Single Copy Research Articles

Unveiling the complete chloroplast genome of tribulus macropterus var. Arabicus (hosni) al-hemaid & j. Thomas: genome structure, comparative analysis and phylogeny

The present investigation unveiled the first complete chloroplast (cp) genome of Tribulus macropterus var. arabicus (Hosni) Al-Hemaid & J. Thomas (Zygophyllaceae), a medicinal herb, indigenous to Saudi Arabia. The cp genome, comprising a length of 158,179 bp and a GC content of 35.8%, exhibited the typical circular quadripartite arrangement of flowering plants, including two inverted repeat regions (25842 bp) separated by a large single-copy (88873 bp) and a small single-copy region (17622 bp). Genome annotation unraveled 132 genes, comprising of 87 protein-coding genes, 37 tRNAs and eight rRNAs. A comparative plastomics approach demonstrated a very similar genome structure and gene organization in closely related taxa. Whole-genome alignment indicated that the inverted repeat regions exhibited greater conservation when compared to the single copy zones. Repeat analysis of the cp genome identified 80 simple sequence repeats, with the majority (64) being mononucleotides. Among the longer repeats, forward repeats were frequent (20) followed by palindromic and reverse repeats. The nucleotide diversity endeavor identified some hypervariable sites (rpl22, cemA, trnL-UAG) in the small and large single copies which would offer opportunities to design molecular markers for potential application in taxonomic identification. Phylogenetic analysis with rbcL barcode elucidated the distinct position of T. macropterus var. arabicus compared to T. macropterus within Zygophyllaceae and further validated the assembly. The findings of this investigation provide significant insights into biological fields, particularly enhancing the current understanding of the genetic and evolutionary variations within Zygophyllaceae. Bangladesh J. Plant Taxon. 31(1): 1-14, 2024 (June)

The Construction of an Environmentally Friendly Super-Secreting Strain of Bacillus subtilis through Systematic Modulation of Its Secretory Pathway Using the CRISPR-Cas9 System.

Achieving commercially significant yields of recombinant proteins in Bacillus subtilis requires the optimization of its protein production pathway, including transcription, translation, folding, and secretion. Therefore, in this study, our aim was to maximize the secretion of a reporter α-amylase by overcoming potential bottlenecks within the secretion process one by one, using a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system. The strength of single and tandem promoters was evaluated by measuring the relative α-amylase activity of AmyQ integrated into the B. subtilis chromosome. Once a suitable promoter was selected, the expression levels of amyQ were upregulated through the iterative integration of up to six gene copies, thus boosting the α-amylase activity 20.9-fold in comparison with the strain harboring a single amyQ gene copy. Next, α-amylase secretion was further improved to a 26.4-fold increase through the overexpression of the extracellular chaperone PrsA and the signal peptide peptidase SppA. When the final expression strain was cultivated in a 3 L fermentor for 90 h, the AmyQ production was enhanced 57.9-fold. The proposed strategy allows for the development of robust marker-free plasmid-less super-secreting B. subtilis strains with industrial relevance.

A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

Common genetic variants associated with urinary phthalate levels in children: A genome-wide study

IntroductionPhthalates, or dieters of phthalic acid, are a ubiquitous type of plasticizer used in a variety of common consumer and industrial products. They act as endocrine disruptors and are associated with increased risk for several diseases. Once in the body, phthalates are metabolized through partially known mechanisms, involving phase I and phase II enzymes. ObjectiveIn this study we aimed to identify common single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) associated with the metabolism of phthalate compounds in children through genome-wide association studies (GWAS). MethodsThe study used data from 1,044 children with European ancestry from the Human Early Life Exposome (HELIX) cohort. Ten phthalate metabolites were assessed in a two-void pooled urine collected at the mean age of 8 years. Six ratios between secondary and primary phthalate metabolites were calculated. Genome-wide genotyping was done with the Infinium Global Screening Array (GSA) and imputation with the Haplotype Reference Consortium (HRC) panel. PennCNV was used to estimate copy number variants (CNVs) and CNVRanger to identify consensus regions. GWAS of SNPs and CNVs were conducted using PLINK and SNPassoc, respectively. Subsequently, functional annotation of suggestive SNPs (p-value < 1E-05) was done with the FUMA web-tool. ResultsWe identified four genome-wide significant (p-value < 5E-08) loci at chromosome (chr) 3 (FECHP1 for oxo-MiNP_oh-MiNP ratio), chr6 (SLC17A1 for MECPP_MEHHP ratio), chr9 (RAPGEF1 for MBzP), and chr10 (CYP2C9 for MECPP_MEHHP ratio). Moreover, 115 additional loci were found at suggestive significance (p-value < 1E-05). Two CNVs located at chr11 (MRGPRX1 for oh-MiNP and SLC35F2 for MEP) were also identified. Functional annotation pointed to genes involved in phase I and phase II detoxification, molecular transfer across membranes, and renal excretion. ConclusionThrough genome-wide screenings we identified known and novel loci implicated in phthalate metabolism in children. Genes annotated to these loci participate in detoxification, transmembrane transfer, and renal excretion.

Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.

Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes and distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) that dramatically improves the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely-matched control promoters, integrated together into the genome at single copy. To test our ability to capture post-transcriptional and post-translational regulation through sequencing, we screened for genes that affected nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq screens accurately capture the known components of well-studied RNA and protein quality control pathways with minimal background. These results demonstrate the precision and versatility of CiBER-seq for dissecting the genetic networks controlling cellular behaviors.

The Importance of Copy Number Variant Analysis in Patients with Monogenic Kidney Disease

IntroductionGenetic testing can reveal monogenic causes of kidney diseases, offering diagnostic, therapeutic, and prognostic benefits. While single nucleotide variants (SNVs) and copy number variants (CNVs) can result in kidney disease, CNV analysis is not always included in genetic testing. MethodsWe investigated the diagnostic value of CNV analysis in 2,432 kidney disease patients genetically tested at the University Medical Centre Utrecht between 2014 and May 2022. We combined previous diagnostic testing results, encompassing SNVs and CNVs, with newly acquired results based on retrospective CNV analysis. The reported yield considers both ACMG classification and whether the genotype actually results in disease. ResultsWe report a diagnostic yield of at least 23% for our complete diagnostic cohort. The total diagnostic yield based solely on CNVs was 2.4%. The overall contribution of CNV analysis, defined as the proportion of positive genetic tests requiring CNV analysis, was 10.5% and varied among different disease subcategories, with the highest impact seen in congenital anomalies of the kidney and urinary tract and chronic kidney disease at a young age. We highlight the efficiency of exome-based CNV calling, which reduces the need for additional diagnostic tests. Furthermore, a complex structural variant, likely a COL4A4 founder variant, was identified. Additional findings unrelated to kidney diseases were reported in a small percentage of cases. ConclusionIn summary, this study demonstrates the substantial diagnostic value of CNV analysis, providing insights into its contribution to the diagnostic yield and advocating for its routine inclusion in genetic testing of kidney disease patients.

Quantum Nonlocality: Multicopy Resource Interconvertibility and Their Asymptotic Inequivalence.

Quantum nonlocality, pioneered in Bell's seminal work and subsequently verified through a series of experiments, has drawn substantial attention due to its practical applications in various protocols. Evaluating and comparing the extent of nonlocality within distinct quantum correlations holds significant practical relevance. Within the resource theoretic framework this can be achieved by assessing the interconversion rate among different nonlocal correlations under free local operations and shared randomness. In this study we, however, present instances of quantum nonlocal correlations that are incomparable in the strongest sense. Specifically, when starting with an arbitrary many copies of one nonlocal correlation, it becomes impossible to obtain even a single copy of the other correlation, and this incomparability holds in both directions. Such incomparable quantum correlations can be obtained even in the simplest Bell scenario, which involves two parties, each having two dichotomic measurements setups. Notably, there exist an uncountable number of such incomparable correlations. Our result challenges the notion of a "unique gold coin," often referred to as the "maximally resourceful state," within the framework of the resource theory of quantum nonlocality. To this end, we provide examples of isotropic quantum correlations that cannot be distilled up to the Tsirelson point, and thus partially answer a long-standing open question in nonlocality distillation.

First Chromosome-Level Genome Assembly of a Ribbon Worm from the Hoplonemertea Clade, Emplectonema gracile, and Its Structural Annotation.

Genome-wide information has so far been unavailable for ribbon worms of the clade Hoplonemertea, the most species-rich class within the phylum Nemertea. While species within Pilidiophora, the sister clade of Hoplonemertea, possess a pilidium larval stage and lack stylets on their proboscis, Hoplonemertea species have a planuliform larva and are armed with stylets employed for the injection of toxins into their prey. To further compare these developmental, physiological, and behavioral differences from a genomic perspective, the availability of a reference genome for a Hoplonemertea species is crucial. Such data will be highly useful for future investigations toward a better understanding of molecular ecology, venom evolution, and regeneration not only in Nemertea but also in other marine invertebrate phyla. To this end, we herein present the annotated chromosome-level genome assembly for Emplectonema gracile (Nemertea; Hoplonemertea; Monostilifera; Emplectonematidae), an easily collected nemertean well suited for laboratory experimentation. The genome has an assembly size of 157.9 Mb. Hi-C scaffolding yielded chromosome-level scaffolds, with a scaffold N50 of 10.0 Mb and a score of 95.1% for complete BUSCO genes found as a single copy. Annotation predicted 20,684 protein-coding genes. The high-quality reference genome reaches an Earth BioGenome standard level of 7.C.Q50.

Rice Big Grain1 improves grain yield in ectopically expressing rice and heterologously expressing tobacco plants.

Functional genomics through transgenesis has provided faster and more reliable methods for identifying, characterizing, and utilizing genes or quantitative trait loci linked to agronomic traits to target yield. The present study explored the role of Big Grain1 (BG1) gene of rice (Oryza sativa L.) in yield improvement of crop plants. We aimed to identify the genetic variation of OsBG1 in various indica rice cultivars by studying the allelic polymorphism of the gene, while also investigating the gene's potential to increase crop yield through the transgenic approach. Our study reports the presence of an extra 393bp sequence having two 6bp enhancer elements in the 3' regulatory sequence of OsBG1 in the large-grain cultivar IR64 but not in the small-grain cultivar Badshahbhog. A single copy of the OsBG1 gene in both the cultivars and a 4.1-fold higher expression of OsBG1 in IR64 than in Badshahbhog imply that the grain size is positively correlated with the level of OsBG1 expression in rice. The ectopic expression of OsBG1 under the endosperm-specific glutelin C promoter in Badshahbhog enhanced the flag leaf length, panicle weight, and panicle length by an average of 33.2%, 33.7%, and 30.5%, respectively. The length of anthers, spikelet fertility, and grain yield per plant increased in transgenic rice lines by an average of 27.5%, 8.3%, and 54.4%, respectively. Heterologous expression of OsBG1 under the constitutive 2xCaMV35S promoter improved the number of seed pods per plant and seed yield per plant in transgenic tobacco lines by an average of 2.2-fold and 2.6-fold, respectively. Improving crop yield is crucial to ensure food security and socio-economic stability, and identifying suitable genetic factor is the essential step towards this endeavor. Our findings suggest that the OsBG1 gene is a promising candidate for improving the grain yield of monocot and dicot plant systems by molecular breeding and genetic engineering.

Comparative Analysis of Chloroplast Genome Variation and Evolutionary Analysis of the Endangered Species Dendrobium flexicaule and its Relative Species

Globally, the genus Dendrobium (Family: Orchidaceae) exhibits widespread distribution, predominantly occupying the tropical and subtropical areas of Asia. Although the chloroplast genomes (cpDNAs) of certain Dendrobium species have been documented, the evolutionary relationships among these species remain largely unexplored. In this pioneering investigation, a comprehensive analysis of the complete cpDNAs from Dendrobium species is presented, including Dendrobium flexicaule along with three closely related taxa (D. nobile, D. officinale, and D. huoshanense). The size of the novel complete cpDNAs ranged from 150,529 to 152,588 bp, and their cpDNA structure exhibits a unique quadrifoliate structure reminiscent of female quadrivalents, including large single copy, small single copy (SSC), and inverted repeat regions. A total of 175 simple sequence repeats were identified in the D. flexicaule cpDNA. A few pseudogenes of the NA(D)H dehydrogenase-like (NDH) genes were found in the annotated cpDNAs of the four Dendrobium species. Within the shared pseudogenes ndhF and ndhG, ndhF frequently undergoes mutations. Additionally, the phylogenetic analysis showed that D. flexicaule and D. scoriarum were close sister species. The research findings provide crucial genetic information for the molecular phylogenetic system of D. flexicaule.

The reference genome of an endangered Asteraceae, Deinandra increscens subsp. villosa, endemic to the Central Coast of California.

We present a reference genome for the federally endangered Gaviota tarplant, Deinandra increscens subsp. villosa (Madiinae, Asteraceae), an annual herb endemic to the Central California coast. Generating PacBio HiFi, Oxford Nanopore Technologies, and Dovetail Omni-C data, we assembled a haploid consensus genome of 1.67 Gb as 28.7 K scaffolds with a scaffold N50 of 74.9 Mb. We annotated repeat content in 74.8% of the genome. Long terminal repeats (LTRs) covered 44.0% of the genome with Copia families predominant at 22.9% followed by Gypsy at 14.2%. Both Gypsy and Copia elements were common in ancestral peaks of LTRs, and the most abundant element was a Gypsy element containing nested Copia/Angela sequence similarity, reflecting a complex evolutionary history of repeat activity. Gene annotation produced 33,257 genes and 68,942 transcripts, of which 99% were functionally annotated. BUSCO scores for the annotated proteins were 96.0% complete of which 77.6% was single copy and 18.4% duplicates. Whole genome duplication synonymous mutation rates of Gaviota tarplant and sunflower (Helianthus annuus) shared peaks that correspond to the last Asteraceae polyploidization event and subsequent divergence from a common ancestor at ∼27 MYA. Regions of high-density tandem genes were identified, pointing to potentially important loci of environmental adaptation in this species.

Comparative and phylogenetic analysis of the complete chloroplast genomes of 10 Artemisia selengensis resources based on high-throughput sequencing

BackgroundArtemisia selengensis, classified within the genus Artemisia of the Asteraceae family, is a perennial herb recognized for its dual utility in culinary and medicinal domains. There are few studies on the chloroplast genome of A. selengensis, and the phylogeographic classification is vague, which makes phylogenetic analysis and evolutionary studies very difficult.ResultsThe chloroplast genomes of 10 A. selengensis in this study were highly conserved in terms of gene content, gene order, and gene intron number. The genome lengths ranged from 151,148 to 151,257 bp and were typical of a quadripartite structure with a total GC content of approximately 37.5%. The chloroplast genomes of all species encode 133 genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Due to the contraction and expansion of the inverted repeats (IR), the overlap of ycf1 and ndhF genes occurred at the inverted repeats B (IRB) and short single copy sequence (SSC) boundaries. According to a codon use study, the frequent base in the chloroplast genome of A. selengensis’ third codon position was A/T. The number of SSR repeats was 42–44, most of which were single nucleotide A/T repeats. Sequence alignment analysis of the chloroplast genome showed that variable regions were mainly distributed in single copy regions, nucleotide diversity values of 0 to 0.009 were calculated by sliding window analysis, 8 mutation hotspot regions were detected, and coding regions were more conserved than non-coding regions. Analysis of non-synonymous substitution (Ka) and synonymous substitution (Ks) revealed that accD, rps12, petB, and atpF genes were affected by positive selection and no genes were affected by neutral selection. Based on the findings of the phylogenetic analysis, Artemisia selengensis was sister to the genus Artemisia Chrysanthemum and formed a monophyletic group with other Artemisia genera.ConclusionsIn this research, the present study systematically compared the chloroplast genomic features of A. selengensis and provided important information for the study of the chloroplast genome of A. selengensis and the evolutionary relationships among Asteraceae species.

CHARACTERIZATION OF THE CHLOROPLAST GENOME OF AQUILARIA CRASSNA PIERRE EX LECOMTE DISTRIBUTED IN VIETNAM USING LONG-READ DATA (OXFORD NANOPORE TECHNOLOGIES)

Aquilaria crassna Pierre ex Lecomte is a critically endangered species that is famous for its fragrance compounds for perfume and incense production. In this study, the complete chloroplast genome of A. crassna was sequenced using a long-read sequencing method of Oxford Nanopore Technologies. The complete chloroplast genome of A. crassna was 174,766 bp in length, which comprised a large single copy region of 87,231 bp, a small single copy region of 3,343 bp, and two inverted repeat regions of 42,096 bp. This genome contained 95 protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. The analysis ofsmall single repeats revealed 39 mononucleotide and seven dinucleotide repeats, which are located mainly in non-coding regions. Comparative genomic analysis exhibited high conservation of the chloroplast genome within the Aquilaria genus regarding gene content, gene order, and genomic structure. This study provides useful genomic data for further studies examining the genetic population, genetic conservation, molecular markers, and phylogeny of Aquilaria crassna and related species within Thymelaeaceae.

The complete chloroplast genome of the first registered Paeonia Itoh hybrid cv. Hexie in China

The first registered Paeonia Itoh hybrid cv. Hexie in China is a naturally occurring intersectional hybrid of Sect. Paeonia and Sect. Moutan. In this study, we sequenced, assembled, and analyzed the complete chloroplast genome of Paeonia Itoh hybrid cv. Hexie. The result showed that the chloroplast genome of Hexie, with a typical circular tetrad structure, is 152,958 bp in length, comprising a large single copy (LSC) region of 84,613 bp, a small single copy (SSC) region of 17,051 bp, and two reverse complementary sequences (IRs) of 25,647 bp. The chloroplast genome encoded 116 genes, including 80 protein-coding genes, 32 tRNA genes, and 4 rRNA genes. Phylogenetic analysis inferred from the shared protein-coding genes showed that the Paeonia Itoh hybrid cv. Hexie had the closest phylogenetic relationship with P. suffruticosa, followed by P. ostii, indicating that P. suffruticosa was its maternal parent. This study provides a molecular resource for phylogenetic and maternal parent studies of Paeonia Itoh hybrid, contributing to a basis for Paeonia Itoh hybrid breeding strategies in the future.

Complete chloroplast genome of Saxifraga fortunei var. pilosissima (Saxifragaceae) endemic to Ulleung-do island, Korea

The Saxifraga fortunei var. pilosissima Nakai is an endemic species to Ulleung-do island. Here we report the complete chloroplast genome sequence of S. fortunei var. pilosissima, which was 151,186 bp in total length with the large single copy (LSC) region of 83,536 bp, the small single copy (SSC) region of 17,644 bp, and two inverted repeat (IR) regions of 25,003 bp. The chloroplast genome contained 131 genes, including 86 protein-coding, 37 tRNA, 8 rRNA genes, and GC (guanine-cytosine) content was 37.8%. Additionally, we found that the chloroplast genome of S. fortunei var. pilosissima exhibited intron loss in the rpl2 gene. Within the IR region, six protein-coding genes, seven tRNA genes, and four rRNA genes were duplicated. In the chloroplast genome of S. fortunei var. pilosissima, there are a total of 17 genes containing introns. Among them, 15 genes contain one intron each, while two genes contain two introns. Phylogenetic analysis based on 37 representative chloroplast genomes of the Saxifragaceae suggested that genus Saxifraga is monophyletic and S. fortunei var. pilosissima is placed in sect. Irregulares Haw. and grouped with S. fortunei, S. rufescens, and S. stolonifera with a strong maximum likelihood bootstrap support value.

A method to comprehensively identify germline SNVs, INDELs and CNVs from whole exome sequencing data of BRCA1/2 negative breast cancer patients.

In the rapidly evolving field of genomics, understanding the genetic basis of complex diseases like breast cancer, particularly its familial/hereditary forms, is crucial. Current methods often examine genomic variants-such as Single Nucleotide Variants (SNVs), insertions/deletions (Indels), and Copy Number Variations (CNVs)-separately, lacking an integrated approach. Here, we introduced a robust, flexible methodology for a comprehensive variants' analysis using Whole Exome Sequencing (WES) data. Our approach uniquely combines meticulous validation with an effective variant filtering strategy. By reanalyzing two germline WES datasets from BRCA1/2 negative breast cancer patients, we demonstrated our tool's efficiency and adaptability, uncovering both known and novel variants. This contributed new insights for potential diagnostic, preventive, and therapeutic strategies. Our method stands out for its comprehensive inclusion of key genomic variants in a unified analysis, and its practical resolution of technical challenges, offering a pioneering solution in genomic research. This tool presents a breakthrough in providing detailed insights into the genetic alterations in genomes, with significant implications for understanding and managing hereditary breast cancer.

Whole-exome-based single nucleotide variants and copy number analysis for prenatal diagnosis of compound heterozygosity of SMPD4.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder. These variants have been identified in a group of children with neurodevelopmental disorders with microcephaly, arthrogryposis, and structural brain anomalies. SMPD4 encodes a sphingomyelinase that hydrolyzes sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes. For the efficient prenatal diagnosis of rare and undiagnosed diseases, the parallel detection of copy number variants (CNVs) and single nucleotide variants using whole-exome analysis is required. A physical examination of the parents was performed. Karyotype and whole-exome analysis were performed for the fetus and the parents. A fetus with microcephaly and arthrogryposis; biallelic null variants (c.387-1G>A; Chr2[GRCh38]: g.130142742_130202459del) were detected by whole-exome sequencing (WES). We have reported for the first time the biallelic loss-of-function mutations in SMPD4 in patients born to unrelated parents in China. WES could replace chromosomal microarray analysis and copy number variation sequencing as a more cost-effective genetic test for detecting CNVs and diagnosing highly heterogeneous conditions.

Effect of plasma-based molecular testing in patients with newly diagnosed advanced NSCLC on initiation of targeted agents in patients with targetable genomic alterations: A real world experience from the Middle East.

e20514 Background: The treatment of metastatic non-small cell lung cancer ( m-NSCLC) is personalized and driven by mutational profiling of the tumour tissue at diagnosis. This study evaluates the additional clinical utility of plasma based molecular testing towards shortening time to initiation of treatment and detection of targetable mutations in a cohort of patients with newly diagnosed m-NSCLC. Methods: This is a retrospective analysis of patients with newly diagnosed advanced NSCLC referred to the Thoracic Oncology Program between September 2021 to December 2023, who underwent “paired” plasma based circulating tumour ( ct-DNA ) and tissue based next generation sequencing (NGS) testing (done less than 4 weeks apart). ct-DNA testing was performed using the Oncomine Pancancer NGS Assay (Thermo Scientific) that screens for single nucleotide variants (SNV) in hotspot regions of 52 and common fusions involving 12 genes respectively. Tissue NGS testing was performed using Oncomine Comprehensive Assay Plus (Thermo Scientific); a panel covering ~500 genes capable of identifying all single nucleotide variants, indels, fusions and copy number variants (CNV). Patient demographics and clinicopathological characteristics were collected by retrospective review of electronic medical records (EMR). Results: Among 36 patients, the median age was 66.0 years (range: 32-66), 25 (69.4%) were males, and 16 (44.4%) were never smokers. Thirty (83.3%) had adenocarcinoma and 33 (91.67%) had stage IV NSCLC. The mutation detection rate of ct-DNA was 66.7% with a concordance of 58.3% between ct-DNA and tissue NGS testing. The median turnaround time (TAT) from sample collection to reporting were12 days (IQR:8-15) and 14 days (IQR:9-24.50) for ct-DNA and tissue NGS testing respectively. Among 31 patients who started treatment, the median time to initiation of 1st line treatment was 26 days from date of first visit (IQR:18-42). Fifteen(41.7%), 6(16.7%) and 10(27.8%) started first line targeted therapy, chemotherapy and chemoimmunotherapy respectively. Twenty-three(63.89%) had targetable mutations (EGFR Exon 19 or 21, EGFR Exon 20 insertion, ALK, ROS1, MET Exon1 4 skipping, ROS1 or KRAS12C detected either in ct-DNA or tissue). Fifteen (65.21%) out of these 23 started targeted therapy as their 1st line treatment and of these 8 (53.3%) were initiated on targeted agents prior to availability of tissue NGS reports. Conclusions: Though our TAT for ct-DNA and tissue based NGS testing was similar, ct-DNA based NGS testing enables earlier initiation of targeted treatment for newly diagnosed advanced NSCLC patients with targetable mutations. In the real world, this increases access to targeted agents and may translate to a subsequent improvement in survival outcomes.

Association of a germline single nucleotide polymorphism (SNP) in the interleukin-7 (IL7) gene with immune-related adverse events (irAEs) in the CheckMate 025 trial.

12140 Background: Immune checkpoint inhibitors (ICIs) have dramatically improved outcomes of patients (pts) with metastatic renal cell carcinoma (mRCC). Unfortunately, adverse events (AEs) can limit treatment efficacy and worsen patient outcomes. Recently, a germline IL7 SNP (rs16906115) was identified as a potential biomarker for prediction of irAEs. We aimed to characterize the association between a germline IL7 SNP (rs16906115) and AEs in a prospective clinical trial of patients with mRCC treated with nivolumab (NIVO) or everolimus (EVE). Methods: Whole-exome sequencing (WES) data of tumor and peripheral blood samples from CheckMate 025 (NCT01668784) were used to infer somatic alterations using the Cancer Genome Analysis pipeline, as well as SNP carrier status using the STITCH pipeline. Within each treatment arm, time to incident adverse events (AEs) were compared between carriers (SNP+) and non-carriers (SNP-) via multivariable Cox regression, controlling for age, sex and sample purity, followed by a SNP´treatment interaction term in the whole cohort. Overall survival (OS) and progression-free survival (PFS) were also assessed. Finally, a recurrent event analysis for AEs was conducted using the Andersen-Gill model, controlling for the same variables. Results: In total, 382 pts were included (NIVO: n=189, EVE: n=193), among which 56 (14.7%) were SNP+. There were no differences in clinical and pathological characteristics between SNP+ and SNP-, except for sex (SNP+ 16.1% vs. SNP- 30.1% females, P=0.046). Similarly, no differences in somatic alterations, including single nucleotide and copy number variants were seen between SNP+ and SNP-. SNP carrier status had no effect on OS nor PFS in both treatment arms (all P≥0.47). Regarding AEs, 63 pts (33.3%) in the NIVO arm experienced at least 1 grade 2+ AE, compared to 78 pts (40.4%) in the EVE arm. The most common types of grade 2+ AEs were cutaneous (21.0%), hepatobiliary (16.8%) and endocrine (15.8%) in the NIVO arm, and respiratory (30.1%), cutaneous (22.3%) and gastrointestinal (19.4%) in the EVE arm. The rate of grade 2+ AEs was significantly higher in SNP+ vs. SNP- in the NIVO arm (HR=2.91[1.48-5.72]), but not in the EVE arm (HR=0.63[0.3-1.29], SNP´treatment Pinteraction=0.002). The rate of recurrent grade 2+ AEs was also significantly higher in SNP+ vs. SNP- in the NIVO arm (HR=3.43[1.83-6.43]), whereas a trend for fewer recurrent grade 2+ AEs was seen in SNP+ vs. SNP- in the EVE arm (HR=0.46[0.17-1.25], SNP´treatment Pinteraction=0.0005). Conclusions: The IL7 SNP (rs16906115) is associated with significantly higher rates of grade 2+ AEs, including recurrent events, in pts with mRCC treated with NIVO but not with EVE, with no effect on survival outcomes. These results affirm the SNP’s predictive potential as a biomarker for irAEs to guide therapeutic decisions in pts treated with ICIs.

Differential genomic profiling of the progesterone receptor (PR) negative and estrogen receptor (ER) low metastatic breast cancer subtypes through circulating tumor DNA: A TNBC-like disease subtype?

1039 Background: ER positive/HER2 negative metastatic breast cancer (MBC) is typically regarded as a single entity, although significant clinical differences are often observed, especially in the ER low subgroup (ER&lt;10% regardless of PR status). This study aims to investigate genomic differences among HER2 negative subtypes (i.e., ERlow, PRpos [ER &gt;10%/PRpositve], PRneg [ER &gt;10%/PRnegative], triple negative [TNBC]) through circulating tumor DNA (ctDNA) next-generation sequencing (NGS). Methods: The study analyzed a retrospective cohort of 879 patients (pts) with HER2 negative MBC with ctDNA testing using the Guardant360 NGS panel within a large multicenter academic consortium. ER, PR and HER2 status was defined based on the most updated biopsy. Associations across single nucleotide and copy number variations (SNVs and CNVs) and ER/PR status were tested by multinomial logistic regression in terms of Relative Risk Ratio (RRR), adjusting for significant clinical characteristics (i.e., lines of treatment, metastatic sites, histology). Oncogenic pathway was defined based on reference (1). Prognosis was analyzed through Cox regression for overall survival (OS) defined from time of ctDNA collection. Results: Among the 879 analyzed pts, PRpos was the most prevalent subtype (N:420, 48%), followed by PRneg (N:216, 24%), TNBC (N:209, 24%), and ERlow (N:34, 4%). Compared to PRpos, lobular MBC was less common in TNBC (RRR: 0.33, P=0.001), while lung involvement was more frequent in TNBC (RRR: 1.53, P=0.019), and liver involvement in PRneg (RRR: 1.44, P=0.035). In contrast, bone metastases were less common in TNBC and ERlow (RRR: 0.26, P&lt;0.001 and RRR: 0.45, P=0.03, respectively). After multivariable analysis, ERlow and TNBC were associated with a higher incidence of TP53 SNVs (RRR: 5.42, P&lt;0.001 and RRR: 3.08, P=0.007, respectively) and a lower incidence of ESR1 SNVs (RRR: 0.23, P=0.025 and not detected, respectively). TNBC exhibited significantly fewer PIK3CA SNVs (RRR: 0.41, P=0.005) and CCND1 CNVs (RRR: 0.20, P=0.009), along with a higher frequency of CCNE1 CNVs (RRR: 6.47, P&lt;0.001). PRneg had a lower incidence of ESR1 SNVs (RRR: 0.52, P=0.013) and more frequent FGFR1 CNVs (RRR: 2.61, P=0.004). Similar results were observed across oncogenic pathways. Compared to PRpos, OS analysis indicated poorer outcomes across all subgroups (PRneg HR: 1.29 P = 0.035, TNBC HR: 1.96 P&lt;0.001, ERlow HR: 2.32 P&lt;0.001). Conclusions: This study highlights several clinical and molecular similarities between TNBC and ERlow MBC, while suggesting specific endocrine resistance mechanisms in PRneg that are less reliant on ESR1 and more exposed to alternative mechanisms such as FGFR1 amplification. These findings underscore the importance of more precise disease subtyping to inform future drug development trials. 1. Sanchez-Vega F et al, Cell. 2018.