Nonsense Mutation Research Articles

536. TUMOR-INFORMED “LIQUID BIOPSY” FOR ESOPHAGEAL ADENOCARCINOMA FROM MATCHED CANCER ORGANOID CULTURE

Abstract Background The absence of recurrent mutations in esophageal adenocarcinoma (EAC) poses a challenge in detecting circulating tumor DNA (ctDNA) in plasma and may hinder the advancement of liquid biopsy methods. To address this, we cultured patient-derived EAC organoids (PDOs), speculating that they could serve as a guide for identifying ctDNA in the patient's blood samples. This approach aims to leverage organoids as a potential tool to overcome the complexity of identifying ctDNA in EAC, offering a promising avenue for refining liquid biopsy strategies in clinical practice. Methods PDOs were generated from EAC tumor tissue in Matrigel domes and expanded in suspension culture. To isolate mononucleosomes (147 bp), chromatin from PDOs was extracted and digested with micrococcal nuclease (MNase). Fragments larger than 147 bp were removed through size selection. MNase-sequencing was performed to generate a mutation map with preferential coverage of nucleosome-protected DNA for each sample. Matched whole genome sequencing of the tumor for each respective PDO sample was used as a control. Primers were designed for the identified mutations in nucleosome-protected DNA and used to amplify patient cfDNA for sequencing. Results DNA from five different PDOs were collected and MNase digested. MNase concentration and digestion time were optimized for each sample. MNase digestion produced mononucleosomes of approximately 147 bp for all samples. MNase-sequencing identified 24 mutations in peaks (mononucleosomes) in 24 genes, including known oncogenes. Among these were 16 missense, 2 frameshift, and 1 nonsense mutations, and 5 mutations in splice regions. To date, amplicons of expected size were detected by PCR for six genes using either total PDO DNA or normal cell-free DNA, confirming the detectability of these genes. PCR amplification using patient ctDNA and next-generation sequencing is ongoing. Conclusion These findings show that we are able to isolate and detect somatic mutations in nucleosomes from different PDOs, allowing us to generate a nucleosome SNV map for each sample. Preliminary data indicate these regions can be PCR amplified from normal cfDNA. Amplification and sequence verification of mutated regions from corresponding patient blood ctDNA is ongoing. The mapping of patient-specific variants will enable the development of targeted personalized PCR panels, aiding in recurrence prediction and enhancing drug screening accuracy. This advancement holds promise for early cancer detection and improving prognoses for individuals with EAC by addressing gaps in recurrence prediction.

124P Incidence of activating frameshift and nonsense mutations in clinically actionable oncogenes

124P Incidence of activating frameshift and nonsense mutations in clinically actionable oncogenes

Characterization of 19 novel gene mutation sites associated with autosome-dominant polycystic kidney disease

By analyzing the of genetic testing data of patients with renal polycystic kidney disease and their relatives, this study aims to identify unreported novel gene mutation sites associated with autosomal dominant polycystic kidney disease (ADPKD). Structural prediction software was employed to investigate protein structural changes before and after mutations, explore genotype-phenotype correlations, and enrich the ADPKD gene database. In this single-center retrospective study, patients with multiple renal cysts diagnosed from January 2019 to February 2023 at the Zhong Da Hospital Southeast University were included. Genetic and clinical data of patients and their families were collected. Unreported novel gene mutation sites associated with ADPKD were identified. The AlphaFold v2.3.1 software was used to predict protein structures. Changes in protein structure before and after mutations were compared to explore genotype-phenotype correlations and enrich the ADPKD gene database. Twelve mutated genes associated with renal cysts were detected in 52 families. Nineteen novel gene mutation sites associated with ADPKD were identified, including 17 mutations in the PKD1 gene (one splicing mutation, seven frameshift mutations, four nonsense mutations, one whole-codon insertion, and four missense mutations); one ALG9 missense mutation; and one chromosomal structural variation. Truncating mutations in the PKD1 gene were correlated with a more severe clinical phenotype, while non-truncating mutations were associated with greater clinical heterogeneity. Numerous novel gene mutation sites associated with ADPKD remain unreported. Therefore, it is essential to analyze the pathogenicity of these novel mutation sites, establish genotype-phenotype correlations, and enrich the ADPKD gene database.

Site-specific bioorthogonal regulation of bone morphogenetic protein 2 expression for effective bone regeneration

Growth factor holds great promise for bone regeneration, and spatiotemporal control of their expressing through site-specific reactions is crucial but challenging for on-demand therapy. In this study, we present the development of a novel unnatural amino acids (UAAs)-triggered therapeutic switch (UATS) system, composed of an orthogonal aminoacyl-tRNA-synthase (aaRS)-tRNA pair and a bone morphogenetic protein 2 (BMP2) gene harboring premature stop codon, which enable in situ and on-demand initiation of the expression of BMP2. The resulting UATS system allowed specifically control of base expressing on the BMP2 mRNA that switched to the BMP2 protein with complete structure and function to facilitate bone regeneration. Our investigations showed that the UATS system exhibits remarkable attributes of rapid, sensitive, reversible, and sustained BMP2 expression both in vitro and in vivo settings. Moreover, the implantation of microencapsulated cells with UATS system is applied to a mouse femur defect model, demonstrating high effciency in controlled expressing of BMP2 protein and substantial repair of bone defect following oral administration of UAAs. Therefore, our findings underscore the great potential of UATS system for on-demand awakening of functional growth factor, thus offering promising prospects in the realm of regenerative medicine.

Nonredundant Role of Leishmanolysin-Like (Lmln) Zinc-Metallopeptidase in Retinal Homeostasis

To determine if Lmln, a Zn-metallopeptidase, is important for retinal homeostasis. Combining an unbiased N-ethyl-N-nitrosourea mutagenesis pipeline in mice with optical coherence tomography (OCT) screening and automated meiotic mapping, we identified an allele (nemeth) that seemed to be associated with outer nuclear layer (ONL) thinning. Since nemeth was predicted to lead to a nonsense mutation of the Lmln gene, we targeted Lmln using CRISPR/Cas-9 technology and characterized the impact on retinal anatomy and function. OCT imaging demonstrated an outer retinal degeneration in Lmln-/- mice (P = 7.3 × 10-9 for ONL at 2 m) that progressed over the first 6 months of life and then stabilized. Light microscopy showed loss of ONL nuclei (P ranged between .00033 and .0097 for posterior measurements), and a TUNEL assay revealed a small but significant increase in apoptosis (P = .034). Lmln-/- mice accumulated fundus spots (P = .0030 by 2 m of age) and activated subretinal microglia (p ranged from .0007 to 8 × 10-13 for Gal3+ cells). Scotopic electroretinography demonstrated a decrease in retinal function in Lmln-/- mice both at 6 m (only a-wave, P < .01 for all stimuli) and at 10 m of age (P < .01 for both a-wave and b-wave with all stimuli). Our work revealed a previously unknown essential requirement for Lmln in maintaining retinal anatomy and function. Further studies using this new model will be aimed at determining the cellular expression of Lmln and its mechanisms of action within the retina.

Clinical and molecular findings in children with retinitis pigmentosa

ABSTRACT Purpose To study the clinical and genetic features of a cohort of RP children. Methods We identified 46 RP patients with pathogenic or likely pathogenic mutations among 96 patients with a clinical diagnosis of retinitis pigmentosa. All of the patients underwent comprehensive clinical examinations and genetic testing. A retrospective study was conducted on 46 children with retinitis pigmentosa. The genetic and clinical characteristics of children with different genotypes were analyzed. Results Among the 46 children, 13 inherited X-linked gene mutations, including 9 RPGR and 4 RP2 mutations. There were 10 cases of autosomal dominant genes and 23 cases of autosomal recessive genes. XLRP accounted for a larger proportion of children, as observed in previous studies on RP. We found that RPGR genes were the most commonly mutated genes in RP children. The most frequently mutated gene was RPGR (9.3%), followed by RP2 (4.2%) and RPE65 (4.2%). Forty-six patients had mutations in 21 different genes, 19 of which were novel mutations. Most children with XLRP have a high degree of myopia, poor vision, and severe clinical symptoms. Frameshift mutations were more common in XLRP, followed by nonsense mutations. The onset of XLRP is relatively serious since childhood. Most children with ADRP have relatively good visual acuity and mild clinical symptoms, and missense mutations are common. The clinical manifestations of ARRP in children are more severe than those of ADRP in children but milder than those of XLRP in children, and missense mutations are common. The manifestations of RPE65 mutations are also severe and appear early. Conclusions Our results revealed that XLRP gene mutations were more common in children than in adults, as observed in previous studies on RP. The proportion of RP children with ADRP is relatively small. The new findings in our study polished the spectrum of novel mutations and the proportions of different genotypes in pediatric patients. The onset of XLRP occurred earlier. The genes with a high incidence in children were all relatively severe gene types of RP. This comprehensive database may provide essential information regarding the initial stage of RP.

Deciphering the Genetic Basis of Kernel Composition in a Maize Association Panel.

The primary objective in contemporary maize breeding is to pursue high quality alongside high yield. Deciphering the genetic basis of natural variation in starch, protein, oil, and fiber contents is essential for manipulating kernel composition, thereby enhancing the kernel quality and meeting growing demands. Here, we identified 12 to 88 statistically significant loci associated with kernel composition traits through a genome-wide association study (GWAS) using a panel of 212 diverse inbred lines. A regional association study pinpointed numerous causal candidate genes at these loci. Coexpression and protein-protein interaction network analyses of candidate genes revealed several causal genes directly or indirectly involved in the metabolic processes related to kernel composition traits. Subsequent mutant experiment revealed that nonsense mutations in ZmTIFY12 affect starch, protein, and fiber content, whereas nonsense mutations in ZmTT12 affect starch, protein, and oil content. These findings provide valuable guidance for improving kernel quality in maize breeding efforts.

Application of Flow Cytometry for Viability Assessment of Mutants for Translation Termination Factors in the Yeast Saccharomyces cerevisiae

Nonsense mutations in the essential SUP45 and SUP35 genes, encoding translation termination factors, affect the viability of Saccharomyces cerevisiae cells. Flow cytometry revealed that the viability of mutants was 3.5‒4 times lower compared to the wild-type. Moreover, the mutants were found to have higher sensitivity to ultrasonic treatment.

Analysis of gene mutation in a family with Muir-Torre syndrome accompanied with extraorbital cystic sebaceous carcinoma

This study reported a family of MLH1 mutation-induced Muir-Torre syndrome (MTS) and evaluated it's clinical and genetic characteristics. A 51 year-old patient with extraorbital cystic sebaceous and colon adenocarcinoma diagnosed in November 2021 in Zhongshan Hospital of Xiamen University was included. The clinical data of the family were collected and a pedigree chart was drawn, which was in line with the Chinese Lynch syndrome diagnostic criteria and was a typical MTS family. NM_000249.4:c.298C>T(p.R100*) of MLH1 gene in exon 3 was detected by whole exome sequencing and multiplex ligation dependent amplification, which is a pathogenic mutation. After the pathogenic mutation was identified, Sanger sequencing was performed on 4 direct members of the family for MLH1 gene, and 3 family members were found to have detected the mutation and included in MTS risk control. Until December 25 2023, follow-up showed the proband patients were not suffered from recurrence or new occurrence of skin or gastrointestinal tumors. The study reported a typical MTS family and found a possible pathogenic nonsense mutation in the MLH1 gene, which provides new evidence for the pathogenicity of this mutation.

Pre-term Familial Exudative Vitreoretinopathy: A Novel Nonsense LRP5 Mutation.

This case report details the diagnosis and management of a pre-term infant with aggressive bilateral retinal pathology. A 4-week-old preterm baby girl, born at 28 weeks and 6 days to consanguineous parents, was referred for suspected aggressive posterior retinopathy of prematurity (ROP). She had a family history of bilateral retinal detachments and intellectual disability in an older sister. Clinical assessment included retinal examination, fluorescein angiography, optical coherence tomography, dual-energy X-ray absorptiometry (DEXA), and genetic testing. The genetic testing involved sequence analysis and copy number variation analysis of 25 genes related to vitreoretinopathy. Retinal examination and fluorescein angiography revealed extensive non-perfusion and telangiectatic vessels in both eyes, and a macula-involving tractional retinal detachment in the left eye. Despite treatment with intravitreal bevacizumab and laser photocoagulation, they progressed to total retinal detachment and no light perception in both eyes. Genetic testing revealed a pathogenic homozygous nonsense mutation in the LRP5 gene (c.3259C>T, p.(Gln1087*)), a mutation not previously reported in association with familial exudative vitreoretinopathy (FEVR). At 10 months of age, DEXA demonstrated normal bone density, diverging from the typical presentation of osteoporosis pseudoglioma syndrome associated with LRP5 mutations. This case describes a novel mutation in a complex retinal disease and underscores the necessity of considering pre-term FEVR in the differential diagnosis of atypical or aggressive ROP in preterm infants. The overlap in clinical features between ROP and FEVR highlights the complexity of diagnosis and management and the importance of genetic testing in preterm infants with retinal vascular abnormalities.

The ACTN3 p.Arg577Ter Genetic Variants Biomarker of the Fitness Performance, in Oaxaca Amerindian Population

Backgrond. ACTN3 is primarily expressed in fast skeletal muscle fibres. A common nonsense polymorphism in this gene is ACTN3 R577X (rs1815739:C&gt;T) results in replacement of an arginine (R) with a premature stop codon (X) at amino acid 577 in the fast muscle protein α-actinin-3, which causes an absolute deficiency of α-actinin-3 protein and alterations in muscle metabolism. A common null polymorphism in the ACTN3 gene The ACTN3 p.Arg577Ter allele (p.R577* or R577X) has undergone positive selection, with an increase in the X allele frequency as modern humans migrated out of Africa into the colder, less species-rich Eurasian climates Aim. Analized the frequency of allele or genotypes from ACTN3 p.Arg577Ter genetic variants biomarker of the fitness performance, in Oaxaca Amerindian population. Methods. From people of different ethnic groups the State of Oaxaca, DNA was extracted from peripheral blood to extract DNA using the geneKatcher kit (invitrogene), to amplify the polymorphism p.Arg577Ter was realized by qRT_PCR, using tqman probes by determined by allelic discrimination. Results. The more frequency allele is wild type R in all populations, more tan 50%. The Genotype more prevalent is heterocygote RX. In afromexican population Genotype XX o ter-X/ter-X is a allelic variant. Distribution allelic and genotype is verry heterogenus in the populations. In conclusión the ACTN3 R577X gene variant can be used as a clinical marker for the selection of sports talents in the state of Oaxaca, considering that it has already been validated at the population level, with the R allele being more frequent in the state, which is the one associated with a greater selective advantage in sports that require muscular strength and greater physical performance.

De Novo Deep Intron ELANE Mutation Resulting in Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) comprises a diverse range of rare hematological disorders characterized by recurrent, often life-threatening infections that manifest within the first months of life. Mutations in the ELANE gene are the most prevalent cause of SCN. While over 230 mutations in ELANE have been documented, including substitutions, frameshifts, nonsense mutations, and splice site alterations, the occurrence of deep intronic mutations has not been previously reported. Herein, we present the case of a young girl who exhibited recurrent fever, respiratory infections, skin abscesses, and gingivitis shortly after birth. Laboratory analysis revealed markedly diminished neutrophil levels alongside elevated monocyte and eosinophil counts. Bone marrow examination disclosed a halt in myelopoiesis maturation. ELANE gene full-length sequencing identified a novel de novo deep intron mutation in ELANE (c.598 + 79G > T), subsequently confirmed by Sanger sequencing. cDNA sequencing of the patient demonstrated aberrant gene splicing. Utilizing a mini-gene splicing assay for ELANE intronic variants, we identified a mutant ELANE allele (c.597 + 1_597 + 83ins) leading to the creation of a premature termination codon (p.Gly200ValfsTer40). Confocal microscopy revealed heightened expression of myeloperoxidase and neutrophil elastase in the patient, suggesting a potential role for the unfolded protein response in the pathogenesis of the deep intron ELANE mutation. In summary, our findings illustrate the first reported instance of de novo deep intron ELANE mutations associated with SCN, underscoring the importance of exploring deep intronic regions in SCN patients lacking identifiable disease-causing gene mutations.

Irregular green netting of eggplant fruit peel: a domestication trait controlled by SmGLK2 with potential for fruit colour diversification.

The distribution of chlorophylls in eggplant (Solanum melongena) peel exhibits either a uniform pattern or an irregular green netting. The latter, manifested as a gradient of dark green netting intensified in the proximal part of the fruit on a pale green background, is common in wild relatives and some eggplant landraces. Despite the selection of uniform chlorophylls during domestication, the netting pattern contributes to a greater diversity of fruit colours. Here, we have used over 2,300 individuals from different populations, including a multi-parental MAGIC population for candidate genomic region identification, an F2 population for BSA-Seq, and advanced backcrosses for edges-to-core fine-mapping, to identify SmGLK2 gene as responsible for the irregular netting in eggplant fruits. We have also analysed the gene sequence of 178 S. melongena accessions and 22 wild relative species for tracing the evolutionary changes that the gene has undergone during domestication. Three different mutations were identified leading to the absence of netting. The main causative indel induces a premature stop codon disrupting the protein conformation and function, which was confirmed by western blotting analysis and confocal microscopy observations. SmGLK2 has a major role in regulating chlorophyll biosynthesis in eggplant fruit peel.

Mar1, a high mobility group box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

Molecular insights into the oleic acid accumulation in safflower

AbstractMost of the Indian safflower (Carthamus tinctorius L.) varieties produce oil rich in linoleic acid (LA, ~75%) and low in oleic acid (OA, ~15%). In the fatty acid biosynthetic pathway, the fatty acid desaturase 2 (FAD2) enzyme converts OA to LA. Safflower is reported to have 12–20 FAD2 genes. Gene expression analysis of four FAD2 genes during seed development in a high LA variety, PBNS‐12, revealed high expression of FAD2‐1 at 21 days after flowering (DAF), correlating with high LA accumulation. Fatty acid profiling of 448 Indian safflower germplasm accessions revealed four lines to have high (58%–77%) OA content, with NASF‐39 having the highest OA content. Interestingly, all four high OA lines showed the same mutation in the FAD2‐1 gene. The DNA sequence of FAD2‐1 from the four high OA lines showed a deletion of C at the +606 position, resulting in a premature stop codon at the +733 position and a truncated protein of 244 amino acids. Hence, despite the high expression levels of FAD2‐1 in NASF‐39 at 18–21 DAF, it exhibited high OA (77%). The dysfunctional nature of the truncated FAD2‐1 in NASF‐39 was evident in molecular docking studies with 1‐stearoyl‐2‐oleoyl phosphatidylcholine. We also sequenced FATB, a thioesterase responsible for releasing stearic acid from acyl carrier protein for further desaturation to oleic acid, where an A773G substitution was observed. This resulted in E258G substitution in NASF‐39 FATB compared to that of PBNS‐12. This probably made the acyl‐binding pocket of NASF‐39 FATB unstable, contributing to high OA accumulation. Thus, the outcomes of this study can help develop super and ultra‐high oleic safflower varieties through various genetics and genomics approaches.

A lmod1a mutation causes megacystis microcolon intestinal hypoperistalsis in a CRISPR/Cas9-modified zebrafish model.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is defined as a congenital visceral myopathy with genetic mutations. However, the etiology and pathophysiology are not fully understood. We aimed to generate a gene leiomodin-1a (lmod1a) modification technique to establish a zebrafish model of MMIHS. We targeted lmod1a in zebrafish using CRISPR/Cas9. After confirming the genotype, we measured the expression levels of the target gene and protein associated with MMIHS. A gut transit assay and spatiotemporal mapping were conducted to analyze the intestinal function. Genetic confirmation showed a 5-base-pair deletion in exon 1 of lmod1a, which caused a premature stop codon. We observed significant mRNA downregulation of lmod1a, myh11, myod1, and acta2 and the protein expression of Lmod1 and Acta2 in the mutant group. A functional analysis of the lmod1a mutant zebrafish showed that its intestinal peristalsis was fewer, slower, and shorter in comparison to the wild type. This study showed that targeted deletion of lmod1a in zebrafish resulted in depletion of MMIHS-related genes and proteins, resulting in intestinal hypoperistalsis. This model may have the potential to be utilized in future therapeutic approaches, such as drug discovery screening and gene repair therapy for MMIHS.

The spectrum of novel ABCB11 gene variations in children with progressive familial intrahepatic cholestasis type 2 in Pakistani cohorts

Progressive familial intrahepatic cholestasis (PFIC) is a rare childhood manifested disease associated with impaired bile secretion with severe pruritus yellow stool, and sometimes hepatosplenomegaly. PFIC is caused by mutations in ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, SLC51A, USP53, KIF12, ZFYVE19, and MYO5B genes depending on its type. ABCB11 mutations lead to PFIC2 that encodes the bile salt export pump (BSEP). Different mutations of ABCB11 have been reported in different population groups but no data available in Pakistani population being a consanguineous one. We sequenced coding exons of the ABCB11 gene along with its flanking regions in 66 unrelated Pakistani children along with parents with PFIC2 phenotype. We identified 20 variations of ABCB11: 12 in homozygous form, one compound heterozygous, and seven heterozygous. These variants include 11 missenses, two frameshifts, two nonsense mutations, and five splicing variants. Seven variants are novel candidate variants and are not detected in any of the 120 chromosomes from normal ethnically matched individuals. Insilico analysis revealed that four homozygous missense variations have high pathogenic scores. Minigene analysis of splicing variants showed exon skipping and the addition of exon. This data is a useful addition to the disease variants genomic database and would be used in the future to build a diagnostic algorithm.

Variation characteristics and clinical significance of TP53 in patients with myeloid neoplasms

ABSTRACT Objectives: MDS and AML characterized by TP53 variations have a poor prognosis in general. However, specifically, differences in prognosis have also been observed in patients with different TP53 variants and VAFs. Methods: Here, we retrospectively analyzed datasets of patients with MDS, MPN, and AML who underwent targeted DNA sequencing from February 2018 to December 2023, and patients with reportable TP53 variations were screened. Demographic data and clinical data were collected, and the relationship between TP53 alterations and patient prognosis (AML/MDS) was analyzed using the cBioPortal and Kaplan–Meier Plotter databases. The relationship between the VAFs of TP53 variations and prognoses was analyzed using data from the present study. Results: Sixty-two variants of TP53 were identified in 58 patients. We mainly identified single mutations (79.31%, 46/58), followed by double (17.24%, 10/58) and triple (3.45%, 2/58) mutations. The variations were mainly enriched in exon4–exon8 of TP53. Missense (72.58%, 45/62) mutations were the main type of variations, followed by splice-site (9.68%, 6/62), nonsense (9.68%, 6/62), frameshift (6.45%, 4/62), and indel (1.61%, 1/62) mutations. In this study, p.Arg175His and p.Arg273His were high-frequency TP53 mutations, and DNMT3A and TET2 were commonly co-mutated genes in the three types of myeloid neoplasms; However, we reported some new TP53 variants in MPN that have not been found in the public database. Moreover, MDS or AML characterized by altered TP53 had a shorter OS than patients in the unaltered group (P<0.01), low TP53 mRNA levels were associated with shorter OS in patients with AML (P<0.01). Data from our center further found higher VAF (≥10%) associated with shorter OS in patients with MDS (median 2.75 vs. 24 months) (P<0.01). Conclusion: TP53 mutations are mainly enriched in exon4–exon8, are missense and single mutations in myeloid neoplasms, and are associated with poor prognosis of MDS/AML, and higher VAF (≥10%) of TP53 mutations associated with a shorter OS in patients with MDS.

A novel deep intronic variant in the DMD gene causes Duchenne muscular dystrophy by pseudoexon activation encoding a nonsense codon

Dystrophinopathies are a group of neuromuscular disorders, inherited in an X-linked recessive manner, caused by pathogenic variants in the DMD gene. Copy number variation detection and next generation sequencing allow the detection of around 99 % of the pathogenic variants. However, some patients require mRNA studies from muscle biopsies to identify deep intronic pathogenic variants. Here, we report a child suspected of having Duchenne muscular dystrophy, with a muscle biopsy showing dystrophin deficiency, and negative molecular testing for deletions, duplications, and small variants. mRNA analysis from muscle biopsy revealed a pseudoexon activation that introduce a premature stop codon into the reading frame. gDNA sequencing allowed to identified a novel variant, c.832–186 T>G, which creates a cryptic donor splice site, recognizing the underlying mechanism causing the pseudoexon insertion. This case highlights the usefulness of the mRNA analysis from muscle biopsy when routine genetic testing is negative and clinical suspicion of dystrophinopathies remains the main clinical diagnosis suspicion.

Exploration and genetic analyses of canopy leaf pigmentation changes in soybean (Glycine max L.): unveiling a novel phenotype

Key messagePigmentation changes in canopy leaves were first reported, and subsequent genetic analyses identified a major QTL associated with levels of pigmentation changes, suggesting Glyma.06G202300 as a candidate gene.An unexpected reddish-purple pigmentation in upper canopy leaves was discovered during the late reproductive stages in soybean (Glycine max L.) genotypes. Two sensitive genotypes, ‘Uram’ and PI 96983, exhibited anomalous canopy leaf pigmentation changes (CLPC), while ‘Daepung’ did not. The objectives of this study were to: (i) characterize the physiological features of pigmented canopy leaves compared with non-pigmented leaves, (ii) evaluate phenotypic variation in a combined recombinant inbred line (RIL) population (N = 169 RILs) under field conditions, and (iii) genetically identify quantitative trait loci (QTL) for CLPC via joint population linkage analysis. Comparison between pigmented and normal leaves revealed different Fv/Fm of photosystem II, hyperspectral reflectance, and cellular properties, suggesting the pigmentation changes occur in response to an undefined abiotic stress. A highly significant QTL was identified on chromosome 6, explaining ~ 62.8% of phenotypic variance. Based on the QTL result, Glyma.06G202300 encoding flavonoid 3′-hydroxylase (F3′H) was identified as a candidate gene. In both Uram and PI 96983, a 1-bp deletion was confirmed in the third exon of Glyma.06G202300 that results in a premature stop codon in both Uram and PI 96983 and a truncated F3′H protein lacking important domains. Additionally, gene expression analyses uncovered significant differences between pigmented and non-pigmented leaves. This is the first report of a novel symptom and an associated major QTL. These results will provide soybean geneticists and breeders with valuable knowledge regarding physiological changes that may affect soybean production. Further studies are required to elucidate the causal environmental stress and the underlying molecular mechanisms.