Deletion Of Exon Research Articles

STK11 Causative Variants and Copy Number Variations Identified in Thai Patients With Peutz-Jeghers Syndrome.

Introduction Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disorder caused by germline mutations in the serine-threonine kinase 11 (STK11) tumor suppressor gene. This syndrome is characterized by hamartomatous gastrointestinal polyps, mucocutaneous melanin pigmentation, and a higher risk of developing various cancers. Methods We summarized the clinical and molecular characteristics of five unrelated Thai patients with PJS. Denaturing high-performance liquid chromatography (DHPLC) screening, coupled with direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA), were applied for the molecular analysis of STK11. Results A total of four STK11 pathogenic changeswere identified in the five PJS patients, including two frameshift variants (a novel c.199dup, p.Leu67ProfsTer96 and a known c.834_835del, p.Cys278TrpfsTer6) and two types of copy number variations (CNV), exon 1 deletion and exons 2-3 deletion. Among reported STK11 exonic deletions, exon 1 and exons 2-3 deletions were found to be the two most commonly deleted exons. Conclusion All identified STK11 mutations were null mutations that were associated with more severe PJS phenotypes and cancers. This study broadens the phenotypic and mutational spectrum of STK11 in PJS.

The complexity of EGFR exon 19 deletion and L858R mutant cells as assessed by proteomics, transcriptomics, and metabolomics

Most lung adenocarcinoma-associated EGFR tyrosine kinase mutations are either an exon 19 deletion (19Del) or L858R point mutation in exon 21. Although patients whose tumors contain either of these mutations exhibit increased sensitivity to tyrosine kinase inhibitors, progression-free and overall survival appear to be longer in patients with 19Del than in those with L858R. In mutant-transfected Ba/F3 cells, 19Del and L858R were compared by multi-omics analyses including proteomics, transcriptomics, and metabolomics. Proteome analysis identified increased plastin-2, TKT, PDIA5, and ENO1 expression in L858R cells, and increased EEF1G expression in 19Del cells. RNA sequencing showed significant differences between 19Del and L858R cells in 112 genes. Metabolome analysis showed that amino acids, adenylate, guanylate, NADPH, lactic acid, pyruvic acid glucose 6-phosphate, and ribose 5-phosphate were significantly different between the two mutant cells. Because GSH was increased with L858R, we combined osimertinib with the GSH inhibitor buthionine sulfoximine in L858R cells and observed synergistic effects. The complexity of EGFR 19Del and L858R mutant cells was demonstrated by proteomics, transcriptomics, and metabolomics analyses. Therapeutic strategies for lung cancer with different EGFR mutations could be considered because of their different metabolic phenotypes.

G2 checkpoint targeting via Wee1 inhibition radiosensitizes EGFRvIII-positive glioblastoma cells

BackgroundThe gene of the Epidermal growth factor receptor (EGFR) is one of the most frequently altered genes in glioblastoma (GBM), with deletions of exons 2–7 (EGFRvIII) being amongst the most common genomic mutations. EGFRvIII is heterogeneously expressed in GBM. We already showed that EGFRvIII expression has an impact on chemosensitivity, replication stress, and the DNA damage response. Wee1 kinase is a major regulator of the DNA damage induced G2 checkpoint. It is highly expressed in GBM and its overexpression is associated with poor prognosis. Since Wee1 inhibition can lead to radiosensitization of EGFRvIII-negative (EGFRvIII−) GBM cells, we asked, if Wee1 inhibition is sufficient to radiosensitize also EGFRvIII-positive (EGFRvIII+) GBM cells.MethodsWe used the clinically relevant Wee1 inhibitor adavosertib and two pairs of isogenetic GBM cell lines with and without endogenous EGFRvIII expression exhibiting different TP53 status. Moreover, human GBM samples displaying heterogenous EGFRvIII expression were analyzed. Expression of Wee1 was assessed by Western blot and respectively immunohistochemistry. The impact of Wee1 inhibition in combination with irradiation on cell cycle and cell survival was analyzed by flow cytometry and colony formation assay.ResultsAnalysis of GBM cells and patient samples revealed a higher expression of Wee1 in EGFRvIII+ cells compared to their EGFRvIII− counterparts. Downregulation of EGFRvIII expression by siRNA resulted in a strong decrease in Wee1 expression. Wee1 inhibition efficiently abrogated radiation-induced G2-arrest and caused radiosensitization, without obvious differences between EGFRvIII− and EGFRvIII+ GBM cells.ConclusionWe conclude that the inhibition of Wee1 is an effective targeting approach for the radiosensitization of both EGFRvIII− and EGFRvIII+ GBM cells and may therefore represent a promising new therapeutic option to increase response to radiotherapy.

Germline heterozygous exons 8–11 pathogenic BARD1 gene deletion reported for the first time in a family with suspicion of a hereditary colorectal cancer syndrome: more than an incidental finding?

BackgroundColorectal cancer (CRC) is a highly prevalent disease in developed countries. Inherited Mendelian causes account for approximately 5% of CRC cases, with Lynch syndrome and familial adenomatous polyposis being the most prevalent forms. Scientific efforts are focused on the discovery of new candidate genes associated with CRC and new associations of phenotypes with well-established cancer-related genes. BRCA1-associated ring domain (BARD1) gene deleterious germline variants are associated with a moderate increase in the relative risk of breast cancer, but their association with other neoplasms, such as CRC, remains unclear.Case presentationWe present the case of a 49-year-old male diagnosed with rectal adenocarcinoma whose maternal family fulfilled Amsterdam clinical criteria for Lynch syndrome. Genetic test confirmed the presence in heterozygosis of a germline pathogenic deletion of exons 8–11 in BARD1 gene. The predictive genetic study of the family revealed the presence of this pathogenic variant in his deceased cancer affected relatives, confirming co-segregation of the deletion with the disease.ConclusionsTo the best of our knowledge, this is the first published work in which this BARD1 deletion is detected in a family with familial colorectal cancer type X (FCCTX) syndrome, in which the clinical criteria for Lynch syndrome without alteration of the DNA mismatch repair (MMR) system are fulfilled. Whether this incidental germline finding is the cause of familial colorectal aggregation remains to be elucidated in scientific forums. Patients should be carefully assessed in specific cancer genetic counseling units to account for hypothetical casual findings in other genes, in principle unrelated to the initial clinical suspicion, but with potential impact on their health.

DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation

The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.

Is Your Kid Actin Out? A Series of Six Patients With Inherited Actin-Related Protein 2/3 Complex Subunit 1B Deficiency and Review of the Literature

Hereditary actin-related protein 2/3 complex subunit 1B deficiency is characterized clinically by ear, skin, and lung infections, bleeding, eczema, food allergy, asthma, skin vasculitis, colitis, arthritis, short stature, and lymphadenopathy. We aimed to describe the clinical, laboratory, and genetic features of six patients from four Mexican families. We performed exome sequencing in patients of four families with suspected actinopathy, collected their data from medical records, and reviewed the literature for reports of other patients with actin-related protein 2/3 complex subunit 1B deficiency. Six patients from four families were included. All had recurrent infections, mainly bacterial pneumonia, and cellulitis. A total of 67% had eczema whereas 50% had food allergies, failure to thrive, hepatomegaly, and bleeding. Eosinophilia was found in all; 84% had thrombocytopenia, 67% had abnormal-size platelets and anemia. Serum levels of IgG, IgA, and IgE were highly increased in most; IgM was normal or low. T cells were decreased in 67% of patients, whereas B and NK cells were increased in half of patients. Two of the four probands had compound heterozygous variants. One patient was successfully transplanted. We identified 28 other patients whose most prevalent features were eczema, recurrent infections, failure to thrive, bleeding, diarrhea, allergies, vasculitis, eosinophilia, platelet abnormalities, high IgE/IgA, low T cells, and high B cells. Actin-related protein 2/3 complex subunit 1B deficiency has a variable and heterogeneous clinical spectrum, expanded by these cases to include keloid scars and Epstein-Barr virus chronic hepatitis. A novel deletion in exon 8 was shared by three unrelated families and might be the result of a founder effect.

Novel pathogenic variants in the androgen receptor gene associated with androgen insensitivity syndrome identified through exome sequencing and in silico analysis

Androgen insensitivity syndrome (AIS) is a common disorder/differences of sex development with a 46, XY karyotype, but diverse genital phenotypes. Various pathogenic variants within the androgen receptor (AR) gene on the X chromosome are the primary pathogenesis of AIS. However, some patients with AIS still lack a definitive molecular diagnosis. Here, molecular diagnosis of eight patients with the clinical phenotype of AIS was performed using exome sequencing. We found eight variants of the AR gene, including p.(C131*), p.(W435*), p.(T653Lfs*8), c.2318+1G>T, p.(S397R), p.(Y572C), p.(S648G), and p.(D691G), and a pathogenic copy number variation covering a deletion of exon 2 of AR gene. Patient pedigree validation confirmed that the discovered variants conformed to the X-linked recessive inheritance patterns of AIS. In silico analysis indicated that the splice site variant (c.2318+1G>T) could lead to loss of the original 5′ splice donor site and exon skipping. Missense variants, including p.(S397R), p.(S648G), and p.(D691G), may affect the structure and function of the AR protein. Our results highlight the applicability of exome sequencing for molecular diagnosis of AIS. The novel variants found in this study enrich the pathogenic variant spectrum of the AR gene and provide a basis for the diagnosis and management of patients with AIS. A definite molecular diagnosis will provide accurate guidance for genetic counseling of proband’s family members.

HLA-C*07:1044N a novel allele, caused by a single nucleotide deletion in exon 3 of HLA-C*07:01:01:01.

HLA-C*07:1044N differs from HLA-C*07:01:01:01 by a single nucleotide deletion in exon 3, codon 124, nt. 442.

Identifying a novel frameshift pathogenic variant in a Chinese family with neurofibromatosis type 1 and review of literature.

To detect the pathogenic gene variant in a family with neurofibromatosis type 1 (NF1). This patient with NF1 was sequenced using target sequence capture and high-throughput sequencing technology. After detecting the suspicious pathogenic variant type, the pathogenic variant sites of the patient and the patient's family members were verified by multiple ligation dependent probe amplification and Sanger sequencing. Sift, polyphen-2, Mutation Taster and GERP++ software were used to predict the pathogenicity of the unknown loci. The clinical data, diagnosis and treatment process of the patients were reviewed. Using the keyword "NF1; frameshift pathogenic variant", relevant literature was gathered for analysis from Chinese and international databases, with articles dating from the establishment of each database to April 2022. A heterozygous frameshift pathogenic variant of NF1 in exon 33 was detected in the patient. The insertion of adenine in coding region 4486 resulted in the replacement of isoleucine with asparagine in protein 1497. Sanger sequencing validation and segregation analysis were performed, which demonstrated that the NF1 gene was cosegregated with the disease phenotype in this family. This study identified a novel NF1 heterozygous frameshift mutation c.4486dupA (p.I1497Nfs*12). Relevant literature retrieval found 7 Chinese articles and 12 foreign articles. With NF1 gene mutation, mutation types are diverse, including point mutation, frameshift mutation, splice site mutation, exon mutation, chimeric mutation and de novo mutation. Foreign reports are based on autosomal dominant inheritance. This study's results demonstrate that a novel deletion in exon 33 caused NF1 in this Chinese family, expanding the mutational spectrum of the NF1 gene.

Infertility control of transgenic fluorescent zebrafish with targeted mutagenesis of the dnd1 gene by CRISPR/Cas9 genome editing.

Transgenic technology and selective breeding have great potential for the genetic breeding in both edible fish and ornamental fish. The development of infertility control technologies in transgenic fish and farmed fish is the critical issue to prevent the gene flow with wild relatives. In this study, we report the genome editing of the dead end (dnd1) gene in the zebrafish model, using the CRISPR/Cas9 technology to achieve a loss-of-function mutation in both wild-type zebrafish and transgenic fluorescent zebrafish to develop complete infertility control technology of farmed fish and transgenic fish. We effectively performed targeted mutagenesis in the dnd1 gene of zebrafish with a single gRNA, which resulted in a small deletion (-7 bp) or insertion (+41 bp) in exon 2, leading to a null mutation. Heterozygotes and homozygotes of dnd1-knockout zebrafish were both selected by genotyping in the and generations. Based on a comparison of histological sections of the gonads between wild-type, heterozygous, and homozygous dnd1 zebrafish mutants, the dnd1 homozygous mutation (aa) resulted in the loss of germ cells. Still, there was no difference between the wild-type (AA) and dnd1 heterozygous (Aa) zebrafish. The homozygous dnd1 mutants of adult zebrafish and transgenic fluorescent zebrafish became all male, which had normal courtship behavior to induce wild-type female zebrafish spawning. However, they both had no sperm to fertilize the spawned eggs from wild-type females. Thus, all the unfertilized eggs died within 10h. The targeted mutagenesis of the dnd1 gene using the CRISPR/Cas9 technology is stably heritable by crossing of fertile heterozygous mutants to obtain sterile homozygous mutants. It can be applied in the infertility control of transgenic fluorescent fish and genetically improved farmed fish by selective breeding to promote ecologically responsible aquaculture.

Technical feasibility of newborn screening for spinal muscular atrophy by next-generation DNA sequencing.

Newborn screening (NBS) assays for spinal muscular atrophy (SMA) typically use a polymerase chain reaction (PCR) based assay to identify individuals with homozygous deletion in exon 7 of the SMN1 gene. Due to high DNA sequence homology between SMN1 and SMN2, it has previously been difficult to accurately bioinformatically map short reads from next-generation DNA sequencing (NGS) to SMN1, resulting in low analytical performance and preventing NGS being used for SMA screening. Advances in bioinformatics have allowed NGS to be used in diagnostic settings, but to date these assays have not reached the scale required for high volume population newborn screening and have not been performed on the dried blood spot samples that NBS programs currently use. Here we integrate an NGS assay using hybridisation-based capture with a customised bioinformatics algorithm and purpose designed high throughput reporting software into an existing NBS program to achieve a laboratory workflow for population SMA screening. We tested the NGS assay on over 2500 newborns born over 2weeks in a NBS program in a technical feasibility study and show high sensitivity and specificity. Our results suggest NGS may be an alternate method for SMA screening by NBS programs, providing a multiplex testing platform on which potentially hundreds of inherited conditions could be simultaneously tested.

Mutational Profiling of Lung Cancer Using Next Generation Sequencing: A Malaysian Real-World Clinical Diagnostic Experience

Lung cancer is one of the most common cancers and a leading cause of cancer-related mortality in Malaysia. This analysis aimed to evaluate the prevalence of actionable and common mutations, as well as co-mutations frequently occurring with EGFR variants in lung cancer. Mutational profiling of lung tumour samples was performed using next generation sequencing (NGS) panels at the Subang Jaya Medical Centre laboratory. A total of 469 lung tumour samples referred from several medical facilities in Malaysia were analysed and 84% were of the adenocarcinoma subtype. The three most frequent mutations found were EGFR (46.5%), TP53 (37.5%) and KRAS (14.3%). Actionable mutations with approved drug targets for lung cancer were detected in 63.5% of patient samples. Among patients with EGFR mutations, deletions in exon 19 were detected in 44.5% and p.L858R in 38.5% of samples. The most common co-mutations for samples with EGFR mutations were found in the TP53 gene (38.1%). A median turnaround time (TAT) of 3 working days was achievable with an automated NGS platform. NGS testing can provide valuable information on the mutational landscape and the prevalence of common or actionable mutations present in lung cancer patients. This real-world experience demonstrates the high percentage of actionable mutations detected and highlights the value of NGS testing in a clinical diagnostic setting.

The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45-47.

Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy(DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P<0.0001), wire hang (P=0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P=0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P<0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P=0.0017) and centrally located nuclei indicating degeneration/regeneration (P<0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P<0.0001), collagen deposition (+1.4-fold, P=0.0217), and sarcolemmal damage measured by intracellular IgM (P=0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Osimertinib in NSCLC With Atypical EGFR-Activating Mutations: A Retrospective Multicenter Study

IntroductionEGFR mutations drive a subset of NSCLC. Patients harboring the common EGFR mutations, deletion of exon 19 and L858R, respond well to osimertinib, a third-generation tyrosine kinase inhibitor. Nevertheless, the effect of osimertinib on NSCLC with atypical EGFR mutations is not well described. This multicenter retrospective study evaluates the efficacy of osimertinib among patients with NSCLC harboring atypical EGFR mutations. MethodsPatients with metastatic NSCLC treated with osimertinib, harboring at least one atypical EGFR mutation, excluding concurrent deletion of exon 19, L858R, or T790M mutations, from six U.S. academic cancer centers were included. Baseline clinical characteristics were collected. The primary end point was the time to treatment discontinuation (TTD) of osimertinib. Objective response rate by the Response Evaluation Criteria in Solid Tumors version 1.1 was also assessed. ResultsA total of 50 patients with NSCLC with uncommon EGFR mutations were identified. The most frequent EGFR mutations were L861Q (40%, n = 18), G719X (28%, n = 14), and exon 20 insertion (14%, n = 7). The median TTD of osimertinib was 9.7 months (95% confidence interval [CI]: 6.5–12.9 mo) overall and 10.7 months (95% CI: 3.2–18.1 mo) in the first-line setting (n = 20). The objective response rate was 31.7% (95% CI: 18.1%–48.1%) overall and 41.2% (95% CI: 18.4%–67.1%) in the first-line setting. The median TTD varied among patients with L861Q (17.2 mo), G719X (7.8 mo), and exon 20 insertion (1.5 mo) mutations. ConclusionsOsimertinib has activity in patients with NSCLC harboring atypical EGFR mutations. Osimertinib activity differs by the type of atypical EGFR-activating mutation.

Identification and characterization of two SERPINC1 mutations causing congenital antithrombin deficiency

BackgroundAntithrombin (AT) is the main physiological anticoagulant involved in hemostasis. Hereditary AT deficiency is a rare autosomal dominant thrombotic disease mainly caused by mutations in SERPINC1, which was usually manifested as venous thrombosis and pulmonary embolism. In this study, we analyzed the clinical characteristics and screened for mutant genes in two pedigrees with hereditary AT deficiency, and the functional effects of the pathogenic mutations were evaluated.MethodsCandidate gene variants were analyzed by next-generation sequencing to screen pathogenic mutations in probands, followed by segregation analysis in families by Sanger sequencing. Mutant and wild-type plasmids were constructed and transfected into HEK293T cells to observe protein expression and cellular localization of SERPINC1. The structure and function of the mutations were analyzed by bioinformatic analyses.ResultsThe proband of pedigree A with AT deficiency carried a heterozygous frameshift mutation c.1377delC (p.Asn460Thrfs*20) in SERPINC1 (NM000488.3), a 1377C base deletion in exon 7 resulting in a backward shift of the open reading frame, with termination after translation of 20 residues, and a different residue sequence translated after the frameshift. Bioinformatics analysis suggests that the missing amino acid sequence caused by the frameshift mutation might disrupt the disulfide bond between Cys279 and Cys462 and affect the structural function of the protein. This newly discovered variant is not currently included in the ClinVar and HGMD databases. p.Arg229* resulted in a premature stop codon in exon 4, and bioinformatics analysis suggests that the truncated protein structure lost its domain of interaction with factor IX (Ala414 site) after the deletion of nonsense mutations. However, considering the AT truncation protein resulting from the p.Arg229* variant loss a great proportion of the molecule, we speculate the variant may affect two functional domains HBS and RCL and lack of the corresponding function. The thrombophilia and decreased-AT-activity phenotypes of the two pedigrees were separated from their genetic variants. After lentiviral plasmid transfection into HEK293T cells, the expression level of AT protein decreased in the constructed c.1377delC mutant cells compared to that in the wild-type, which was not only reduced in c.685C > T mutant cells but also showed a significant band at 35 kDa, suggesting a truncated protein. Immunofluorescence localization showed no significant differences in protein localization before and after the mutation.ConclusionsThe p.Asn460Thrfs*20 and p.Arg229* variants of SERPINC1 were responsible for the two hereditary AT deficiency pedigrees, which led to AT deficiency by different mechanisms. The p.Asn460Thrfs*20 variant is reported for the first time.

Identification of COL4A4 variants in Chinese patients with familial hematuria.

Background: Benign familial hematuria and Alport syndrome are common causes of familial hematuria among children and young adults, which are attributable to variants in the collagen type IV alpha chain genes, COL4A3, COL4A4, or COL4A5. The study was conducted to identify the underlying genetic causes in patients with familial hematuria. Methods: Two unrelated Han-Chinese pedigrees with familial hematuria were recruited for this study. Whole exome sequencing was combined with in silico analysis to identify potential genetic variants, followed by variant confirmation by Sanger sequencing. Reverse transcription, PCR, and Sanger sequencing were performed to evaluate the effect of the detected splicing variant on mRNA splicing. Results: A novel heterozygous splicing c.595-1G>A variant and a known heterozygous c.1715G>C variant in the collagen type IV alpha 4 chain gene (COL4A4) were identified and confirmed in patients of pedigree 1 and pedigree 2, respectively. Complementary DNA analysis indicated this splicing variant could abolish the canonical splice acceptor site and cause a single nucleotide deletion of exon 10, which was predicted to produce a truncated protein. Conclusions: The two COL4A4 variants, c.595-1G>A variant and c.1715G>C (p.Gly572Ala) variant, were identified as the genetic etiologies of two families with familial hematuria, respectively. Our study broadened the variant spectrum of the COL4A4 gene and explained the possible pathogenesis, which will benefit clinical management and genetic counseling.

Predictive value of p53 and AXL immunostaining for the efficacy of immune checkpoint inhibitor-based therapy after osimertinib treatment in patients with epidermal growth factor-mutant non-small cell lung cancer.

Current evidence indicates that immune checkpoint inhibitors (ICIs) have a limited efficacy in patients with lung cancer harboring epidermal growth factor receptor (EGFR) mutations. However, there is a lack of data on the efficacy of ICIs after osimertinib treatment, and the predictors of ICI efficacy are unclear. We retrospectively assessed consecutive patients with EGFR-mutant NSCLC who received ICI-based therapy after osimertinib treatment at 10 institutions in Japan, between March 2016 and March 2021. Immunohistochemical staining was used to evaluate the expression of p53 and AXL. The deletions of exon 19 and the exon 21 L858R point mutation in EGFR were defined as common mutations; other mutations were defined as uncommon mutations. A total of 36 patients with advanced or recurrent EGFR-mutant NSCLC were analyzed. In multivariate analysis, p53 expression in tumors was an independent predictor of PFS after ICI-based therapy (p = 0.002). In patients with common EGFR mutations, high AXL expression was a predictor of shorter PFS and overall survival after ICI-based therapy (log-rank test; p = 0.04 and p = 0.02, respectively). The levels of p53 in pretreatment tumors may be a predictor of ICI-based therapy outcomes in patients with EGFR-mutant NSCLC after osimertinib treatment. High levels of AXL in tumors may also be a predictor of ICI-based therapy outcomes, specifically for patients with common EGFR mutations. Further prospective large-scale investigations on the predictors of ICI efficacy following osimertinib treatment are warranted.

Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Although next-generation sequencing (NGS) technology can be used to sequence tens of thousands of DNA molecules simultaneously. It has poor capture efficiency for the six PKD1 pseudogenes and GC-rich regions. Multiplex ligation-dependent probe amplification (MLPA) technology can detect consecutive deletions of exons, but it is less sensitive for single-base mutations. However, pathogenic genes might not be detected in some patients, even when using the above methods. Improving the detection rate of pathogenic genes is an important technical problem hindering clinical diagnosis of ADPKD. Four pedigrees of ADPKD patients with mutation sites not identified by NGS were examined by other methods. First, MLPA was performed. Then, pedigrees in which MLPA did not identify pathogenic genes were subjected to multiplex polymerase chain reaction (MPCR) and targeted region sequencing. Finally, the detected mutation sites were verified by Sanger sequencing. The results showed that MLPA detected the following PKD1 exonic deletion mutations in three pedigrees: PKD1-18nt-290nt, PKD1-up-257nt, PKD1-up-444nt and PKD1-3nt-141nt. A new mutation site was identified through targeted region sequencing in one pedigree: PKD1 NM_001009944: c.151T > C at the protein level, described as p. Cys51Arg. In summary, we established a system of genetic detection and analytical methods, from NGS to MLPA to targeted region sequencing and finally to Sanger sequencing. We combined MPCR and targeted region sequencing for the first time in ADPKD diagnosis, which further improved diagnosis accuracy. Moreover, we identified one new missense mutation and four new deletion mutations.

Deletion induced splicing in RIC3 drives nicotinic acetylcholine receptor regulation with implications for endoplasmic reticulum stress in human astrocytes.

Nicotinic acetylcholine receptor (nAChR) dysregulation in astrocytes is reported in neurodegenerative disorders. Modulation of nAChRs through agonists confers protection to astrocytes from stress but regulation of chaperones involved in proteostasis with pathological implications is unclear. Resistance to inhibitors of cholinesterase 3 (RIC3), a potential chaperone of nAChRs is poorly studied in humans. We characterized RIC3 in astrocytes derived from an isogenic wild-type and Cas9 edited "del" human iPSC line harboring a 25 bp homozygous deletion in exon2. Altered RIC3 transcript ratio due to deletion induced splicing and an unexpected gain of α7nAChR expression were observed in "del" astrocytes. Transcriptome analysis showed higher expression of neurotransmitter/G-protein coupled receptors mediated by cAMP and calcium/calmodulin-dependent kinase signaling with increased cytokines/glutamate secretion. Functional implications examined using tunicamycin induced ER stress in wild-type astrocyte stress model showed cell cycle arrest, RIC3 upregulation, reduction in α7nAChR membrane levels but increased α4nAChR membrane expression. Conversely, tunicamycin-treated "del" astrocytes showed a comparatively higher α4nAChR membrane expression and upsurged cAMP signaling. Furthermore, reduced expression of stress markers CHOP, phospho-PERK and lowered XBP1 splicing in western blot and qPCR, validated by proteome-based pathway analysis indicated lowered disease severity. Findings indicate (i) a complex RNA regulatory mechanism via exonic deletion induced splicing; (ii) RIC-3 as a disordered protein having contrasting effects on co-expressed nAChR subtypes under basal/stress conditions; and (iii) RIC3 as a potential drug target against ER stress in astrocytes for neurodegenerative/nicotine-related brain disorders. Cellular rescue mechanism through deletion induced exon skipping may encourage ASO-based therapies for tauopathies.

New Insights Into The Evolution of Chloroplast Genomes in Ochna Species (Ochnaceae, Malpighiales).

Ochnaceae DC. includes more than 600 species that exhibit potential values for environmental ecology, ornamental, pharmaceutical, and timber industries. Although studies on phylogeny and phytochemicals have been intensively conducted, chloroplast genome data of Ochnaceae species have not been fully explored. In this study, the next-generation sequencing method was used to sequence the chloroplast genomes of Ochna integerrima and Ochna serrulata which were 157 329 and 157 835 bp in length, respectively. These chloroplast genomes had a quadripartite structure and contained 78 protein-coding genes, 30 tRNAs, and 4 rRNAs. Comparative analysis revealed 8 hypervariable regions, including trnK_UUU-trnQ_UUG, rpoB-psbM, trnS_GGA-rps4, accD-psaI, rpl33-rps18, rpl14-rpl16, ndhF-trnL_UAG, and rps15-ycf1 among 6 Ochnaceae taxa. Additionally, there were shared and unique repeats among 6 examined chloroplast genomes. The notable changes were the loss of rpl32 in Ochna species and the deletion of rps16 exon 2 in O. integerrima compared to other taxa. This study is the first comprehensive comparative genomic analysis of complete chloroplast genomes of Ochna species and related taxa in Ochnaceae. Consequently, the current study provides initial results for further research on genomic evolution, population genetics, and developing molecular markers in Ochnaceae and related taxa.