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BackgroundGlanzmann's thrombasthenia (GT) is a congenital platelet disorder affecting approximately 1:1 000 000 people globally and characterized by impaired platelet aggregation and clot retraction. Autosomal recessive, loss-of-function, variants in ITGA2B or ITGB3 of the αIIbβ3 receptor cause the disease in humans. A cat affected by Glanzmann's and macrothrombocytopenia was presented to the UC Davis VMTH.Hypothesis/objectivesSevere thrombopathia in this cat has an underlying genetic etiology.AnimalsA single affected patient, 2 age-matched clinically healthy controls, and a geriatric population (n = 20) of normal cats.MethodsPhysical examination and clinical pathology tests were performed on the patient. Flow cytometry and platelet aggregometry analyses for patient phenotyping were performed. Patient and validation cohort gDNA samples were extracted for Sanger sequencing of a previously identified ITGA2B (c.1986delC) variant. Reverse transcriptase PCR was performed on patient and healthy control PRP samples to verify ITGA2B variant consequence.ResultsA novel c.1986_1987insCC autosomal recessive variant in ITGA2B was identified. This variant was absent in a population of 194 unrelated cats spanning 44 different breeds. Complete loss of ITGA2B transcript and protein expression was verified by RT-PCR and flow cytometry, explaining the underlying etiology of GT, and likely macrothrombocytopenia, in this cat.Conclusions and clinical importanceThis study emphasizes the role of precision medicine in cardiovascular disease of cats and identified yet another variant that may be of utility for screening in the feline population. This study provides a small-volume, standardized, successful protocol for adequate platelet RNA isolation and subsequent molecular assessment of gene expression in cats.

Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications. Synopsis: While off-target mutations have been carefully addressed, CRISPR/Cas9 mediated genome editing often causes harmful on-target chromosomal alterations. CasPlus combines Cas9 genome editing with an engineered phage T4 DNA polymerase and reduces the incidence of large deletions and chromosomal translocations. CasPlus can be applied to correct disease-causing mutations such as in Duchenne muscular dystrophy (DMD), but also for generating engineered T cells for cell therapy. CasPlus editing inhibits undesired on-target large deletions while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing. CasPlus efficiently corrects the frameshift mutations in DMD (exon 52 deletions) with fewer on-target DNA damage. CasPlus greatly reduces the frequency of on-target large deletions in mouse germline editing. CasPlus represses chromosomal translocation while maintaining gene disruption efficiency that is higher or comparable to Cas9 in multiplex gene-edited human primary T cells. CasPlus combines Cas9 genome editing with an engineered phage T4 DNA polymerase and reduces the incidence of large deletions and chromosomal translocations. [ABSTRACT FROM AUTHOR]

Introduction: Pathogenic variants in the XK gene are associated with dysfunction or loss of XK protein leading to McLeod syndrome (MLS), a rare X-linked neuroacanthocytosis syndrome with multisystemic manifestation. Here we present clinical, genetic and immunological data on a patient originally admitted to our clinic for presumed post-COVID-19 syndrome, where thorough clinical work-up revealed a novel frameshift deletion in XK causal for the underlying phenotype. We additionally review the clinicogenetic spectrum of reported McLeod cases in the literature. Methods: We performed in-depth clinical characterization and flow cytometry of cerebrospinal fluid (CSF) in a patient where multi-gene panel sequencing identified a novel hemizygous frameshift deletion in XK. Additionally, Kell (K) and Cellano (k) antigen expression was analysed by Fluorescence-activated Cell Sorting (FACS). KEL gene expression was examined by RNA sequencing. Results: A novel hemizygous frameshift deletion in the XK gene resulting in premature termination of the amino acid chain was identified in a 46-year old male presenting with decrease in physical performance and persisting fatigue after COVID-19 infection. Examinations showed raised creatine kinase (CK) levels, neuropathy and clinical features of myopathy. FACS revealed the K-k+ blood type and reduced Cellano density. CSF flow cytometry showed elevation of activated T Cells. Conclusion: In-depth clinical, genetic, immunological and ribonucleic acid (RNA) expression data revealed axonal neuropathy, myopathy and raised levels of activated CSF-T-lymphocytes in a patient with a previously unpublished frameshift deletion in the XK gene. [ABSTRACT FROM AUTHOR]

Carbon dioxide-dependent Proteus mirabilis has been isolated from clinical specimens. It is not clear whether mutations in carbonic anhydrase are responsible for the carbon dioxide dependence of P. mirabilis. The pathogenicity of carbon dioxide-dependent P. mirabilis also remains unclear. The purpose of this study was to determine the cause carbon dioxide dependence of P. mirabilis and its pathogenicity. The DNA sequence of can encoding carbonic anhydrase of a carbon dioxide-dependent P. mirabilis small colony variant (SCV) isolate was analyzed. To confirm that impaired carbonic anhydrase activity is responsible for the formation of the carbon dioxide-dependent SCV phenotype of P. mirabilis , we performed complementation experiments using plasmids with intact can. Additionally, mouse infection experiments were performed to confirm the change in virulence due to the mutation of carbonic anhydrase. We found that the can gene of the carbon dioxide-dependent P. mirabilis SCV isolate showed had a frameshift mutation with a deletion of 1 bp (c. 173delC). The can of P. mirabilis encodes carbonic anhydrase was also found to function in Escherichia coli. The cause of the carbon dioxide-dependent SCV phenotype of P. mirabilis was an abnormality in carbonic anhydrase. Nevertheless, no changes were observed in virulence due to the mutation of carbonic anhydrase in mouse infection experiments. The can gene is essential for the growth of P. mirabilis in ambient air. The mechanisms underlying this fitness advantage in terms of infection warrant further investigation. [ABSTRACT FROM AUTHOR]

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-β-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2(N=3), and blaNDM-1(N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones. [ABSTRACT FROM AUTHOR]

Clarithromycin (CLR) is currently a key antibiotic for Helicobacter pylori infection treatment, however, the data on CLR resistance patterns in Russia are missing. Here, we applied WGS-based approach to H. pylori clinical isolates from Russia to comprehensively investigate sequence variation, identify putative markers of CLR resistance and correlate them with phenotypic susceptibility testing. The phenotypic susceptibility of 44 H. pylori isolates (2014–2022) to CLR was determined by disc diffusion method: 23 isolates were CLR-resistant and 21-CLR-susceptible. All isolates were subjected to WGS and submitted to GenBank. Based on complete sequence analysis, we showed that among all sequence variants, the combination of mutations A2146G/A2147G in the 23S rRNA gene is the most reliable for prediction of phenotypic susceptibility. For the first time, the average number of mutations in 106 virulence-associated genes between resistant and susceptible groups were compared. Moreover, this study presents the first WGS insight into genetic diversity of H. pylori in Russia with a particular focus on the molecular basis of drug resistance: the novel mutations were described as potential markers for the resistance development. Of these, the most prominent was a frameshift deletion (252:CGGGT) in HP0820 coding region, which is a good candidate for further investigation. [ABSTRACT FROM AUTHOR]

Background: Ovarian mucinous tumor with a mural nodule is a rare and special type of ovarian surface epithelial–stromal tumor. Mural nodules are morphologically classified into three types: sarcoma-like, anaplastic carcinomatous, and true sarcomatous nodules. Ovarian mucinous tumors with true sarcomatous mural nodules are rare and challenging to diagnose, with only 10 cases reported worldwide. Currently, liposarcoma mural nodules remain unreported. Case presentation: A 91-year-old woman was hospitalized for postmenopausal vaginal bleeding for 3 weeks. Imaging revealed a large cystic mass (20.0 cm × 17.7 cm × 12.8 cm) on the right ovary. The mass was multilocular cystic, with a mural nodule (1.4 cm × 1.2 cm × 1.0 cm) in the focal cyst wall. Based on histological morphology, immunohistochemical staining, and MDM2/CDK4 fluorescence in situ hybridization testing, the diagnosis was ovarian mucinous cystadenoma with a mural nodule of well-differentiated liposarcoma. To the best of our knowledge, this has never been reported before. High-throughput sequencing identified KRAS mutations in the ovarian mucinous cystadenoma. However, the liposarcoma mural nodule did not exhibit KRAS mutations but displayed copy number amplifications of CDK4 and DDR2, as well as a frameshift mutation in exon 13 of ASXL1 (p. A627Gfs*8). Conclusions: This case broadens the morphological spectrum of mural nodules in ovarian mucinous tumors, deepening our knowledge of this rare morphology. Meanwhile, through high-throughput sequencing, we found no overlapping genetic evidence between the liposarcoma mural nodule and associated ovarian mucinous cystadenoma. [ABSTRACT FROM AUTHOR]

Background: Whole-genome sequencing (WGS)-based prediction of drug resistance in Mycobacterium tuberculosis has the potential to guide clinical decisions in the design of optimal treatment regimens. Methods: We utilized WGS to investigate drug resistance mutations in a 32-year-old Tanzanian male admitted to Kibong'oto Infectious Diseases Hospital with a history of interrupted multidrug-resistant tuberculosis treatment for more than three years. Before admission, he received various all-oral bedaquiline-based multidrug-resistant tuberculosis treatment regimens with unfavourable outcomes. Results: Drug susceptibility testing of serial M. tuberculosis isolates using Mycobacterium Growth Incubator Tubes culture and WGS revealed resistance to first-line anti-TB drugs, bedaquiline, and fluoroquinolones but susceptibility to linezolid, clofazimine, and delamanid. WGS of serial cultured isolates revealed that the Beijing (Lineage 2.2.2) strain was resistant to bedaquiline, with mutations in the mmpR5 gene (Rv0678. This study also revealed the emergence of two distinct subpopulations of bedaquiline-resistant tuberculosis strains with Asp47f and Glu49fs frameshift mutations in the mmpR5 gene, which might be the underlying cause of prolonged resistance. An individualized regimen comprising bedaquiline, delamanid, pyrazinamide, ethionamide, and para-aminosalicylic acid was designed. The patient was discharged home at month 8 and is currently in the ninth month of treatment. He reported no cough, chest pain, fever, or chest tightness but still experienced numbness in his lower limbs. Conclusion: We propose the incorporation of WGS in the diagnostic framework for the optimal management of patients with drug-resistant and extensively drug-resistant tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Mutations in PDE6A and PDE6B are known to cause autosomal recessive RP in humans, On the other hand, mutations in PDE6G are rare but can lead to severe early-onset RP. Case presentation: An 8-year-old Chinese boy was referred to our hospital for poor vision issues. Refraction with cycloplegia showed high hyperopia with astigmatism both eyes. Funduscopic examination revealed typical bone spicule-type pigment deposits in the periphery and midperiphery. The patient was given glasses and a whole exome sequencing containing mitochondrial genes was performed. The results of genetic testing showed that there was a heterozygous frameshift mutation and a segment deletion in the proband's PDE6G gene. Analysis of the parental genes showed that frameshift mutation was inherited from the proband's mother and segment deletion from his father. Conclusions: In this paper, we give a firsthand report that the complex heterozygous mutations of PDE6G gene can causes autosomal recessiveRP (arRP), which expands the understanding of the pathogenic genes of RP. [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum is a rare autosomal recessive genodermatoses characterized by a deficiency in nucleotide excision repair. Erythropoietic protoporphyria is a rare inherited metabolic disease caused by the perturbation of heme. Xeroderma pigmentosum-erythropoietic protoporphyria is exceedingly rare. Hereby, we firstly report a young Chinese patient of xeroderma pigmentosum Group A with erythropoietic protoporphyria carrying an XPA Met214AsnfsTer7 frameshift mutation and a homozygous splicing mutation, c.315-48T>C, in the proband's intron3 of FECH. [ABSTRACT FROM AUTHOR]

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations. [ABSTRACT FROM AUTHOR]

To improve the amylose content (AC) and resistant starch content (RSC) of maize kernel starch, we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I (SBEI) and starch branching enzyme IIb (SBEIIb). A frameshift mutation in SBEI (E1, a nucleotide insertion in exon 6) led to plants with higher RSC (1.07%), lower hundred-kernel weight (HKW, 24.71 ± 0.14 g), and lower plant height (PH, 218.50 ± 9.42 cm) compared to the wild type (WT). Like the WT, E1 kernel starch had irregular, polygonal shapes with sharp edges. A frameshift mutation in SBEIIb (E2, a four-nucleotide deletion in exon 8) led to higher AC (53.48%) and higher RSC (26.93%) than that for the WT. E2 kernel starch was significantly different from the WT regarding granule morphology, chain length distribution pattern, X-ray diffraction pattern, and thermal characteristics; the starch granules were more irregular in shape and comprised typical B-type crystals. Mutating SBEI and SBEIIb (E12) had a synergistic effect on RSC, HKW, PH, starch properties, and starch biosynthesis-associated gene expression. SBEIIa, SSI, SSIIa, SSIIIa, and SSIIIb were upregulated in E12 endosperm compared to WT endosperm. This study lays the foundation for rapidly improving the starch properties of elite maize lines. [ABSTRACT FROM AUTHOR]

Purpose: Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes. Methods: In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation. Results: Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors. Conclusions: The results demonstrated that PATL2A496Sfs*4 and PATL2R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function. [ABSTRACT FROM AUTHOR]

In patients with neurodevelopmental disorders (NDDs), exome sequencing (ES), the diagnostic gold standard, reveals an underlying monogenic condition in only approximately 40% of cases. We report the case of a female patient with profound NDD who died 30 years ago at the age of 3 years and for whom genome sequencing (GS) now identified a single-exon deletion in TBCK previously missed by ExomeDepth, the copy number variation (CNV) detection algorithm in ES. Deoxyribonucleic acid (DNA) was extracted from frozen muscle tissue of the index patient and the parents' blood. Genome data were analyzed for structural variants and single nucleotide variants (SUVs)/indels as part of the Bavarian Genomes consortium project. Biallelic variants in TBCK , which are linked to the autosomal recessive disorder TBCK syndrome, were detected in the affected individual: a novel frameshift variant and a deletion of exon 23, previously established as common but underrecognized pathogenic variant in individuals with TBCK syndrome. While in the foregoing ES analysis, calling algorithms for (SNVs)/indels were able to identify the frameshift variant, ExomeDepth failed to call the intragenic deletion. Our case illustrates the added value of GS for the detection of single-exon deletions for which calling from ES data remains challenging and confirms that the deletion of exon 23 in TBCK may be underdiagnosed in patients with NDDs. Furthermore, it shows the importance of "molecular or genetic autopsy" allowing genetic risk counseling for family members as well as the end of a diagnostic odyssey of 30 years. [ABSTRACT FROM AUTHOR]

AbstractWe reported a rare β-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged β-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the β-globin predicted that the novel prolonged β-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families. [ABSTRACT FROM AUTHOR]

TNF-α-induced protein 3 (TNFAIP3), commonly referred to as A20, is an integral part of the ubiquitin-editing complex that significantly influences immune regulation, apoptosis, and the initiation of diverse immune responses. The A20 protein is characterized by an N-terminal ovarian tumor (OTU) domain and a series of seven zinc finger (ZNF) domains. Mutations in the TNFAIP3 gene are implicated in various immune-related diseases, such as Behçet's disease, polyarticular juvenile idiopathic arthritis, autoimmune thyroiditis, autoimmune hepatitis, and rheumatoid arthritis. These mutations can lead to a spectrum of symptoms, including, but not limited to, recurrent fever, ulcers, rashes, musculoskeletal and gastrointestinal dysfunctions, cardiovascular issues, and respiratory infections. The majority of these mutations are either nonsense (STOP codon) or frameshift mutations, which are typically associated with immune dysfunctions. Nonetheless, missense mutations have also been identified as contributors to these conditions. These genetic alterations may interfere with several biological pathways, notably abnormal NF-κB signaling and dysregulated ubiquitination. Currently, there is no definitive treatment for A20 haploinsufficiency; however, therapeutic strategies can alleviate the symptoms in patients. This review delves into the mutations reported in the TNFAIP3 gene, the clinical progression in affected individuals, potential disease mechanisms, and a brief overview of the available pharmacological interventions for A20 haploinsufficiency. Mandatory genetic testing of the TNFAIP3 gene should be performed in patients diagnosed with autoinflammatory disorders to better understand the genetic underpinnings and guide treatment decisions. [ABSTRACT FROM AUTHOR]

Key Clinical Message: This study presents a family with nine children, two of them diagnosed with RTS2 using genetic testing. The other siblings show some of the RTS2 criteria and are suggestive of the syndrome. Such reports help physicians be more alert in dealing with cases of rare syndromes. Timely initiation of genetic counseling and testing once the first child was diagnosed with the syndrome could have prevented the birth of affected siblings by RTS2. Since RTS2 patients could have a severe clinical manifestation as osteosarcoma which probably leads to death at a young age, the importance of genetic testing is even more underlined. [ABSTRACT FROM AUTHOR]

We recently discovered that the Manech Tête Rousse (MTR) deficient homozygous haplotype 2 (MTRDHH2) probably carries a recessive lethal mutation in sheep. In this study, we fine‐mapped this region through whole‐genome sequencing of five MTRDHH2 heterozygous carriers and 95 non‐carriers from various ovine breeds. We identified a single base pair duplication within the SLC33A1 gene, leading to a frameshift mutation and a premature stop codon (p.Arg246Alafs*3). SLC33A1 encodes a transmembrane transporter of acetyl‐coenzyme A that is crucial for cellular metabolism. To investigate the lethality of this mutation in homozygous MTR sheep, we performed at‐risk matings using artificial insemination (AI) between heterozygous SLC33A1 variant carriers (SLC33A1_dupG). Pregnancy was confirmed 15 days post‐AI using a blood test measuring interferon Tau‐stimulated MX1 gene expression. Ultrasonography between 45 and 60 days post‐AI revealed a 12% reduction in AI success compared with safe matings, indicating embryonic/fetal loss. This was supported by the MX1 differential expression test suggesting fetal losses between 15 and 60 days of gestation. We also observed a 34.7% pre‐weaning mortality rate in 49 lambs born from at‐risk matings. Homozygous SLC33A1_dupG lambs accounted for 47% of this mortality, with deaths occurring mostly within the first 5 days without visible clinical signs. Therefore, appropriate management of SLC33A1_dupG with an allele frequency of 0.04 in the MTR selection scheme would help increase overall fertility and lamb survival. [ABSTRACT FROM AUTHOR]

Background and aims: SYNGAP1-related disorder (SYNGAP1-RD) is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. Methods: We used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild-type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. Results: We show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 α1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab mutant larvae are hyperactive compared to wild-type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, and hyperactivity was proportional to the number of mutant syngap1 alleles. Limitations: Syngap1 loss-of-function mutations produce relatively subtle phenotypes in zebrafish compared to mammals. For example, while mouse Syngap1 homozygotes die at birth, zebrafish syngap1ab-/- survive to adulthood and are fertile, thus some aspects of symptoms in people with SYNGAP1-Related Disorder are not likely to be reflected in zebrafish. Conclusion: Our data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments. [ABSTRACT FROM AUTHOR]

Canine circovirus (CanineCV) is a recently identified member of the Circoviridae family. Since its discovery in 2011, CanineCV has been detected in different countries worldwide, infecting both domestic and wild canids. The virus is potentially associated with gastrointestinal and respiratory illnesses. In 2016, CanineCV was reported in the southwestern region of Guangxi, China. However, its prevalence in other provinces in the Southwest region remained unknown. This study collected a total of 208 serum samples from domestic dogs in Sichuan, China in 2022 to investigate the prevalence of CanineCV. Among these samples, 26 tested positive for CanineCV, resulting in a positivity rate of 12.5%. Additionally, 12 strains were sequenced, 9 of which had a sequence length of 2,063 nucleotides (nt), 2 of the other threes had a length of 2,062 nts and another was 2,064 nt. Notably, a frameshift mutation was identified, resulting in a truncated ORF1 and the occurrence of a novel sequence comprised of 13 amino acids at the end of the replicate protein (Rep). This mutation could affect the replication cycle of the virus. Phylogenetic and evolutionary analyses revealed that the isolates belonged to the CanineCV-3 genotype and were prevalent in the Southeast and the Southwest regions of China, as well as in the neighboring countries alongside other strains of the same genotype. Collectively, this epidemiological investigation widens our understanding of the genetic diversity of CanineCV in Southwest China and provides insights into viral evolution. [ABSTRACT FROM AUTHOR]

Juvenile polyposis syndrome (JPS) is an inherited autosomal dominant condition that predisposes to the development of juvenile polyps throughout the gastrointestinal (GI) tract, and it poses an increased risk of GI malignancy. Germline causative variants were identified in the SMAD4 gene in a subset (20%) of JPS cases. Most SMAD4 germline genetic variants published to date are missense, nonsense, and frameshift mutations. SMAD4 germline alterations predicted to result in aberrant splicing have rarely been reported. Here, we report two unrelated Italian families harboring two different SMAD4 intronic variants, c.424+5G>A and c.425-9A>G, which are clinically associated with colorectal cancer and/or juvenile GI polyps. In silico prediction analysis, in vitro minigene assays, and RT-PCR showed that the identified variants lead to aberrant SMAD4 splicing via the exonization of intronic nucleotides, resulting in a premature stop codon. This is expected to cause the production of a truncated protein. This study expands the landscape of SMAD4 germline genetic variants associated with GI polyposis and/or cancer. Moreover, it emphasizes the importance of the functional characterization of SMAD4 splicing variants through RNA analysis, which can provide new insights into genetic disease variant interpretation, enabling tailored genetic counseling, management, and surveillance of patients with GI polyposis and/or cancer. [ABSTRACT FROM AUTHOR]

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11–q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116 , SNORD115 , and SNORD109A , as well as protein-coding genes MKRN3 and SNRPN , thus bridging the gap between PWS and SYS. [Display omitted] Mutations within MAGEL2 from chromosomal region 15q11–q13 cause Schaaf-Yang syndrome, which is phenotypically related to Prader-Willi syndrome, caused by deletion of the SNORD116 cluster within the same locus. We correlate mutations within MAGEL2 to spinal muscular atrophy and autism and also demonstrate its influence on the abundance of SNORD116. [ABSTRACT FROM AUTHOR]

Background: Alport syndrome (AS) is an inherited nephropathy caused by mutations in the type IV collagen genes. It is clinically characterized by damage to the eyes, ears and kidneys. Diagnosis of AS is hampered by its atypical clinical picture, particularly when the typical features, include persistent hematuria and microscopic changes in the glomerular basement membrane (GBM), are the only clinical manifestations in the patient. Methods: We screened 10 families with suspected AS using whole exome sequencing (WES) and analyzed the harmfulness, conservation, and protein structure changes of mutated genes. In further, we performed in vitro functional analysis of two missense mutations in the COL4A5 gene (c.2359G > C, p.G787R and c.2605G > A, p.G869R). Results: We identified 11 pathogenic variants in the type IV collagen genes (COL4A3, COL4A4 and COL4A5). These pathogenic variants include eight missense mutations, two nonsense mutations and one frameshift mutation. Notably, Family 2 had digenic mutations in the COL4A3 (p.G1170A) and UMOD genes (p.M229K). Family 3 had a digenic missense mutation (p.G997E) in COL4A3 and a frameshift mutation (p.P502L fs*151) in COL4A4. To our knowledge, four of the 11 mutations are novel mutations. In addition, we found that COL4A5 mutation relation mRNA levels were significantly decreased in HEK 293 T cell compared to control, while the cellular localization remained the same. Conclusions: Our research expands the spectrum of COL4A3-5 pathogenic variants, which is helpful for clinical and scientific research. [ABSTRACT FROM AUTHOR]

BackgroundMethodsResultsConclusionsBCL6 co-repressor (BCOR) gene variants are involved in oculofaciocardiodental (OFCD) syndrome, acute myeloid leukaemia, renal tumours, and photoreceptor degenerative diseases. Here, we describe a British family with a pathogenic heterozygous variant in the BCOR gene causing congenital nuclear cataract.Whole-exome sequencing was conducted on an individual affected by X-linked dominant congenital cataract in a three-generation family to establish the underlying genetic basis. Bioinformatics analysis confirmed the variants with damaging pathogenicity scores.A novel likely pathogenic frameshift variant BCOR NM_001123385.1: c.3621del; p.Lys1207AsnfsTer31, was identified and found to co-segregate with the disease in this family.This is apparently the first report of a variant in BCOR causing X-linked dominant congenital cataract which is potentially isolated or presenting with a remarkably mild systemic phenotype. Our findings extend the genetic basis for congenital cataract and add to the phenotypic spectrum of BCOR variants. [ABSTRACT FROM AUTHOR]

The current work aimed to identify genomic regions and candidate genes associated with resilience in pigs. In previous work, we proposed the body weight deviation from the expected growth curve (ΔBW) and the increase of the positive acute‐phase protein haptoglobin (ΔHP) after a vaccine challenge as resilience indicators which may be improved through selective breeding in pigs. Individuals with steady growth rate and minor activation of haptoglobin (high ΔBW and low ΔHP values) were considered resilient. In contrast, pigs with perturbed growth rate and high activation of haptoglobin (low ΔBW and high ΔHP values) were considered susceptible. Both ∆BW and ∆HP were simultaneously considered to select the most resilient (N = 40) and susceptible (N = 40) pigs. A genome‐wide association study was carried out for the pigs' response classification to the challenge test using whole‐genome sequence data (7,760,720 variants). Eleven associated genomic regions were identified, harbouring relevant candidate genes related to the immune response (such as pro‐ and anti‐inflammatory responses) and growth pathways. These associated genomic regions harboured 41 potential functional mutations (frameshift, splice donor, splice acceptor, start loss and stop loss/gain) in candidate genes. Overall, this study advances our knowledge about the genetic determinism of resilience, highlighting its polygenic nature and strong relationship with immunity and growth. [ABSTRACT FROM AUTHOR]

Spastic paraplegia type 4 (SPG4), caused by SPAST mutations, is the most predominant subtype of hereditary spastic paraplegia. Most documented SPG4 patients present as pure form, with the complex form rarely reported. We described the clinical and genetic features of 20 patients with complex phenotypes of SPG4 and further explored the genotype–phenotype correlations. We collected detailed clinical data of all SPG4 patients and assessed their phenotypes. SPAST gene mutations were identified by Multiplex ligation‐dependent probe amplification in combination with whole exome sequencing. We further performed statistical analysis in genotype and phenotype among patients with various manifestations and different variants. Out of 90 SPG4 patients, 20 patients (male:female = 16:4) with additional neurologic deficits, namely complex form, were included in our study. The bimodal distribution of age of onset at 0–10 and 21–40 years old is concluded. On cranial MRI, obvious white matter lesions can be observed in five patients. We identified 9 novel and 8 reported SPAST mutations, of which 11 mutations were located in AAA (ATPase associated with various cellular activities) domain. The AAA cassette of spastin is the hottest mutated region among complex SPG4. All patients with cognitive impairment (CI) are males (n = 9/9). Additionally, 80% patients with ataxia are due to frameshift mutations (n = 4/5). Overall, our study summarized and analyzed the genetic and phenotypic characteristics of complex SPG4, making up over 1/5 of in‐house SPG4 cohort, among which CI and ataxia are the most common features. Further studies are expected to explore the underlying mechanisms. [ABSTRACT FROM AUTHOR]

To date, only 10 of the more than 30 fur colours that had been observed in American mink (Neogale vison) have been linked to specific genes. The Royal pastel fur colour is part of a large family of brownish colours that are quite similar to one another, making breeding and selecting processes more difficult. Here we carried out whole‐genome sequencing of five American minks with Royal pastel (b/b) phenotypes originating from two distinct mink populations. We identified an insertion of endogenous retroviral element type 1 (ERV1) into the first intron of the gene encoding the HPS3 protein, which regulates the trafficking of tyrosinase‐containing vesicles to maturing melanosomes. With Cas9‐targeted nanopore sequencing, we reconstructed the full‐length sequence of the 11.7 Kb ERV1 insertion and observed hypermethylation that spread to the HPS3 gene promoter region. These findings highlight the role of HPS3 in the formation of melanosomes and melanin, as well as the genetic process regulating the intensity and spectrum of hair colour. Moreover, in mink breeding projects, these data are also useful for tracking economically important fur qualities. [ABSTRACT FROM AUTHOR]

POLE and POLD1 proofreading deficiency (POLE/D1pd) define a rare subtype of ultramutated metastatic colorectal cancer (mCRC; over 100 mut/Mb). Disease-specific data about the activity and efficacy of immune checkpoint inhibitors (ICIs) in POLE/D1pd mCRC are lacking and it is unknown whether outcomes may be different from mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRCs treated with ICIs. In this global study, we collected 27 patients with mCRC harboring POLE/D1 mutations leading to proofreading deficiency and treated with anti-programmed cell death-ligand 1 alone +/− anti-cytotoxic T-lymphocyte antigen-4 agents. We collected clinicopathological and genomic characteristics, response, and survival outcomes after ICIs of POLE/D1pd mCRC and compared them with a cohort of 610 dMMR/MSI-H mCRC patients treated with ICIs. Further genomic analyses were carried out in an independent cohort of 7241 CRCs to define POLE and POLD1pd molecular profiles and mutational signatures. POLE/D1pd was associated with younger age, male sex, fewer RAS/BRAF driver mutations, and predominance of right-sided colon cancers. Patients with POLE/D1pd mCRC showed a significantly higher overall response rate (ORR) compared to dMMR/MSI-H mCRC (89% versus 54%; P = 0.01). After a median follow-up of 24.9 months (interquartile range: 11.3-43.0 months), patients with POLE/D1pd showed a significantly superior progression-free survival (PFS) compared to dMMR/MSI-H mCRC [hazard ratio (HR) = 0.24, 95% confidence interval (CI) 0.08-0.74, P = 0.01] and superior overall survival (OS) (HR = 0.38, 95% CI 0.12-1.18, P = 0.09). In multivariable analyses including the type of DNA repair defect, POLE/D1pd was associated with significantly improved PFS (HR = 0.17, 95% CI 0.04-0.69, P = 0.013) and OS (HR = 0.24, 95% CI 0.06-0.98, P = 0.047). Molecular profiling showed that POLE/D1pd tumors have higher tumor mutational burden (TMB). Responses were observed in both subtypes and were associated with the intensity of POLE/D1pd signature. Patients with POLE/D1pd mCRC showed more favorable outcomes compared to dMMR/MSI-H mCRC to treatment with ICIs in terms of tumor response and survival. • POLE/D1pd is associated with male sex, younger age, right-sided primary tumor, and uncommon KRAS mutations in mCRC. • A significantly higher ORR is observed for POLE/D1pd compared to dMMR/MSI-H mCRC treated with ICIs. • POLE/D1pd mCRC exposed to ICIs have a higher PFS and OS than dMMR/MSI-H. • POLE/D1pd mCRCs are enriched in single nucleotide variant while dMMR/MSI-H are enriched in frameshift mutations. • Non-responder POLEpd mCRCs have lower TMB than responders and lower intensity of the POLE signature. [ABSTRACT FROM AUTHOR]

This article presents a case report of a 12-year-old girl with atypical Mowat-Wilson syndrome (aMOWS), a milder form of the rare developmental disorder characterized by distinct facial features, intellectual disability, and language impairment. The girl in the case had mild intellectual disability, dystonia, and choreoathetosis, which are movement disorders not typically associated with MOWS. The study emphasizes the need to consider aMOWS in patients with mild intellectual disability and movement disorders, challenging previous assumptions about the behavioral patterns of MOWS. [Extracted from the article]

PURA syndrome is a congenital developmental disorder caused by de novo mutations in the PURA gene, which encodes a DNA/RNA-binding protein essential for transcriptional and translational regulation. We present the case of an 11-year-old patient with a de novo frameshift variant in the PURA gene, identified through whole exome sequencing (WES). In addition to the classical PURA deficiency phenotype, our patient exhibited pronounced sialorrhea and seizures, which were effectively treated with the ketogenic diet (KD). Our integrative approach, combining a literature review and bioinformatics data, has led to the first documented clinical case showing improvement in both sialorrhea and seizures with KD treatment, a phenomenon not previously reported. Although a direct relationship between the de novo PURA mutation and the KD was not established, we identified a novel frameshift deletion associated with a new clinical phenotype. [ABSTRACT FROM AUTHOR]

Introduction: Myeloid neoplasms with germline RUNX1 mutation, an autosomal dominant disease characterized by thrombocytopenia and risk of hematologic malignancy, usually have a single nucleotide variant or insertions/deletions in RUNX1. Here, we present a patient with double nucleotide variant (DNV) in RUNX1. Case Presentation: The patient was a woman with progressive thrombocytopenia. She had a strong family history of thrombocytopenia and hematologic malignancies with a high fatality rate. We detected that genomic RUNX1 had consecutive DNV (c.352G>T and c.352-1 G>C) at its splice site, resulting in a frameshift mutation in exon 5. She rapidly progressed to a high-risk myeloid neoplasm owing to a germline RUNX1 mutation. Allogeneic transplantation successfully resulted in durable complete remission. Conclusion: To our knowledge, this is the first report of hereditary myeloid neoplasm carrying DNV in RUNX1. A family history of thrombocytopenia and the rapid malignant transformation suggests the potential pathogenic significance of the DNV. [ABSTRACT FROM AUTHOR]

In Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and gold standard phenotypic drug sensitivity testing (DST) questions the significance of embB406 mutations used in molecular DST. This study tabulates embB mutations found in Canadian M. tuberculosis isolates and evaluates the impact of specific mutations on ethambutol resistance. The National Reference Centre for Mycobacteriology culture collection (n = 2796) was screened for isolates with embB mutations. Phenotypic DST was performed on the BACTEC™ MGIT™ 960 at ethambutol concentrations of 2–5 μg/mL. Whole genome sequencing was used for drug resistance predictions, phylogenomics and single nucleotide polymorphism analysis. Detection of resistance-associated embB mutations corresponded to a positive predictive value of 64.3%, negative predictive value of 99.2%, 98.7% specificity, and 73.3% sensitivity compared to phenotypic DST. Two embB406 mutation subtypes (Gly406Asp, Gly406Ala) were found among 16 isolates, of which 12 were sensitive at 5 µg/mL ethambutol with variable resistance between 2–4 µg/mL. A novel frameshift mutation in regulator embR (Gln258fs) was found in nine isolates. Mutations in embB406 were associated with low-level ethambutol resistance undetectable at the recommended critical concentration (5 μg/mL). These novel mutations may exacerbate variability in ethambutol resistance. [ABSTRACT FROM AUTHOR]

The clinical characteristics, genetic mutation spectrum, treatment strategies and prognoses of 15 children with Dent disease were retrospectively analyzed to improve pediatricians' awareness of and attention to this disease. We analyzed the clinical and laboratory data of 15 Chinese children with Dent disease who were diagnosed and treated at our hospital between January 2017 and May 2023 and evaluated the expression of the CLCN5 and OCRL1 genes. All 15 patients were male and complained of proteinuria, and the incidence of low-molecular-weight proteinuria (LMWP) was 100.0% in both Dent disease 1 (DD1) and Dent disease 2 (DD2) patients. The incidence of hypercalciuria was 58.3% (7/12) and 66.7% (2/3) in DD1 and DD2 patients, respectively. Nephrocalcinosis and nephrolithiasis were found in 16.7% (2/12) and 8.3% (1/12) of DD1 patients, respectively. Renal biopsy revealed focal segmental glomerulosclerosis (FSGS) in 1 patient, minimal change lesion in 5 patients, and small focal acute tubular injury in 1 patient. A total of 11 mutations in the CLCN5 gene were detected, including 3 missense mutations (25.0%, c.1756C > T, c.1166T > G, and c.1618G > A), 5 frameshift mutations (41.7%, c.407delT, c.1702_c.1703insC, c.137delC, c.665_666delGGinsC, and c.2200delG), and 3 nonsense mutations (25.0%, c.776G > A, c.1609C > T, and c.1152G > A). There was no significant difference in age or clinical phenotype among patients with different mutation types (p > 0.05). All three mutations in the OCRL1 gene were missense mutations (c.1477C > T, c.952C > T, and c.198A > G). Pediatric Dent disease is often misdiagnosed. Protein electrophoresis and genetic testing can help to provide an early and correct diagnosis. [ABSTRACT FROM AUTHOR]

Chorea-acanthocytosis (ChAc) is a rare autosomal recessive inherited syndrome with heterogeneous symptoms, which makes it a challenge for early diagnosis. The mutation of VPS13A is considered intimately related to the pathogenesis of ChAc. To date, diverse mutation patterns of VPS13A, consisting of missense, nonsense, and frameshift mutations, have been reported. In this study, we first report a clinical case that was misdiagnosed as epilepsy due to recurrent seizures accompanied by tongue bite for 9 months, which was not rectified until seizures were controlled and involuntary orolingual movements with awareness became prominent and were confirmed to be orolingual dyskinesia. The patient was eventually diagnosed as ChAc based on whole-exome sequencing revealing novel homozygous c.2061dup (frameshift mutation) and c.6796A > T dual mutations in VPS13A. The patient from a family with consanguineous marriage manifested epileptic seizures at onset, including both generalized tonic-- clonic seizures and absence but normal long-term electroencephalography, and gradually developed orofacial dyskinesia, including involuntary tongue protrusion, tongue biting and ulcers, involuntary open jaws, occasionally frequent eye blinks, and head swings. The first test of the peripheral blood smear was negative, and repeated checks confirmed an elevated percentage of acanthocytes by 15--21.3%. Structural brain MRI indicated a mildly swollen left hippocampus and parahippocampal gyrus and a progressively decreased volume of the bilateral hippocampus 1 year later, along with atrophy of the head of the caudate nucleus but no progression in 1 year. We deeply analyzed the reasons for long-term misdiagnosis in an effort to achieve a more comprehensive understanding of ChAc, thus facilitating early diagnosis and treatment in future clinical practice. [ABSTRACT FROM AUTHOR]

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position. [ABSTRACT FROM AUTHOR]

Simple Summary: The live attenuated infectious bronchitis virus vaccine strain QXL87 was derived from the parental virulent strain JS/2010/12 through serial passages in specific pathogen-free (SPF) chicken embryos. However, the molecular mechanism underlying its attenuation remains unknown. In this study, the complete genome sequences of both strains were obtained through next-generation sequencing technology. By comparing amino acids, protein structures, and functional sites, three potential virulence genes (Nsp2, Nsp3, and S) were identified as likely contributors to altering viral pathogenicity. Furthermore, analysis of the effects of single amino acid mutations on protein function and stability revealed three potential functional mutation sites: P106S (Nsp2), A352T (Nsp2), and L472F (Nsp2). This research provides an initial insight into the molecular basis of IBV attenuation and is expected to facilitate the development of new vaccines. The QXL87 live attenuated vaccine strain for infectious bronchitis represents the first approved QX type (GI-19 lineage) vaccine in China. This strain was derived from the parental strain CK/CH/JS/2010/12 through continuous passage in SPF chicken embryos. To elucidate the molecular mechanism behind its attenuation, whole-genome sequencing was conducted on both the parental and attenuated strains. Analysis revealed 145 nucleotide mutations in the attenuated strain, leading to 48 amino acid mutations in various proteins, including Nsp2 (26), Nsp3 (14), Nsp4 (1), S (4), 3a (1), E (1), and N (1). Additionally, a frameshift mutation caused by a single base insertion in the ORFX resulted in a six-amino-acid extension. Subsequent comparison of post-translational modification sites, protein structure, and protein–protein binding sites between the parental and attenuated strains identified three potential virulence genes: Nsp2, Nsp3, and S. The amino acid mutations in these proteins not only altered their conformation but also affected the distribution of post-translational modification sites and protein–protein interaction sites. Furthermore, three potential functional mutation sites—P106S, A352T, and L472F, all located in the Nsp2 protein—were identified through PROVEAN, PolyPhen, and I-Mutant. Overall, our findings suggest that Nsp2, Nsp3, and S proteins may play a role in modulating IBV pathogenicity, with a particular focus on the significance of the Nsp2 protein. This study contributes to our understanding of the molecular mechanisms underlying IBV attenuation and holds promise for the development of safer live attenuated IBV vaccines using reverse genetic approaches. [ABSTRACT FROM AUTHOR]

Background: Biallelic pathogenic variants in USH2A lead to Usher syndrome or non-syndromic retinitis pigmentosa, and shown to have geographical and ethnical distribution in previous studies. This study provided a deeper understanding of the detailed clinical features using multimodal imaging, genetic spectrum, and genotype–phenotype correlations of USH2A-related retinal dystrophies in Taiwan. Results: In our cohort, the mean age at first visit was 47.66 ± 13.54 years, and the mean age at symptom onset, which was referred to the onset of nyctalopia and/or visual field constriction, was 31.21 ± 15.24 years. Among the variants identified, 23 (50%) were missense, 10 (22%) were splicing variants, 8 (17%) were nonsense, and 5 (11%) were frameshift mutations. The most predominant variant was c.2802T>G, which accounted for 21% of patients, and was located in exon 13. Patients with truncated alleles had significantly earlier symptom onset and seemly poorer disease progression regarding visual acuity, ellipsoid zone line length, and hypofluorescent lesions in the macula than those who had the complete gene. However, the clinical presentation revealed similar progression between patients with and without the c.2802T>G variant. During long-term follow-up, the patients had different ellipsoid zone line progression rates and were almost evenly distributed in the fast, moderate, and slow progression subgroups. Although a younger onset age and a smaller baseline intact macular area was observed in the fast progression subgroup, the results showed no significant difference. Conclusions: This is the first cohort study to provide detailed genetic and longitudinal clinical analyses of patients with USH2A-related retinal dystrophies in Taiwan. The mutated allele frequency in exon 13 was high in Taiwan due to the predominant c.2802T>G variant. Moreover, truncated variants greatly impacted disease progression and determined the length of therapeutic windows. These findings provide insight into the characteristics of candidates for future gene therapies. [ABSTRACT FROM AUTHOR]

Background Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes, which lead to high microsatellite instability and frameshift mutations at coding mononucleotide repeats in the genome. Recurrent frameshift mutations in these regions are thought to play a central role in the increased risk of various cancers, but no biomarkers are currently available for the surveillance of high microsatellite instability-associated cancers. Methods A frameshift mutation-based biomarker panel was developed and validated by targeted next-generation sequencing of supernatant DNA from cultured high microsatellite instability colorectal cancer cells. This panel supported selection of 122 frameshift mutation targets as potential biomarkers. This biomarker panel was then tested using matched tumor, adjacent normal tissue, and buffy coat samples (53 samples) and blood-derived cell-free DNA (cfDNA) (38 samples) obtained from 45 high microsatellite instability and mismatch repair-deficient patients. We also sequenced cfDNA from 84 healthy participants to assess background noise. Results Recurrent frameshift mutations at coding mononucleotide repeats were detectable not only in tumors but also in cfDNA from high microsatellite instability and mismatch repair-deficient patients, including a Lynch syndrome carrier, with a varying range of target detection (up to 85.2%), whereas they were virtually undetectable in healthy participants. Receiver operating characteristic curve analysis showed high sensitivity and specificity (area under the curve = 0.94) of the investigated panel. Conclusions We demonstrated that frameshift mutations can be detected in cfDNA from high microsatellite instability and mismatch repair-deficient patients and asymptomatic carriers. The 122-target frameshift mutation panel described here has promise as a tool for improved surveillance of high microsatellite instability and mismatch repair-deficient patients, with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort. [ABSTRACT FROM AUTHOR]

Background: Hereditary sensory and autonomic neuropathies (HSANs) are rare heterogeneous group of neurological disorders caused by peripheral nerve deterioration. The HSANs sub-clinical classes have clinical and genetic overlap which often lead to misdiagnosis. In the present study a Pakistani family with five affected members suffering from severe neuropathy were genetically analyzed to identify the disease causative element in the family. Methods: Genome wide high-density single nucleotide polymorphism (SNP) microarray analysis was carried out followed by whole exome sequencing of the affected proband and another affected sibling. Shared homozygous regions in all severely affected members were identified through homozygosity mapping approach. Results: The largest homozygous region of 14.1 Mb shared by the five severely affected members of the family was identified on chromosome 2. Subsequent exome sequencing identified a novel single nucleotide deletion c.2658del; p.(Ser887Profs*64) in KIF1A. Segregation analysis revealed that this mutation was homozygous in all five affected individuals of the family with severe clinical manifestation, while members of the family that were heterozygous carriers shared abnormal skin features (scaly skin) only with the homozygous affected members. Conclusions: A novel frameshift mutation p.(Ser887Profs*64) in KIF1A is the potential cause of severe HSANIIC in a Pakistani family along with incomplete penetrance in mutation carriers. We demonstrate that using a combination of different techniques not only strengthens the gene finding approach but also helps in proper sub-clinical characterization along with identification of mutated alleles exhibiting incomplete penetrance leading to intrafamilial clinical variability in HSAN group of inherited diseases. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 11 (SCA11) is a rare disease and the tau tubulin kinase 2 (TTBK2) gene was the causative gene. To date, only six SCA11 families have been reported. Here, we reported a Chinese SCA11 pedigree with cerebellar ataxia. Both patients in the family demonstrated typical clinical features of cerebellar ataxia and cerebellar atrophy on brain MRI. A novel heterozygous duplication mutation (c.1211_1217dupAGGAGAA) of the TTBK2 gene was identified in the proband using whole-exome sequencing (WES), which resulted in a frameshift mutation and formed a premature stop codon (p. N406Kfs*47). The mutation was detected in the proband's affected brother, and his unaffected mother, who with a lower percentage of the mutation and considered as an asymptomatic mutation carrier. Our study delineated the genotypic spectrum of SCA11. [ABSTRACT FROM AUTHOR]

Hypogammaglobulinemia without B-cells is a subgroup of inborn errors of immunity (IEI) which is characterized by a significant decline in all serum immunoglobulin isotypes, coupled with a pronounced reduction or absence of B-cells. Approximately 80 to 90% of individuals exhibit genetic variations in Bruton's agammaglobulinemia tyrosine kinase (BTK), whereas a minority of cases, around 5–10%, are autosomal recessive agammaglobulinemia (ARA). Very few cases are grouped into distinct subcategories. We evaluated phenotypically and genetically 27 patients from 13 distinct families with hypogammaglobinemia and no B-cells. Genetic analysis was performed via whole-exome and Sanger sequencing. The most prevalent genetic cause was mutations in BTK. Three novel mutations in the BTK gene include c.115 T > C (p. Tyr39His), c.685-686insTTAC (p.Asn229llefs5), and c.163delT (p.Ser55GlnfsTer2). Our three ARA patients include a novel homozygous stop-gain mutation in the immunoglobulin heavy constant Mu chain (IGHM) gene, a novel frameshift mutation of the B-cell antigen receptor complex-associated protein (CD79A) gene, a novel bi-allelic stop-gain mutation in the transcription factor 3 (TCF3) gene. Three patients with agammaglobulinemia have an autosomal dominant inheritance pattern, which includes a missense variant in PIK3CD, a novel missense variant in PIK3R1 and a homozygous silent mutation in the phosphoinositide-3-kinase regulatory subunit (RASGRP1) gene. This study broadens the genetic spectrum of hypogammaglobulinemia without B-cells and presented a few novel variants within the Iranian community, which may also have implications in other Middle Eastern populations. Notably, disease control was better in the second affected family member in families with multiple cases. [ABSTRACT FROM AUTHOR]

Objective: Mutations in the gene encoding for optineurin (OPTN) have been reported in the context of different neurodegenerative diseases including the amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum. Based on single case reports, neuropathological data in OPTN mutation carriers have revealed transactive response DNA‐binding protein 43 kDa (TDP‐43) pathology, in addition to accumulations of tau and alpha‐synuclein. Herein, we present two siblings from a consanguineous family with a homozygous frameshift mutation in the OPTN gene and different clinical presentations. Methods: Both affected siblings underwent (i) clinical, (ii) neurophysiological, (iii) neuropsychological, (iv) radiological, and (v) laboratory examinations, and (vi) whole‐exome sequencing (WES). Postmortem histopathological examination was conducted in the index patient, who deceased at the age of 41. Results: The index patient developed rapidly progressing clinical features of upper and lower motor neuron dysfunction as well as apathy and cognitive deterioration at the age of 41. Autopsy revealed an ALS‐FTLD pattern associated with prominent neuronal and oligodendroglial TDP‐43 pathology, and an atypical limbic 4‐repeat tau pathology reminiscent of argyrophilic grain disease. The brother of the index patient exhibited behavioral changes and mnestic deficits at the age of 38 and was diagnosed with behavioral FTD 5 years later, without any evidence of motor neuron dysfunction. WES revealed a homozygous frameshift mutation in the OPTN gene in both siblings (NM_001008212.2: c.1078_1079del; p.Lys360ValfsTer18). Interpretation: OPTN mutations can be associated with extensive TDP‐43 pathology and limbic‐predominant tauopathy and present with a heterogeneous clinical phenotype within the ALS‐FTD spectrum within the same family. [ABSTRACT FROM AUTHOR]

Introduction: Pseudohypoparathyroidism type IA (PHP1A) is characterized by end-organ resistance to multiple hormones and Albright's hereditary osteodystrophy (AHO). PHP1A is caused by inactivating mutations of the GNAS gene encoding the α-subunit of the stimulatory G protein (Gsα). In line with the underlying genetic defect, impaired inhibition of platelet aggregation has been demonstrated in some patients. However, no PHP1A case with thrombotic events has been described. Also, PHP1A cases typically have subcutaneous ossifications, but soft tissue calcifications are another common finding. Treatment options for those and other nonhormonal features of PHP1A are limited. Case Presentation: A female patient presented with short stature, fatigue, and exercise-induced carpopedal spasms at age 117/12 years. Diagnosis of PHP1A was made based on hypocalcemia, hyperphosphatemia, elevated serum parathyroid hormone, and AHO features, including short stature and brachydactyly. A novel frameshift variant was detected in the last exon of GNAS (c.1065_1068delGCGT, p.R356Tfs*47), showing complete loss of baseline and receptor-stimulated activity in transfected cells. The patient developed venous thrombosis and vascular and subcutaneous calcifications on both forearms after venous puncture on the right and extravasation of calcium gluconate during treatment on the left. The thrombosis and calcifications completely resolved following treatment with low-molecular-weight heparin and acetazolamide for 5 and 8 months, respectively. Conclusions: This case represents the first PHP1A patient displaying thrombosis and the first successful use of acetazolamide for PHP1A-associated soft tissue calcifications, thus providing new insights into the treatment of non-endocrinological features in this disease. Established Facts: Pseudohypoparathyroidism type IA (PHP1A) is caused by inactivating mutations of the GNAS gene encoding the α-subunit of the stimulatory G protein (Gsα), resulting in end-organ resistance to hormone actions and laboratory evidence for impaired inhibition of platelet aggregation. In addition to subcutaneous ossifications, patients with PHP1A commonly exhibit soft tissue calcifications. Acetazolamide has shown successful outcomes in treating soft tissue calcification in patients with tumoral calcinosis. [ABSTRACT FROM AUTHOR]

• RUNX1 mutation and three chimeras were found in ASM without KIT D816V and JAK2 V617F. • The novel KAT6A::NCOA2 and RXRA::NOTCH1 originated from cryptic abnormalities. • The PRKG2::PDGFRB chimera persisted in the evolution of the patients towards SM-AHN. • The combined transcriptional/mutational profiling aids ASM diagnosis and management. Systemic mastocytosis (SM) is a myeloproliferative neoplasm displaying abnormal mast cell proliferation. It is subdivided into different forms, including aggressive systemic mastocytosis (ASM) and systemic mastocytosis with an associated hematologic neoplasm (SM-AHN). Oncogenic genetic alterations include point mutations, mainly the KIT D816V, conferring poor prognosis and therapy resistance, and fusion genes, with those involving PDGFRA / PDGFRB as the most recurrent events. We here describe an ASM case negative to the KIT D816V and JAK2 V617F alterations but showing a RUNX1 frameshift heterozygous mutation and the co-occurrence of three fusion transcripts. The first one, PRKG2::PDGFRB, was generated by a balanced t(4;5)(q24;q32) translocation as the sole abnormality. Other two novel chimeras, KAT6A::NCOA2 and RXRA::NOTCH1, originated from cryptic intra-chromosomal abnormalities. The patient rapidly evolved towards SM-AHN, characterized by the persistence of the PRKG2::PDGFRB chimera, due to the presence of an extra copy of the der(5)t(4;5)(q24;q34) chromosome and an increase in the RUNX1 mutation allelic frequency. The results indicated that the transcriptional landscape and the mutational profile of SM deserve attention to predict the evolution and prognosis of this complex disease, whose classification criteria are still a matter of debate. [ABSTRACT FROM AUTHOR]

The development of the Escherichia coli K‐12 laboratory strains JM83, JM109 and XL1‐Blue was instrumental in early gene technology. We report the comprehensive genome sequence analysis of JM83 and XL1‐Blue using Illumina and Oxford Nanopore technologies and a comparison with both the wild‐type sequence (MG1655) and the genome of JM109 deposited at GenBank. Our investigation provides insight into the way how the genomic background that allows blue/white colony selection—by complementing a functionally inactive ω‐fragment of β‐galactosidase (LacZ) with its α‐peptide encoded on the cloning vector—has been implemented independently in these three strains using classical bacterial genetics. In fact, their comparative analysis reveals recurrent motifs: (i) inactivation of the native enzyme via large deletions of chromosomal regions encompassing the lac locus, or a chemically induced frameshift deletion at the beginning of the lacZ cistron, and (ii) utilization of a defective prophage (ϕ80), or an F′‐plasmid, to provide the lacZ∆M15 allele encoding its ω‐fragment. While the genetic manipulations of the E. coli strains involved repeated use of mobile genetic elements as well as harsh chemical or physical mutagenesis, the individual modified traits appear remarkably stable as they can be found even in distantly related laboratory strains, beyond those investigated here. Our detailed characterization at the genome sequence level not only offers clues about the mechanisms of classical gene transduction and transposition but should also guide the future fine‐tuning of E. coli strains for gene cloning and protein expression, including phage display techniques, utilizing advanced tools for site‐specific genome engineering. [ABSTRACT FROM AUTHOR]

Background: Microsatellite instability (MSI) secondary to mismatch repair (MMR) deficiency is characterized by insertions and deletions (indels) in short DNA sequences across the genome. These indels can generate neoantigens, which are ideal targets for precision immune interception. However, current neoantigen databases lack information on neoantigens arising from coding microsatellites. To address this gap, we introduce The MicrOsatellite Neoantigen Discovery Tool (MONET). Method: MONET identifies potential mutated tumor-specific neoantigens (neoAgs) by predicting frameshift mutations in coding microsatellite sequences of the human genome. Then MONET annotates these neoAgs with key features such as binding affinity, stability, expression, frequency, and potential pathogenicity using established algorithms, tools, and public databases. A userfriendly web interface (https://monet.mdanderson.org/) facilitates access to these predictions. Results: MONET predicts over 4 million and 15 million Class I and Class II potential frameshift neoAgs, respectively. Compared to existing databases, MONET demonstrates superior coverage (>85% vs. <25%) using a set of experimentally validated neoAgs. Conclusion: MONET is a freely available, user-friendly web tool that leverages publicly available resources to identify neoAgs derived from microsatellite loci. This systems biology approach empowers researchers in the field of precision immune interception. [ABSTRACT FROM AUTHOR]

Arrhythmic risk stratification in patients with Lamin A/C gene (LMNA)-related cardiomyopathy influences clinical decisions. An implantable cardioverter defibrillator (ICD) should be considered in patients with an estimated 5-year risk of malignant ventricular arrhythmia (MVA) of ≥10%. The risk prediction score for MVA includes non-missense LMNA mutations, despite their role as an established risk factor for sudden cardiac death (SCD) has been questioned in several studies. The purpose of this study is to investigate cardiac features and find gene–phenotype correlations that would contribute to the evidence on the prognostic implications of non-missense vs. missense mutations in a cohort of LMNA mutant patients. An observational, prospective study was conducted in which 54 patients positive for a Lamin A/C mutation were enrolled, and 20 probands (37%) were included. The median age at first clinical manifestation was 41 (IQR 19) years. The median follow-up was 8 years (IQR 8). The type of LMNA gene mutation was distributed as follows: missense in 26 patients (48%), non-frameshift insertions in 16 (30%), frameshift deletions in 5 (9%), and nonsense in 7 (13%). Among the missense mutation carriers, two (8%) died and four (15%) were admitted onto the heart transplant list or underwent transplantation, with a major adverse cardiovascular event (MACE) rate of 35%. No statistically significant differences in MACE prevalence were identified according to the missense and non-missense mutation groups (p value = 0.847). Our data shift the spotlight on this considerable topic and could suggest that some missense mutations may deserve attention regarding SCD risk stratification in real-world clinical settings. [ABSTRACT FROM AUTHOR]

Cherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disorders. [ABSTRACT FROM AUTHOR]

Bilateral breast cancer (BBC), an infrequent breast cancer subtype, has primarily been studied in terms of incidence, prognosis, and through comparative analysis of synchronous (SBBC) and metachronous (MBBC) manifestations. The advent and application of organoid technology hold profound implications for tumor research and clinical management. This study represents the pioneering use of organoid models in BBC research. We established organoid lines from two surgical tumor specimens of a BBC patient, with one line undergoing detailed pathological and genomic analysis. The BBC organoid from the right breast demonstrated a marker expression profile of ER (-), PR (-), HER-2 (0), and Ki67 index 10%, indicating that it may derived from the TNBC tissue. Whole Exome Sequencing (WES) displayed consistent set of Top10 cancer driver genes affected by missense mutations, frameshift mutation, or splice site mutations in three tumor tissues and the organoid samples. The organoids' single nucleotide polymorphisms (SNPs) were more closely aligned with the TNBC tissue than other tumor tissues. Evolutionary analysis suggested that different tumor regions might evolve from a common ancestral layer. In this case, the development of BBC organoids indicated that simultaneous lesions with diverse molecular profiles shared a high degree of consistency in key tumor-driving mutations. These findings suggest the feasibility of generating BBC organoids representing various molecular types, accurately replicating significant markers and driver mutations of the originating tumor. Consequently, organoids serve as a valuable in vitro model for exploring treatment strategies and elucidating the underlying mechanisms of BBC. [ABSTRACT FROM AUTHOR]

In this study, we report the mutational profiles, pathogenicity, and their association with different clinicopathologic and sociogenetic factors in patients with Pashtun ethnicity for the first time. A total of 19 FFPE blocks of invasive ductal carcinoma (IDC) from the Breast Cancer (BC) tissue and 6 normal FFPE blocks were analyzed by whole-exome sequencing (WES). Various somatic and germline mutations were identified in cancer-related genes, i.e., ATM, CHEK2, PALB2, and XRCC2. Among a total of 18 mutations, 14 mutations were somatic and 4 were germline. The ATM gene exhibited the maximum number of mutations (11/18), followed by CHEK2 (3/18), PALB2 (3/18), and XRCC2 (1/18). Except one frameshift deletion, all other 17 mutations were nonsynonymous single-nucleotide variants (SNVs). SIFT prediction revealed 7/18 (38.8%) mutations as deleterious. PolyPhen-2 and MutationTaster identified 5/18 (27.7%) mutations as probably damaging and 10/18 (55.5%) mutations as disease-causing, respectively. Mutations like PALB2 p.Q559R (6/19; 31.5%), XRCC2 p.R188H (5/19; 26.31%), and ATM p.D1853N (4/19; 21.05%) were recurrent mutations and proposed to have a biomarker potential. The protein network prediction was performed using GeneMANIA and STRING. ISPRED-SEQ indicated three interaction site mutations which were further used for molecular dynamic simulation. An average increase in the radius of gyration was observed in all three mutated proteins revealing their perturbed folding behavior. Obtained SNVs were further correlated with various parameters related to the clinicopathological status of the tumors. Three mutation positions (ATM p. D1853N, CHEK2 p.M314I, and PALB2 p.T1029S) were found to be highly conserved. Finally, the wild- and mutant-type proteins were screened for two drugs: elagolix (DrugBank ID: DB11979) and LTS0102038 (a triterpenoid, isolated from the anticancer medicinal plant Fagonia indica). Comparatively, a higher number of interactions were noted for normal ATM with both compounds, as compared to mutants. [ABSTRACT FROM AUTHOR]