CAR-T cell therapy, and was not inferior to BM-NGS and FCM detection in evaluating efficacy, replase and long-term prognosis. Combination cfDNA-NGS test with traditional MRD detection methods could evaluate the long-term prognosis of CAR-T cell therapy more comprehensively.

Can Emergency Hematopoiesis Due to Sepsis Lead to Clonal Hematopoiesis? Implications for Understanding the Pathogenesis of Clonal Evolution

O.O & T.K are co-first authors. Clonal (myelo) hematopoiesis of indeterminate potential (CHIP) arises from somatic mutations in myeloid genes, providing a proliferative and survival advantage to the affected “CHIP-initiating” early hematopoietic precursor cells. Understanding the conditions that lead to the emergence of detectable clones (early seeding of myeloid neoplasia-MN) is of great interest due to its clinical implications. Among CH facilitating states, aging has been extensively studied, but a clear distinction between the impact of chronological versus biological aging remains challenging. Additionally, other factors contributing to CHIP development may include chemotherapy, genetic predisposition, bone marrow failure states, or immunodeficiency. However, not all CHIP-promoting factors have been identified because they are subtle, pathophysiologically complex, and thus remain cryptic. We hypothesized that episodes of emergency hematopoiesis (EH), such as those caused by acute infections, could potentially result in acquisition of CHIP with repeated episodes of EH resulting in clonal expansion. If this hypothesis is confirmed, one could envision that both instances of significant hematopoietic expansion (high leukocytosis) and repeated cycles of EH could be at play in conjunction with chronological factors. For instance, the older an individual is, the greater is the number of EH episodes possible. To examine our theory, we prospectively collected blood samples from patients admitted at the intensive care unit (ICU) of Cleveland Clinic and Yale University. Inclusion criteria required a white blood cell (WBC) count ≥ 15K/uL. Patients with known or prior malignancies and those exposed to chemotherapy were excluded. For relevant comparisons, we assessed CH incidence in our cohort against a group of patients with solid tumors screened for CH (n=107) but without acute infections, along with historical CHIP cohorts. CH screening employed a targeted NGS myeloid gene panel with a reported VAF cut-off of 2%. A total of 78 individuals admitted to the ICU with possible infections and leukocytosis were enrolled in this study. The median age was 70 years (IQR: 61-77), with a M:F ratio of 1.7 (Table 1). Time from leukocytosis onset to sampling was 3 days (IQR: 1-5), the median WBC at ICU admission was 18 K/uL (IQR: 16-21), and the median absolute neutrophil count (ANC) was 16 K/uL (IQR: 12-20). Overall, 94 variants were identified including 16 (median VAF 50.1) variants of potentially germline origin, and 4 variants of unknown significance. We observed 74 CHIP-associated mutations in 37 (47%) patients at a median VAF of 14% (4-47%). The landscape of CHIP mutations included alterations in DNMT3A (20%), TET2 (12%), ASXL1 (12%), SMC1A, TP53, and PPM1D genes (6% each). Although higher rates of CHIP were expected in the cancer cohort, no difference was found between the two groups (40% vs. 47% in the ICU, p=0.3). DNMT3A, ASXL1 and PPM1D mutations were more frequently encountered in the cancer cohort (Table 1). The prevalence of CHIP in the ICU cohort was higher in older age groups: 30-39 (29%), 50-69 (49%), and ≥70 (51%), compared to the expected frequency in comparable age ranges (and median range) in a historical healthy population, which was 1%, 4%, and 10%, respectively (<0.00001; Fig.1). Notably, CHIP was more prevalent among smokers (57%; p=0.003). No significant differences were found when comparing CHIP + and CHIP - patients of the ICU cohort with regards to WBC (median 18K/uL both). Similarly, CHIP prevalence did not change according to the degree of leukocytosis in an analysis where patients were ranked by WBC (≥18K/uL at 44% vs. <18K/uL at 46%). When looking at the impact of CHIP on critical care outcomes, a logistic regression model accounting for age, gender, and mutation type revealed that the presence of DNMT3A mutations was associated with a higher risk of diagnosis of overt septic shock (log OR= 2.4, 95% CI 0.19-5.4, p<0.05). In summary, our prospective observational study has shown that sepsis is associated with preferential mutant clone expansion, primarily due to EH rather than the extent of leukocytosis and may be contributing to the acquisition of new clones. Whether such episodes result in permanent acquisition of new CHIP mutations with potential for MN progression will be the subject of longitudinal analysis of these samples, and findings will be presented at the ASH meeting.

Computational exploration of the significance of COPS6 in cancer: Functional and clinical relevance across tumor types.

The COP9 signalosome subunit 6 (COPS6) has been implicated in cancer progression, while its precise role in most types of cancer remains elusive. To investigate the functional and clinical relevance of COPS6 across various tumor types using publicly available databases. We used R software and online analysis databases to analyze the differential expression, prognosis, mutation and related functions of COPS6 in pan-cancer. Differential expression analysis and survival analysis demonstrated that COPS6 was highly expressed and associated with high-risk profiles in the majority of cancer types. Possible associations between COPS6 expression level and prognostic outcomes were found using data from public databases. Mutational analysis revealed that missense mutations were the predominant type of COPS6 mutation. Additionally, positive correlations were identified between COPS6 expression level and tumor mutational burden and microsatellite instability in most types of cancer. Immune infiltration analysis demonstrated a negative correlation between COPS6 expression level and CD8+ T cell infiltration in certain types of cancer. The correlation between COPS6 expression level and cancer-associated fibroblast infiltration exhibited heterogeneity, in which a positive correlation was found in head and neck squamous cell carcinoma and tenosynovial giant cell tumor, and a negative correlation was identified in diffuse large B-cell lymphoma and thymoma. The correlation between COPS6 expression level and macrophage infiltration was closely related to macrophage type. Gene co-expression and enrichment analysis highlighted transcription elongation factor B polypeptide 2 and G protein pathway suppressor 1 were significantly and positively associated with COPS6 expression level. These genes were predominantly involved in processes, such as ubiquitin-mediated proteolysis and human immunodeficiency virus 1 infection. In conclusion, this study systematically explored the significance of COPS6 across different tumor types, providing a solid foundation for considering COPS6 as a novel biomarker in cancer research.

Constructing gene similarity networks using co-occurrence probabilities

Gene similarity networks play important role in unraveling the intricate associations within diverse cancer types. Conventionally, gauging the similarity between genes has been approached through experimental methodologies involving chemical and molecular analyses, or through the lens of mathematical techniques. However, in our work, we have pioneered a distinctive mathematical framework, one rooted in the co-occurrence of attribute values and single point mutations, thereby establishing a novel approach for quantifying the dissimilarity or similarity among genes. Central to our approach is the recognition of mutations as key players in the evolutionary trajectory of cancer. Anchored in this understanding, our methodology hinges on the consideration of two categorical attributes: mutation type and nucleotide change. These attributes are pivotal, as they encapsulate the critical variations that can precipitate substantial changes in gene behavior and ultimately influence disease progression. Our study takes on the challenge of formulating similarity measures that are intrinsic to genes' categorical data. Taking into account the co-occurrence probability of attribute values within single point mutations, our innovative mathematical approach surpasses the boundaries of conventional methods. We thereby provide a robust and comprehensive means to assess gene similarity and take a significant step forward in refining the tools available for uncovering the subtle yet impactful associations within the complex realm of gene interactions in cancer.

Clinical and laboratory features of SF3B1-mutated myeloproliferative neoplasms

Objective: To explore the clinical and laboratory characteristics of SF3B1 gene mutations in myeloproliferative neoplasms (MPN) patients. Methods: The clinical data of 273 MPN patients who were diagnosed MPN and treated in the Second Hospital of Tianjin Medical University from November 2017 to March 2023 were retrospectively analyzed. There were 133 males and 140 females, with a median age M(Q1,Q3)of 56(46, 67) years. The molecular biology and cytogenetic characteristics were detected by second-generation sequencing (NGS) and R+G banding techniques, and the clinical and laboratory characteristics of patients with SF3B1 gene mutation were analyzed. Results: SF3B1 gene mutations were found in 13 patients (4.8%, 13/273).The types of SF3B1 mutations included missense (92.3%, 12/13) and nonsense mutations (7.7%, 1/13).Compared to the non-mutant cohort, patients in SF3B1 mutant cohort had older ages [68(51, 76) vs 56(45, 66)years,P=0.025], higher proportion of splenomegaly [46.2%(6/13) vs 15.8%(41/259),P=0.014]and secondary tumor [23.1%(3/13)vs 3.8%(10/260), P=0.018]with higher proportion of bone marrow blast [0.5%(0, 1.5%) vs 0(0, 0.5%),P=0.002] and lower hemoglobin[(104±36) vs (137±40) g/L,P=0.004] and hematocrit [31%(22%, 40%) vs 41%(35%, 52%),P=0.003]. All of the 10 patients in the SF3B1 mutant cohort whose ring sideroblast (RS) could be evaluated showed no RS formation. The overall survival, thrombosis-free survival and leukemia free survival of MPN patients in SF3B1 mutant cohort were 4.0 (2.0, 6.0), 2.0 (0.5, 4.5) and 4.0 (2.0, 6.0) years, respectively, while patients in the non-mutant cohort were 6.0 (3.0, 10.0), 5.0 (1.0, 8.0), 6.0 (3.0, 10.0) years, respectively, there were no statistical significance between two groups (Z=3.69, 1.66, 2.05, all P>0.05).The secondary tumor free survival of SF3B1 mutant cohort patients was 4.0 (2.0, 6.0) years, which was lower than that of non-mutant cohort patients [5.5 (3.0, 10.0) years, Z=18.18, P<0.001). Conclusions: MPN patients with SF3B1 gene mutations are older, more prone to splenomegaly and secondary tumors. They also have a higher proportion of bone marrow blast, lower hemoglobin and hematocrit, and show no RS formation.

Development and phenotypic screening of gamma rays and ethyl methanesulphonate mutant populations in tartary buckwheat (Fagopyrum tartaricum (L.) Gaertn)

Tartary buckwheat (F. tartaricum) is an annual self-pollinated pseudo-cereal crop of the Polygonaceae family which is grown worldwide for its nutritive and medicinal value. In order to increase genetic variability and develop genetic resources for crop improvement, gamma rays and EMS were used to develop mutagenized populations in F. tartaricum. In the present study, the biological effect of different doses and concentration of gamma rays and EMS on F. tartaricum to assess the sensitivity of mutagens to create genetic variability was determined. The results revealed that 219.15 Gy of gamma rays and 0.49 % of EMS were optimum for conducting mutagenesis in buckwheat. Both mutagens showed significant effects on the growth parameters in M1 generation. The mutagenic treatments were efficient in creating numerous types of chlorophyll mutations in M2 generation. The frequency of chlorophyll mutation was 1.67 % and 2.70 % for gamma rays and EMS, respectively. These findings shows that EMS is more efficient and effective at inducing variation than gamma rays. A diverse range of morphological mutants affecting every growth stage were screened with the highest frequency in 150 Gy gamma rays and 0.3 % of EMS treatment. . Six morphological mutant types influencing plant height, leaf morphology, growth habit, days to flowering, maturity period, seed yield and seed morphology emerged from induced phenotypic changes in individual plants of the M2 population were identified and selected. This study demonstrated the effectiveness of gamma rays and EMS in increasing genetic diversity and the successful execution of induced mutagenesis in the buckwheat. These mutants may be a valuable source of many beneficial genes in hybridization programs. It is anticipated that both these populations will be a valuable resource for functional genomics research in buckwheat.

Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.

Computational approach for assessing the involvement of SMYD2 protein in human cancers using TCGA data

BackgroundSMYD2 is a protein of the SET and MYND domain-containing family SMYD. It can methylate the lysine residue of various histone and nonhistone cancer-related proteins and plays a critical role in tumorigenesis. Although emerging evidence supports the association of SMYD2 in the progression of cancers, but its definitive effect is not yet clear. Therefore, further study of the gene in relation with cancer progression needs to be conducted. In the current study, investigators used TCGA data to determine the potential carcinogenic effect of SMYD2 in 11 cancer types. The transcriptional expression, survival rate, mutations, enriched pathways, and Gene Ontology of the SMYD2 were explored using different bioinformatics tools and servers. In addition, we also examined the correlation between SMYD2 gene expression and immunocyte infiltration in multiple cancer types. ResultsFindings revealed that higher expression of SMYD2 was significantly correlated with cancer incidents. In CESC and KIRC, the mRNA expression of SMYD2 was significantly correlated with overall survival (OS). In BRCA, KIRC, COAD, and HNSC, the mRNA expression of SMYD2 was significantly correlated with disease-free survival (DFS). We detected 15 missense, 4 truncating, 4 fusions, and 1 splice type of mutation. The expression of SMYD2 was significantly correlated with tumor purity and immunocyte infiltration in six cancer types. The gene GNPAT was highly associated with SMYD2. Significant pathways and Gene Ontology (GO) terms for co-expressed genes were associated to various processes linked with cancer formation. ConclusionCollectively, our data-driven results may provide reasonably comprehensive insights for understanding the carcinogenic effect of SMYD2. It suggests that SMYD2 might be used as a significant target for identifying new biomarkers for various human tumors.

Mutational screening of GDAP1 in dysphonia associated with Charcot-Marie-Tooth disease: clinical insights and phenotypic effects

IntroductionMutations in GDAP1 (Ganglioside-induced differentiation-associated protein 1) gene are linked to Charcot-Marie-Tooth disease (CMT), a Heterogenous group of disorders with multiple phenotypes, characterized by peripheral nerve dysfunction that can lead to vocal cord paralysis and diaphragmatic dysfunction. Main bodyAll three affected children of this chosen family have manifested the same clinical symptoms with progressive weakness, mild sensory impairment, and absent tendon reflexes in their early years. Electrodiagnostic analysis displayed an axonal type of neuropathy in affected patients. Sequencing of the GDAP1 gene was requested for all members of the family. Diagnostic assessments included pulmonary and vocal cord function tests, as well as phrenic and peripheral nerve conduction studies. Pathogenicity of GDAP1 variant p.Pro419Leu with axonal CMT2 and autosomal recessive inheritance was confirmed via in silico analysis. Patients with GDAP1 mutations showed dysphonia, speech difficulties, and the characteristic symptoms of CMT. The severity of symptoms correlated with the presence of a type of GDAP1 mutation. Patients with normal vocal cords and pulmonary function exhibited milder symptoms compared to those with GDAP1 mutations. Our study provides clinical insights into the phenotypic effects of GDAP1 mutations in CMT patients. The findings highlight the adverse clinical course and severe disability associated with GDAP1 mutations, including weak limb and laryngeal muscles. ConclusionPatients with GDAP1 mutations and autosomal recessive neuropathy present with dysphonia and require interventions such as surgery, braces, physical therapy, and exercise. Early diagnosis and comprehensive clinical evaluations are crucial for managing CMT patients with GDAP1 mutations.

TGFβ1, SMAD2, CTNNβ1, and Wnt3a gene mutational status and serum concentrations in individuals with non-small cell lung cancer.

The objective of the current investigation was to investigate the diagnostic utility of the serum concentrations and mutational status of TGFβ1, SMAD2, CTNNβ1, and Wnt3a. and the expression levels of human‑related genes in patients with non-small cell lung cancer (NSCLC). The serum concentrations were determined using the ELISA technique, and PCR for genotype variations of TGFβ1, SMAD2, CTNNβ1, and Wnt3a were examined using Sanger sequencing in tissue samples obtained from 93 patients with NSCLC and 84 healthy individuals for blood, and 20 Formalin Fixed Paraffin Embedded (FFPE) from normal samples dissected adjacent to the tumour. The findings of the current investigation indicate that individuals diagnosed with NSCLC exhibited significant elevation in the serum levels of CEA and CYFRA21-1, as well as TGFβ1, SMAD2, CTNNβ1, and Wnt3a. In total, 325 mutations in four trialled genes (243 mutations in TGFβ1, 24 mutations in SMAD2,47 mutation Wnt3a and 11 mutations in CTNNβ1) were identified in patients with NSCLC. Furthermore, all mutations were recorded in adenocarcinoma, not squamous and normal adjacent tumour cells. CYFRA21-1 and CEA are more significant between NSCLC and HC, gender, and NSCLC types (p<0.001). In detail, TGFβ1 exhibited the highest rate of mutations among other genes and three types of genomic mutations. Elevated levels and genetic polymorphisms of TGFβ1, SMAD2, CTNNβ1, and Wnt3a may play crucial functions in the pathogenesis and angiogenesis of non-small cell lung cancer (NSCLC). These biomarkers might play a role in future immunologic response and pharmacologically targeted NSCLC therapy.

BRCA-mutated gastric adenocarcinomas are associated with chromosomal instability and responsiveness to platinum-based chemotherapy.

Homologous recombination defect is an important biomarker of chemotherapy in certain tumor types, and the presence of pathogenic or likely pathogenic mutations involving BRCA1 or BRCA2 (p-BRCA) mutations is the most well-established marker for the homologous recombination defect. Gastric cancer, one of the most prevalent tumor types in Asia, also harbors p-BRCA mutations. To investigate the clinical significance of p-BRCA mutations, we analyzed 366 gastric cancer cases through next-generation sequencing. We determined the zygosity of p-BRCA mutations based on the calculated tumor purity through variant allelic fraction patterns and investigated whether the presence of p-BRCA mutations is associated with platinum-based chemotherapy and a certain molecular subtype. Biallelic p-BRCA mutation was associated with better response to platinum-based chemotherapy than heterozygous p-BRCA mutation or wild type BRCA genes. The biallelic p-BRCA mutations was observed only in the chromosomal instability subtype, while all p-BRCA mutations were heterozygous in microsatellite instability subtype. In conclusion, patients with gastric cancer harboring biallelic p-BRCA mutations were associated with a good initial response to platinum-based chemotherapy and those tumors were exclusively chromosomal instability subtype. Further investigation for potential association with homologous recombination defect is warranted.

Detection of TP53 gene mutation in blood of breast cancer patients based on circulating tumor DNA and its application in prognosis.

This experiment was carried out to explore the application value of high throughput gene sequencing technology in detecting TP53 gene mutations in the blood of patients with breast cancer by detecting ctDNA gene mutations, and exploring the relationship between TP53 mutations and clinicopathological characteristics and prognosis of patients. The gene mutation of peripheral blood ctDNA and tissue paraffin DNA (tDNA) of 50 patients was detected by high-throughput sequencing technology. The basic data of 50 cases of Medium to high-risk breast cancer diagnosed and were retrospectively collected, and the clinicopathological characteristics and survival results of TP53 mutant and wild-type patients were compared and analyzed according to the ctDNA detection results and relevant follow-up data. Analyze the impact of TP53 mutations on overall survival and progression-free survival using univariate and multivariate Cox regression models. Among 50 patients, there were 29 cases of 7 kinds of gene mutations detected by ctDNA, and 37 cases of 9 kinds of gene mutations detected by tDNA. Using the gene mutation results detected by tDNA as the gold standard, the sensitivity and specificity of peripheral blood ctDNA in diagnosing TP53 gene mutations are 75% to 100%, 92.31% to 100%, and the overall coincidence rate with tDNA results was 83.33% to 100%. Exon 5 was the most prone to mutation, with a frequency of 24.14% (7/29). The most common type of mutation was the missense mutation of 37.93% (11/29). There was no significant correlation between TP53 mutation and PFS (HR=0.67, 95% CI: 0.41-1.08, P=0.102), while TP53 mutation was a protective factor for OS (HR=0.49, 95% CI: 0.27-0.90, P=0.022). The detection of ctDNA in peripheral blood of breast cancer patients by high-throughput gene sequencing technology can replace tumor tissue sections to understand gene mutation. The TP53 mutation in breast cancer patients is related to tumor size, lymph node metastasis and vascular tumor thrombus, but the prognosis of TP53 mutant patients is similar to that of wild-type patients.

Hairpin amplicon cleavage PCR (HAC-PCR): A novel, highly sensitive and cost-effective approach for detection of low-frequency DNA mutations

Highly sensitive detection of gene variants holds immense significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a substantial challenge when identifying low-frequency mutant circulating tumor DNA (ctDNA) in cancer cases. Here, a novel technique called hairpin amplicon cleavage PCR (HAC-PCR) for detecting low-level DNA mutations with exceptional sensitivity was presented. What sets HAC-PCR apart is its ability to achieve high sensitivity independently of strand-displacement, base modification, or the use of additional reagents. The discrimination between wild-type targets (WT) and mutant targets (MT) is significantly improved by introducing uniquely designed hairpin structures. These hairpins hinder the amplification of WT and are cleaved during the elongation of MT. For the first time, the unique mechanism of hairpin amplicon cleavage by Taq polymerase was exploited for mutation identification. The optimized HAC-PCR consistently demonstrates the capability to discriminate mutant DNA at levels as low as 0.01% variant allele fraction (VAF) for five mutation types. Furthermore, this impressive sensitivity of 0.01% VAF has also been achieved in a multiplex assay. When applied to the testing of tissue DNA from 24 patients with thyroid cancers and plasma ctDNA from 12 patients with non-small cell lung cancers (NSCLCs), HAC-PCR exhibited a 100% concordance rate with the existing commercial kit and droplet digital PCR (ddPCR). Therefore, HAC-PCR stands as a cost-effective and highly effective tool for variant identification in clinical testing.

Clinical and Genetic Characteristics of Limb-Girdle Muscular Dystrophy in Iranian Patients

Background: Limb-girdle muscular dystrophy (LGMD) is a bothersome muscle disease associated with weakness of the shoulder and pelvic girdle. Objectives: The study aimed to determine the genetic diversity and relative frequency of various forms of LGMD in Iranian children. Methods: In this descriptive research, 60 children referred to the neurology or emergency department of the Pediatric Medical Center were studied from April 2019 to April 2020. Additional tests (muscle biopsy and genetic testing) were performed to confirm the diagnosis of LGMDs. Quantitative data such as disease level, motor, respiratory, and cardiac functions, and molecular data underwent statistical analysis. Results: A total of 41 patients with a mean age of 11.1 were studied. Twenty-two patients were diagnosed with genetic tests and 19 with muscle biopsies. Also, there were 26.8% cases of alpha sarcoglycanopathy, 24.4% beta sarcoglycanopathy, 17.1% gamma sarcoglycanopathy, 7.3% calpainopathy, 7.3% dysferlinopathy, 7.3% dystroglycanopathy, 7.3% titinopathy, and one case of laminopathy. Among genetically confirmed individuals, 27.3% had SGCB mutation, and 18.2% had SGCA mutation. A significant relationship was seen between the mutation type and creatine phosphokinase (CPK) levels (P &lt; 0.05). Conclusions: The prevalence of alpha and beta sarcoglycanopathy phenotypes in the study population showed that the severity of clinical involvement may be predicted by SGCB gene mutation and sarcoglycan expression.

A novel homozygous splice-site mutation of JK gene leads to Jk(a-b-) phenotype.

This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient. Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking. In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.

Influence of left ventricular ejection fraction, type of mutation and sex on malignant ventricular arrhythmias in dsp and flnc mutations

Abstract Introduction Pathogenic mutations in FLNC and DSP are a common cause of arrhythmogenic left ventricular cardiomyopathy and "arrhythmic dilated cardiomyopathy". We aimed to study demographic and clinical differences amongst these carriers. Purpose 1. To study the prevalence of malignant ventricular arrhythmias in DSP and FLNC mutation carriers in the studies and reports published in the literature. 2. To analyse the effect of age, left ventricular ejection fraction (LVEF), and sex on ventricular arrhythmias in both groups. Methods A systematic review of the human studies published in Medline and EMBASE until September 2022 was carried out. "Desmoplakin Mutations", "Filamin Mutations", "Arrhythmogenic Cardiomyopathy" and "Dilated Cardiomyopathy" were the keywords included in the search queries. Studies, family reports, and registries reporting on individual data of pathogenic or likely pathogenic mutation carriers were selected for analysis. Results Twenty-four articles published between 2002 and 2020 were selected from a total of 430 entries. 171 DSP carriers (from 16 studies) and 169 FLNC carriers (from 7 studies) were included in the analysis. Mean age was similar in both groups: 39.45±19.23 years in DSP vs 42.02±16.27 in the FLNC group (p:0.201). The proportion of females was higher among DSP carriers than that found among FLNC carriers: 101 (59%) vs 76 (45%), p: 0.012. Non-missense mutations were more frequent in both groups: 98 DSP carriers (57%) and 157 FLNC carriers (97%). LVEF was slightly more depressed amongst FLNC carriers: 47.75±14.94% vs 52.37±14.64%, p:0.014. A total of 81 major arrhythmic events (sudden death, ventricular fibrillation, sustained ventricular tachycardia were reported. The LVEF of the male patients that had suffered a major event was similar in both groups (39.13±18.20% in DSP vs 39.03±12.24% in FLNC, p:0.987). On the contrary, LVEF was significantly lower in female carriers of DSP mutations (32.33±15.65% in DSP vs 47.60±15.88% in FLNC, p:0.050). Male carriers show a worse prognosis than their female counterparts (log-rank 0.005) (Figure). The difference was not significant after adjusting for LVEF and type of mutation (missense vs non-missense). LVEF (HR 0.94 per percentage unit (0.91-0.96), p &amp;lt;0.001) and non-missense mutations (HR 0.21 (0.07-0.66), p:0.008) were inversely associated with adverse outcomes in DSP carriers in a multivariate analysis. LVEF was also associated with a better outcome in FLNC carriers (HR 0.97 (0.95-0.99), p: 0.019. Conclusion Moderate systolic dysfunction is a risk factor for malignant arrhythmias amongst DSP and FLNC mutation carriers. Female FLNC carriers with mild systolic dysfunction are at risk for sustained ventricular arrhythmias. Missense DSP mutations are associated with poor arrhythmic prognosis.Sex differences in survival

Overexpression of cardiac calsequestrin as a novel gene-therapy approach to treat CPVT1: in silico and in vivo proves of principle

Abstract Background More than 200 gain-of-function mutations in the cardiac ryanodine receptor (RyR2) cause catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1). From a molecular point of view, 3 types of mutations exist [1]: Class 1 mutants enhance RyR2 opening in response to sarcoplasmic reticulum (SR) free Ca2+; Class 2 and Class 3 mutants enhance RyR2 opening in response to both cytosolic and SR free Ca2+. Purpose The study's objective was to evaluate in silico and in vivo, in CPVT1 knock-in mice carriers of the Class 1 mutation RyR2(R4496C/+), whether overexpression of cardiac calsequestrin (CASQ2) rescues the arrhythmic phenotype in CPVT1 mice. Rationale: Several studies performed in the 2000’s have collectively demonstrated that an increase in CASQ2 promotes the closure of RyR2 channels [2]: inspired by this evidence, here we test the hypothesis that CASQ2 gene therapy might be a novel treatment for CPVT1. Methods (1) In silico study: Properties of Class 1 and Class 2 RyR2s mutants (Figure, panels A-C) were introduced into an in silico model of the murine ventricular myocyte [3]. Virtual myocytes were paced either at baseline or during simulated delivery of 100 nM Isoproterenol at 1 Hz for 180 seconds. To simulate CASQ2 overexpression, protein levels were progressively increased, and the changes in the arrhythmic burden were documented. (2) In vivo study (performed in compliance with ethical regulations): an adeno-associated viral vector containing the cDNA sequence of CASQ2 was injected into RyR2(R4496C/+) mice (tail iv, 3×10^13 vg/kg) at 4 weeks of age. Eight weeks post-infection, animals were treated with an ip bolus of caffeine (120mg/kg) and epinephrine (2 mg/kg) to elicit arrhythmias (Epi/Caff Test). Results (1) In silico: dose-response curves were simulated to correlate CASQ2 levels with arrhythmia inducibility during virtual Isoproterenol delivery. For the simulated prototypical Class 1 mutation, 1.4-fold CASQ2 expression suppressed arrhythmias. For the prototypical Class 2 mutation, 1.8-fold CASQ2 expression was needed (Figure, panel D). (2) In vivo: At 12 weeks of age, both RyR2(R4496C/+) treated and non-treated littermates received the ip Epi / Caff bolus. Arrhythmias were observed in 17/32 (53%) untreated mice and in 0/12 treated animals (p=0.0012). Conclusion(s) We provide proof-of-principle demonstrating that in mice, the overexpression of CASQ2 may be a powerful and safe therapeutic strategy for CPVT1.