eHSP90α in front-line therapy in EGFR exon 19 deletion and 21 Leu858Arg mutations in advanced lung adenocarcinoma.

PURPOSE: Extracellular heat shock protein 90 AA1(eHSP90α) is intricately linked to tumor progression and prognosis. This study aimed to investigate the difference in the value of eHSP90α in post-treatment response assessment and prognosis prediction between exon 19 deletion(19DEL) and exon 21 Leu858Arg(L858R) mutation types in lung adenocarcinoma(LUAD). METHODS: We analyzed the relationship between the expression of eHSP90α and clinicopathological features in 89 patients with L858R mutation and 196 patients with 19DEL mutation in LUAD. The Kaplan-Meier survival curve was used to determine their respective cut-off values and analyze the relationship between eHSP90α expression and the survival time of the two mutation types. The area under the curve (AUC) was used to evaluate the diagnostic performance of biomarkers. Then, the prognostic model was developed using the univariate-Cox multivariate-Cox and LASSO-multivariate logistic methods. RESULTS: In LUAD patients, eHSP90α was positively correlated with carcinoembryonic antigen(CEA), carbohydrate antigen 125(CA125), and carbohydrate antigen 153(CA153). The truncated values of eHSP90α in L858R and 19DEL patients were 44.5 ng/mL and 40.8 ng/mL, respectively. Among L858R patients, eHSP90α had the best diagnostic performance (AUC = 0.765), and higher eHSP90α and T helper cells(Th cells) expression were significantly related to shorter overall survival(OS) and worse treatment response. Also, high eHSP90a expression and short progression-free survival(PFS) were significantly correlated. Among 19DEL patients, CEA had the best diagnostic efficacy (AUC = 0.734), and CEA and Th cells were independent prognostic factors that predicted shorter OS. Furthermore, high CA125 was significantly associated with short PFS and poor curative effect. CONCLUSIONS: eHSP90α has a better prognostic value in LUAD L858R patients than 19DEL, which provides a new idea for clinical diagnosis and treatment.

Multiple deletions of candidate effector genes lead to the breakdown of partial grapevine resistance to downy mildew.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1-mediated grapevine resistance. We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome-wide association study was conducted to identify genomic variations associated with resistant-breaking phenotypes. We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid-aware reassembly of the P. viticola INRA-Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800-900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY-fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature. This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen.

Concurrent of compound heterozygous variant of a novel in-frame deletion and the common hypomorphic haplotype in TBX6 and inherited 17q12 microdeletion in a fetus.

BACKGROUND: TBX6, a member of the T-box gene family, encodes the transcription factor box 6 that is critical for somite segmentation in vertebrates. It is known that the compound heterozygosity of disruptive variants in trans with a common hypomorphic risk haplotype (T-C-A) in the TBX6 gene contribute to 10% of congenital scoliosis (CS) cases. The deletion of chromosome 17q12 is a rare cytogenetic abnormality, which often leads to renal cysts and diabetes mellitus. However, the affected individuals often exhibit clinical heterogeneity and incomplete penetrance. METHODS: We here present a Chinese fetus who was shown to have CS by ultrasound examination at 17 weeks of gestation. Trio whole-exome sequencing (WES) was performed to investigate the underlying genetic defects of the fetus. In vitro functional experiments, including western-blotting and luciferase transactivation assay, were performed to determine the pathogenicity of the novel variant of TBX6. RESULTS: WES revealed the fetus harbored a compound heterozygous variant of c.338_340del (p.Ile113del) and the common hypomorphic risk haplotype of the TBX6 gene. In vitro functional study showed the p.Ile113del variant had no impact on TBX6 expression, but almost led to complete loss of its transcriptional activity. In addition, we identified a 1.85 Mb deletion on 17q12 region in the fetus and the mother. Though there is currently no clinical phenotype associated with this copy number variation in the fetus, it can explain multiple renal cysts in the pregnant woman. CONCLUSIONS: This study is the first to report a Chinese fetus with a single amino acid deletion variant and a T-C-A haplotype of TBX6. The clinical heterogeneity of 17q12 microdeletion poses significant challenges for prenatal genetic counseling. Our results once again suggest the complexity of prenatal genetic diagnosis.

A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

Evasion of wheat resistance gene Lr15 recognition by the leaf rust fungus is attributed to the coincidence of natural mutations and deletion in AvrLr15 gene.

Employing race-specific resistance genes remains an effective strategy to protect wheat from leaf rust caused by Puccinia triticina (Pt) worldwide, while the newly emerged Pt races, owing to rapid genetic evolution, frequently overcome the immune response delivered by race-specific resistance genes. The molecular mechanisms underlying the newly evolved virulence Pt pathogen remain unknown. Here, we identified an avirulence protein AvrLr15 from Pt that induced Lr15-dependent immune responses. Heterologously produced AvrLr15 triggered pronounced cell death in Lr15-isogenic wheat leaves. AvrLr15 contains a functional signal peptide, localized to the plant nucleus and cytosol and can suppress BAX-induced cell death. Evasion of Lr15-mediated resistance in wheat was associated with a deletion and point mutations of amino acids in AvrLr15 rather than AvrLr15 gene loss in the Lr15-breaking Pt races, implying that AvrLr15 is required for the virulence function of Pt. Our findings identified the first molecular determinant of wheat race-specific immunity and facilitated the identification of the first AVR/R gene pair in the Pt-wheat pathosystem, which will provide a molecular marker to monitor natural Pt populations and guide the deployment of Lr15-resistant wheat cultivars in the field.

Genotypic characterization of novel S-DEL variants of porcine epidemic diarrhea virus identified in South Korea.

The highly pathogenic genotype 2b (HP-G2b) of porcine epidemic diarrhea virus (PEDV), which caused a pandemic in 2013-2014, evolved in South Korea and became endemic, affecting the domestic pig industry. This study describes the genotypic traits of novel HP-G2b PEDV strains identified on affected farms experiencing low disease severity with < 10% neonatal mortality. Nucleotide sequencing revealed common deletion patterns, termed S-DEL2, resulting in a two-amino-acid deletion at positions 60 and 61, 61 and 62, or 63 and 64 in the N-terminal domain of the spike (S) protein of all isolates. The S barcode profiles of S-DEL2 variants differed from each other and shared 96.0-99.4% and 98.5-99.6% nt sequence identity with other South Korean HP-G2b PEDV strains in the S gene and in the complete genome sequence, respectively. Genetic and phylogenetic analysis showed that the S-DEL2 strains belonged to diverse domestic clades: CK, CK.1, CK.2, or NC. The emergence of novel S-DEL2 strains suggests that continuous evolution of PEDV occurs under endemic circumstances, resulting in genetic diversity and distinct clinical presentations. This study advances our knowledge regarding the genetic and pathogenic heterogeneity of PEDV and emphasizes the importance of active monitoring and surveillance to identify novel variants and determine their genotypic and phenotypic characteristics.

Evolution of pfhrp2 and pfhrp3 deletions in Equatorial Guinea between the pre- and post-RDT introduction.

BACKGROUND: Pfhrp2 and pfhrp3 deletions are threatening Plasmodium falciparum malaria diagnosis by rapid diagnostic tests (RDT) due to false negatives. This study assesses the changes in the frequencies of pfhrp2 and pfhrp3 deletions (pfhrp2Del and pfhrp3Del, respectively) and the genes in their flaking regions, before and after RDT introduction in Equatorial Guinea. METHODS: A total of 566 P. falciparum samples were genotyped to assess the presence of pfhrp2 and pfhrp3 deletions and their flanking genes. The specimens were collected 18 years apart from two provinces of Equatorial Guinea, North Bioko (Insular Region) and Litoral Province (Continental Region). Orthologs of pfhrp2 and pfhrp3 genes from other closely related species were used to compare sequencing data to assess pfhrp2 and pfhrp3 evolution. Additionally, population structure was studied using seven neutral microsatellites. RESULTS: This study found that pfhrp2Del and pfhrp3Del were present before the introduction of RDT; however, they increased in frequency after their use, reaching more than 15%. Haplotype networks suggested that pfhrp2Del and pfhrp3Del emerged multiple times. Exon 2 of pfhrp2 and pfhrp3 genes had high variability, but there were no significant changes in amino acid sequences. CONCLUSIONS: Baseline sampling before deploying interventions provides a valuable context to interpret changes in genetic markers linked to their efficacy, such as the dynamic of deletions affecting RDT efficacy.

A Comprehensive Overview of NF1 Mutations in Iranian Patients.

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutations in the NF1 gene. This disorder shows nearly complete penetrance and high phenotypic variability. We used the whole-exome sequencing technique to identify mutations in 32 NF1 cases from 22 Iranian families. A total of 31 variants, including 30 point mutations and one large deletion, were detected. In eight cases, variants were inherited, while they were sporadic in the remaining. Seven novel variants, including c.5576 T > G, c.6658_6659insC, c.2322dupT, c.92_93insAA, c.4360C > T, c.3814C > T, and c.4565_4566delinsC, were identified. The current study is the largest in terms of the sample size of Iranian NF1 cases with identified mutations. The results can broaden the spectrum of NF1 mutations and facilitate the process of genetic counseling in the affected families.

Characterisation of the novel HLA-DQA1*04:14N allele by next-generation sequencing.

HLA-DQA1*04:14N differs from HLA-DQA1*04:01:01:03 by a single nucleotide deletion in codon 113 in exon 3.

Discovery of a novel HLA-A*11 null allele, HLA-A*11:466N, in a Taiwanese individual.

A nucleotide deletion in the residue 371 of HLA-A*11:01:01:01 results in a novel allele HLA-A*11:466N.

Detection of α-thalassemia South-East Asian deletion based on a fully integrated digital polymerase chain reaction system DropXpert S6.

OBJECTIVES: This study aimed to establish a droplet digital polymerase chain reaction (ddPCR) assay for South-East Asian (SEA) deletion based on a fully integrated digital PCR system DropXpert S6. METHODS: A total of 151 whole blood samples, 10 chorionic villus samples, and 17 amniotic fluid samples were collected, including 106 SEA heterozygotes, 43 normal individuals, 10 Hb Bart's hydrops details, and 19 SEA deletions combined with other genotypes.Genotypes of these samples were determined by the Gap-PCR method. We perform a series of optimizations of the ddPCR system to ensure the performance of the entire ddPCR reaction, such as droplet stability, fluorescence clustering, sensitivity, and accuracy. RESULTS: Our assay exhibited 99.4% (177/178) accuracy compared with the Gap-PCR method, and the minimum detection limit of DNA was 0.1 ng/µL.Both targets have reliable linearity, R2 = 0.9999 for the α-thalassemia SEA deletion allele and R2 = 1 for the wild-type allele. The coefficient of variation for α-thalassemia SEA deletion allele detection at 2 and 10 ng/µL concentrations was 5.42% and 1.91%, respectively. In contrast, the coefficient of variation for wild-type allele detection was 4.06% and 1.83%, demonstrating its high quantitative accuracy. In addition, the DropXpert S6 PCR system showed some advantages over other ddPCR instruments, such as reducing testing costs, simplifying and automating the workflow. CONCLUSIONS: The DropXpert S6 PCR system provided a highly accurate diagnosis for α-thalassemia SEA deletion and can be used to detect α-thalassemia as an alternative method.

In vitro phenotypic characterisation of two genotype I African swine fever viruses with genomic deletion isolated from Sardinian wild boars.

African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015-January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.

A Novel 10-Base Pair Deletion in the First Exon of GmHY2a Promotes Hypocotyl Elongation, Induces Early Maturation, and Impairs Photosynthetic Performance in Soybean.

Plants photoreceptors perceive changes in light quality and intensity and thereby regulate plant vegetative growth and reproductive development. By screening a γ irradiation-induced mutant library of the soybean (Glycine max) cultivar "Dongsheng 7", we identified Gmeny, a mutant with elongated nodes, yellowed leaves, decreased chlorophyll contents, altered photosynthetic performance, and early maturation. An analysis of bulked DNA and RNA data sampled from a population segregating for Gmeny, using the BVF-IGV pipeline established in our laboratory, identified a 10 bp deletion in the first exon of the candidate gene Glyma.02G304700. The causative mutation was verified by a variation analysis of over 500 genes in the candidate gene region and an association analysis, performed using two populations segregating for Gmeny. Glyma.02G304700 (GmHY2a) is a homolog of AtHY2a in Arabidopsis thaliana, which encodes a PΦB synthase involved in the biosynthesis of phytochrome. A transcriptome analysis of Gmeny using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed changes in multiple functional pathways, including photosynthesis, gibberellic acid (GA) signaling, and flowering time, which may explain the observed mutant phenotypes. Further studies on the function of GmHY2a and its homologs will help us to understand its profound regulatory effects on photosynthesis, photomorphogenesis, and flowering time.

Identification of a Novel Deletion Variant (c.2999_3005delTGTGTGT/p.Asn1000SerfsTer4) in NPHP4 Associated With Nephronophthisis-4.

BACKGROUND: Nephronophthisis-4 (NPHP4) is an inherited renal ciliopathy described by renal fibrosis and progressive impairment of kidney function. This study aimed to investigate the genetic basis and clinical manifestations of NPHP4 in two Iranian siblings. METHODS: The proband was a 27-year-old male with features of end-stage renal disease, including anemia, uremia, polyuria, and polydipsia. It is worth mentioning that he has a 22-year-old sister with a similar presentation. Clinical diagnosis procedures, such as renal biopsy, brain imaging, blood and urine tests, cardiac evaluation, ophthalmic inspection, and auditory function assessment, were carried out to evaluate organ involvement and potential comorbidities. Whole-exome sequencing (WES) and segregation analysis were performed to identify and confirm genetic variants associated with the condition. Computational variant analysis was conducted to evaluate the pathogenicity of the candidate variant. Furthermore, the SWISS-MODEL server was utilized for protein modeling. RESULTS: The brain, cardiac, ocular, and auditory functions were normal. Renal biopsy of the proband showed chronic interstitial inflammation and fibrosis. We found a novel homozygous 7-base pair deletion (c.2999_3005delTGTGTGT/ p.Asn1000SerfsTer4) in exon 21 of NPHP4 by WES. Segregation analysis confirmed homozygosity for the NPHP4 variant in affected individuals and heterozygous carrier status in parents, supporting autosomal recessive inheritance. 3D protein modeling indicated significant structural changes due to the variant. CONCLUSION: This study expands the genetic causes and phenotypic spectrum of nephronophthisis-4 and reveals the importance of genetic analysis in diagnosing and managing rare inherited kidney disorders, particularly those involving consanguinity.

Unexpected complexity in the molecular diagnosis of spastic paraplegia 11.

BACKGROUND: Spastic paraplegia 11 (SPG11) is the most prevalent form of autosomal recessive hereditary spastic paraplegia, resulting from biallelic pathogenic variants in the SPG11 gene (MIM *610844). METHODS: The proband is a 36-year-old female referred for genetic evaluation due to cognitive dysfunction, gait impairment, and corpus callosum atrophy (brain MRI was normal at 25-years-old). Diagnostic approaches included CGH array, next-generation sequencing, and whole transcriptome sequencing. RESULTS: CGH array revealed a 180 kb deletion located upstream of SPG11. Sequencing of SPG11 uncovered two rare single nucleotide variants: the novel variant c.3143C>T in exon 17 (in cis with the deletion), and the previously reported pathogenic variant c.6409C>T in exon 34 (in trans). Whole transcriptome sequencing revealed that the variant c.3143C>T caused exon 17 skipping. CONCLUSION: We report a novel sequence variant in the SPG11 gene resulting in exon 17 skipping, which, along with a nonsense variant, causes Spastic Paraplegia 11 in our proband. In addition, a deletion upstream of SPG11 was identified in the patient, whose implication in the phenotype remains uncertain. Nonetheless, the deletion apparently affects cis-regulatory elements of the gene, suggesting a potential new pathogenic mechanism underlying the disease in a subset of undiagnosed patients. Our findings further support the hypothesis that the origin of thin corpus callosum in patients with SPG11 is of progressive nature.

Deletion variants calling in third-generation sequencing data based on a dual-attention mechanism.

Deletion is a crucial type of genomic structural variation and is associated with numerous genetic diseases. The advent of third-generation sequencing technology has facilitated the analysis of complex genomic structures and the elucidation of the mechanisms underlying phenotypic changes and disease onset due to genomic variants. Importantly, it has introduced innovative perspectives for deletion variants calling. Here we propose a method named Dual Attention Structural Variation (DASV) to analyze deletion structural variations in sequencing data. DASV converts gene alignment information into images and integrates them with genomic sequencing data through a dual attention mechanism. Subsequently, it employs a multi-scale network to precisely identify deletion regions. Compared with four widely used genome structural variation calling tools: cuteSV, SVIM, Sniffles and PBSV, the results demonstrate that DASV consistently achieves a balance between precision and recall, enhancing the F1 score across various datasets. The source code is available at https://github.com/deconvolution-w/DASV.

A 44-Nucleotide Region in the Chikungunya Virus 3' UTR Dictates Viral Fitness in Disparate Host Cells.

We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.