Large Rearrangements Research Articles

Genome-wide association for agro-morphological traits in a triploid banana population with large chromosome rearrangements

Abstract Banana breeding is hampered by the very low fertility of domesticated bananas and the lack of knowledge about the genetic determinism of agronomic traits. We analysed a breeding population of 2 723 triploid hybrids resulting from crosses between diploid and tetraploid M. acuminata parents, which was evaluated over three successive crop-cycles for 24 traits relating to yield components and plant, bunch and fruit architectures. A subset of 1 129 individuals was genotyped-by-sequencing revealing 205 612 single nucleotide polymorphisms. Most parents were heterozygous for one or several large reciprocal chromosomal translocations, which are known to impact recombination and chromosomal segregation. We applied two linear mixed models to detect associations between markers and traits: (i) a standard model with a kinship calculated using all SNPs and (ii) a model with chromosome-specific kinships that aims at recovering statistical power at alleles carried by long non-recombined haplotypic segments. For 23 of the 24 traits, we identified one to five significant quantitative trait loci (QTLs) for which the origin of favourable alleles could often be determined among the main ancestral contributors to banana cultivars. Several QTLs, located in the rearranged regions, were only detected using the second model. The resulting QTL landscape represents an important resource to support breeding programs. The proposed strategy for recovering power at SNPs carried by long non-recombined rearranged haplotypic segments is an important methodological advance for future association studies in banana and other species affected by chromosomal rearrangements.

Molecular analysis of inherited disorders of cornification in polish patients show novel variants and functional data and provokes questions on the significance of secondary findings.

The Mendelian Disorders of Cornification (MeDOC) comprise a large number of disorders that present with either localised (palmoplantar keratoderma, PPK) or generalised (ichthyoses) signs. The MeDOC are highly heterogenic in terms of genetics and phenotype. Consequently, diagnostic process is challenging and before implementation of the next generation sequencing, was mostly symptomatic, not causal, which limited research on those diseases. The aim of the study was to genetically characterise a cohort of 265 Polish patients with MeDOC and to get insight into the skin lesions using transcriptome and lipid profile analyses. We detected causal variants in 85% (226/265) patients. In addition to the primary gene defect, a pathogenic variant in another gene involved in MeDOC pathology was identified in 23 cases. We found 150 distinct variants in 33 genes, including 32 novel and 16 recurrent (present in > 5 alleles). In 43 alleles large rearrangements were detected, including deletions in the STS, SPINK5, CERS3 and recurrent duplication of exons 10-14 in TGM1. The RNA analysis using samples collected from 18 MeDOC patients and 22 controls identified 1377 differentially expressed genes - DEG. The gene ontology analysis revealed that 114 biological processes were upregulated in the MeDOC group, including i.e. epithelial cell differentiation, lipid metabolic process; homeostasis; regulation of water loss via skin; peptide cross-linking. The DEG between TGM1 and ALOX12B patients, showed that RNA profile is highly similar, though fatty acid profile in epidermal scrapings of those patients showed differences e.g. for the very long chain fatty acids (VLCFAs; FAs ≥ C20), the very long-chain monounsaturated fatty acids (VLC-MUFAs, FAs ≥ C20:1) and the n6 polyunsaturated fatty acids (n6 PUFAs). Our results show that NGS-based analysis is an effective MeDOC diagnostic tool. The Polish MeDOC patients are heterogenic, however recurrent variants are present. The novel variants and high number of TGM1 and SPINK5 copy number variations give further insight into molecular pathology of MeDOC. We show that secondary variants in MeDOC-related genes are present in a significant group of patients, which should be further investigated in the context of phenotype modifiers. Finally, we provide novel RNA and lipid data that characterise molecularly MeDOC epidermis.

Genetics in the Ocean's Twilight Zone: Population Structure of the Glacier Lanternfish Across Its Distribution Range.

The mesopelagic zone represents one of the few habitats that remains relatively untouched from anthropogenic activities. Among the many species inhabiting the north Atlantic mesopelagic zone, glacier lanternfish (Benthosema glaciale) is the most abundant and widely distributed. This species has been regarded as a potential target for a dedicated fishery despite the scarce knowledge of its population genetic structure. Here, we investigated its genetic structure across the North Atlantic and into the Mediterranean Sea using 121 SNPs, which revealed strong differentiation among three main groups: the Mediterranean Sea, oceanic samples, and Norwegian fjords. The Mediterranean samples displayed less than half the genetic variation of the remaining ones. Very weak or nearly absent genetic structure was detected among geographically distinct oceanic samples across the North Atlantic, which contrasts with the low motility of the species. In contrast, a longitudinal gradient of differentiation was observed in the Mediterranean Sea, where genetic connectivity is known to be strongly shaped by oceanographic processes such as current patterns and oceanographic discontinuities. In addition, 12 of the SNPs, in linkage disequilibrium, drove a three clusters' pattern detectable through Principal Component Analysis biplot matching the genetic signatures generally associated with large chromosomal rearrangements, such as inversions. The arrangement of this putative inversion showed frequency differences between open-ocean and more confined water bodies such as the fjords and the Mediterranean, as it was fixed in the latter for the second most common arrangement of the fjord's samples. However, whether genetic differentiation was driven by local adaptation, secondary contact, or a combination of both factors remains undetermined. The major finding of this study is that B. glaciale in the North Atlantic-Mediterranean is divided into three major genetic units, information that should be combined with demographic properties to outline the management of this species prior to any eventual fishery attempt.

Novel rapid molecular diagnosis methods for comprehensive genetic analysis of 21-hydroxylase deficiency

BackgroundMolecular analysis of the CYP21A2 gene is highly important for understanding the aetiology of 21-hydroxylase deficiency (21-OHD). The aim of this study was to use a novel approach named CNVplex, together with the SNaPshot assay and direct sequencing, to identify CYP21A2 mutations efficiently and comprehensively. Targeted CYP21A2 mutation analysis was performed in 113 patients and 226 parents. Large rearrangements of CYP21A2 were characterized by CNVplex; twenty prevalent mutations, including nine common micro-conversions and eleven high-frequency mutations reported in the literature, were detected by SNaPshot; and rare mutations were investigated by direct sequencing.ResultsAmong the 113 21-OHD patients, 95.6% of the affected alleles were detected accurately by SNaPshot and CNVplex. Prevalent mutations were detected in 69.5% of the alleles; 62.4% of alleles contained pseudogene-derived micro-conversions, 1.8% contained nonpseudogene-derived mutations, and 5.3% contained complex variations resulting from multiple recombinations between CYP21A2 and CYP21A1P. Large rearrangements were identified in 27.0% of the alleles, including five types (CH-1, CH-3, CH-4, CH-5 and CH-8) of chimeric CYP21A1P/CYP21A2 genes. Two novel CYP21A2 haplotypes and four de novo CYP21A2 mutations were characterized. A rare haplotype with a c.955 C > T mutation in the duplicated CYP21A2 gene was found in 0.9% of the probands and 33.3% of the parents. In addition, four parents were also diagnosed with 21-OHD.ConclusionCNVplex and SNaPshot appear to be highly efficient and reliable techniques for use in a molecular diagnosis laboratory, and combined with direct sequencing based on locus-specific PCR, they might constitute a definitive way to detect almost all common and rare 21-OHD-related alleles.

The timing of large drainage rearrangement in South America: A study based on morphological and ecological evidence

This study aims to characterize and determine the timing of a major drainage rearrangement between two vast river basins, each exceeding 600,000 km2: the Paraná and São Francisco basins in South America. We used geomorphological analyses and the computational Black Top Hat (BTH) method to identify river captures. By using freshwater fish distribution and evolution data, we performer biogeographical analyses to assess the fish dispersal patterns. Our results show that in the past the upper Paraná River (known locally as the Grande River) once flowed into what is now the São Francisco River Basin. Biogeographical analysis corroborates this past connection between Grande and São Fracisco rivers This analysis reveals also that between 6 and 5 and 2 million years ago, a significant drainage rearrangement occurred, when the Paraná River Basin captured the Grande River and its tributary (the Sapucaí River). In this process the São Francisco Basin losing 50,000 km2 of its area to the Paraná River Basin.

CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model

BackgroundThe development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes.ResultsTo address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout.ConclusionsOur unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

Genome Instability Induced by Topoisomerase Misfunction.

Topoisomerases alter DNA topology by making transient DNA strand breaks (DSBs) in DNA. The DNA cleavage reaction mechanism includes the formation of a reversible protein/DNA complex that allows rapid resealing of the transient break. This mechanism allows changes in DNA topology with minimal risks of persistent DNA damage. Nonetheless, small molecules, alternate DNA structures, or mutations in topoisomerase proteins can impede the resealing of the transient breaks, leading to genome instability and potentially cell death. The consequences of high levels of enzyme/DNA adducts differ for type I and type II topoisomerases. Top1 action on DNA containing ribonucleotides leads to 2-5 nucleotide deletions in repeated sequences, while mutant Top1 enzymes can generate large deletions. By contrast, small molecules that target Top2, or mutant Top2 enzymes with elevated levels of cleavage lead to small de novo duplications. Both Top1 and Top2 have the potential to generate large rearrangements and translocations. Thus, genome instability due to topoisomerase mis-function is a potential pathogenic mechanism especially leading to oncogenic progression. Recent studies support the potential roles of topoisomerases in genetic changes in cancer cells, highlighting the need to understand how cells limit genome instability induced by topoisomerases. This review highlights recent studies that bear on these questions.

Efficacy of PARP inhibitors in advanced high-grade serous ovarian cancer according to BRCA domain mutations and mutation type.

Preclinical studies have emphasized the potential connection between BRCA specific domains defects and the activity of Poly ADP-ribose polymerase inhibitors (PARPi). Nevertheless, real-world evidence regarding the impact of BRCA domain defects and mutations on PARPi efficacy are limited. The aim of his study was to evaluate the efficacy of PARPi in terms of progression free survival (PFS) according to BRCA domains defects and mutation types. A retrospective analysis was performed among 79 BRCA mutated patients, diagnosed with advanced High-grade serous ovarian carcinoma (HGSOC) who received first- and second-line platinum- based chemotherapy followed by PARPi maintenance treatment. PFS was evaluated according to BRCA1 [Really Interesting Gene (RING), DNA Binding (DBD), Serine Cluster (SCD), BRCA1 C-terminal (BRCT)] and BRCA2 [RAD-51 Domain (RAD-51 BD), DBD] specific domain defects and mutation types [missense (MS), nonsense (NS), frameshift (FS), splicing (S), or large rearrangements (LR)]. After a median follow-up of 51 months, no significant difference in PFS was observed between the BRCA functional domains or mutation types in the BRCA1 and BRCA2 subgroups. Patients with BRCA2 DBD and RAD51-BD defects had the longest (39.8 months) and shortest (24.1 months) median PFS, respectively (p = 0.11). Additionally, patients with BRCA1 DBD defects had the greatest benefit (median PFS = 33.8 months) while those with BRCA1 RING domain mutations experienced the worst outcome (median PFS = 30.9 months (p = 0.43). The efficacy of maintenance treatment with PARPi is independent by BRCA domain defects or mutation types. Patients DBD domain defects experienced numerically longer median PFS compared to those with other BRCA1/2 alterations.

Bimodal substrate binding in the active site of the glycosidase BcX.

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.

Phage-mediated resolution of genetic conflict alters the evolutionary trajectory of Pseudomonas aeruginosa lysogens.

The opportunistic human pathogen Pseudomonas aeruginosa is naturally infected by a large class of temperate, transposable, Mu-like phages. We examined the genotypic and phenotypic diversity of P. aeruginosa PA14 lysogen populations as they resolve clustered regularly interspaced short palindromic repeat (CRISPR) autoimmunity, mediated by an imperfect CRISPR match to the Mu-like DMS3 prophage. After 12 days of evolution, we measured a decrease in spontaneous induction in both exponential and stationary phase growth. Co-existing variation in spontaneous induction rates in the exponential phase depended on the way the coexisting strains resolved genetic conflict. Multiple mutational modes to resolve genetic conflict between host and phage resulted in coexistence in evolved populations of single lysogens that maintained CRISPR immunity to other phages and polylysogens that lost immunity completely. This work highlights a new dimension of the role of lysogenic phages in the evolution of their hosts.IMPORTANCEThe chronic opportunistic multi-drug-resistant pathogen Pseudomonas aeruginosa is persistently infected by temperate phages. We assess the contribution of temperate phage infection to the evolution of the clinically relevant strain UCBPP-PA14. We found that a low level of clustered regularly interspaced short palindromic repeat (CRISPR)-mediated self-targeting resulted in polylysogeny evolution and large genome rearrangements in lysogens; we also found extensive diversification in CRISPR spacers and cas genes. These genomic modifications resulted in decreased spontaneous induction in both exponential and stationary phase growth, increasing lysogen fitness. This work shows the importance of considering latent phage infection in characterizing the evolution of bacterial populations.

Structural variants linked to Alzheimer's Disease and other common age-related clinical and neuropathologic traits.

Advances have led to a greater understanding of the genetics of Alzheimer's Disease (AD). However, the gap between the predicted and observed genetic heritability estimates when using single nucleotide polymorphisms (SNPs) and small indel data remains. Large genomic rearrangements, known as structural variants (SVs), have the potential to account for this missing genetic heritability. By leveraging data from two ongoing cohort studies of aging and dementia, the Religious Orders Study and Rush Memory and Aging Project (ROS/MAP), we performed genome-wide association analysis testing around 20,000 common SVs from 1,088 participants with whole genome sequencing (WGS) data. A range of Alzheimer's Disease and Related Disorders (AD/ADRD) clinical and pathologic traits were examined. Given the limited sample size, no genome-wide significant association was found, but we mapped SVs across 81 AD risk loci and discovered 22 SVs in linkage disequilibrium (LD) with GWAS lead variants and directly associated with AD/ADRD phenotypes (nominal P < 0.05). The strongest association was a deletion of an Alu element in the 3'UTR of the TMEM106B gene. This SV was in high LD with the respective AD GWAS locus and was associated with multiple AD/ADRD phenotypes, including tangle density, TDP-43, and cognitive resilience. The deletion of this element was also linked to lower TMEM106B protein abundance. We also found a 22 kb deletion associated with depression in ROSMAP and bearing similar association patterns as AD GWAS SNPs at the IQCK locus. In addition, genome-wide scans allowed the identification of 7 SVs, with no LD with SNPs and nominally associated with AD/ADRD traits. This result suggests potentially new ADRD risk loci not discoverable using SNP data. Among these findings, we highlight a 5.6 kb duplication of coding regions of the gene C1orf186 at chromosome 1 associated with indices of cognitive impairment, decline, and resilience. While further replication in independent datasets is needed to validate these findings, our results support the potential roles of common structural variations in the pathogenesis of AD/ADRD.

Approaches for Lynch syndrome screening and characteristics of subtypes with mismatch repair deficiency in patients with colorectal carcinoma.

To evaluate different Lynch syndrome (LS) screening approaches and establish an efficient and sensitive strategy are critical for clinical practice. In total, 583 patients with colorectal carcinoma (CRC) at Fudan University Shanghai Cancer Center were enrolled. Patient samples were examined by immunohistochemistry (IHC) and next-generation sequencing (NGS), and MLH1 promoter hypermethylation (MPH) was detected in MLH1-deficient cases. Germline genetic testing was performed in cases with deleterious variants and large genomic rearrangements (LGRs) of tumor MMR genes were detected in cases with dMMR or MSI-H cases with no MMR germline variants. Our results showed that triage with IHC and followed by BRAF/MLH1 methylation testing (Strategy 1) identified 93.3% (70/75) of LS cases. IHC followed by germline NGS (Strategy 2) or direct tumor NGS (Strategy 3) both identified 98.7% (74/75) of LS cases. The proportion of LGRs in LS cases was 16.0% (12/75), while 84.0% (63/75) showed SNV/Indel. The average cost per patient was ¥6010.81, ¥6058.48, and ¥8029.98 for Strategy 1, Strategy 2 and Strategy 3, respectively. The average time spent on different strategies was 4.74 days (Strategy 1), 4.89 days (Strategy 2), and 14.50 days (Strategy 3) per patient, respectively. LS and Lynch-like syndrome (LLS) were associated with an earlier onset age than MPH. In conclusion, we compared different workflows for LS screening and IHC plus germline NGS is recommended for LS screening when taking sensitivity, time, and cost into account. Moreover, multiplex ligation-dependent probe amplification made up for the shortcoming of NGS and should be incorporated into routine screening.

Revealing the principles of inter- and intra-domain regulation in a signaling enzyme via scanning mutagenesis.

Multi-domain enzymes can be regulated by both inter-domain interactions and structural features intrinsic to the catalytic domain. The tyrosine phosphatase SHP2 is a quintessential example of a multi-domain protein that is regulated by inter-domain interactions. This enzyme has a protein tyrosine phosphatase (PTP) domain and two phosphotyrosine-recognition domains (N-SH2 and C-SH2) that regulate phosphatase activity through autoinhibitory interactions. SHP2 is canonically activated by phosphoprotein binding to the SH2 domains, which causes large inter-domain rearrangements, but autoinhibition can also be disrupted by disease-associated mutations. Many details of the SHP2 activation mechanism are still unclear, the physiologically-relevant active conformations remain elusive, and hundreds of human variants of SHP2 have not been functionally characterized. Here, we perform deep mutational scanning on both full-length SHP2 and its isolated PTP domain to examine mutational effects on inter-domain regulation and catalytic activity. Our experiments provide a comprehensive map of SHP2 mutational sensitivity, both in the presence and absence of inter-domain regulation. Coupled with molecular dynamics simulations, our investigation reveals novel structural features that govern the stability of the autoinhibited and active states of SHP2. Our analysis also identifies key residues beyond the SHP2 active site that control PTP domain dynamics and intrinsic catalytic activity. This work expands our understanding of SHP2 regulation and provides new insights into SHP2 pathogenicity.

Complete Chloroplast Genome of Megacarpaea megalocarpa and Comparative Analysis with Related Species from Brassicaceae.

Megacarpaea megalocarpa, a perennial herbaceous species belonging to the Brassicaceae family, has potential medicinal value. We isolated and characterized the chloroplast (cp) genome of M. megalocarpa and compared it with closely related species. The chloroplast genome displayed a typical quadripartite structure, spanning 154,877 bp, with an overall guanine-cytosine (GC) content of 36.20%. Additionally, this genome contained 129 genes, 105 simple sequence repeats (SSRs), and 48 long repeat sequences. Significantly, the ycf1 gene exhibited a high degree of polymorphism at the small single copy (SSC) region and the inverted repeat a (IRa) boundary. Despite this polymorphism, relative synonymous codon usage (RSCU) values were found to be similar across species, and no large segment rearrangements or inversions were detected. The large single copy (LSC) and SSC regions showed higher sequence variations and nucleotide polymorphisms compared to the IR region. Thirteen distinct hotspot regions were identified as potential molecular markers. Our selection pressure analysis revealed that the protein-coding gene rpl20 is subjected to different selection pressures in various species. Phylogenetic analysis positioned M. megalocarpa within the expanded lineage II of the Brassicaceae family. The estimated divergence time suggests that M. megalocarpa diverged approximately 4.97 million years ago. In summary, this study provides crucial baseline information for the molecular identification, phylogenetic relationships, conservation efforts, and utilization of wild resources in Megacarpaea.

Germline mutations of breast cancer susceptibility genes through expanded genetic analysis in unselected Colombian patients

BackgroundIn Colombia and worldwide, breast cancer (BC) is the most frequently diagnosed neoplasia and the leading cause of death from cancer among women. Studies predominantly involve hereditary and familial cases, demonstrating a gap in the literature regarding the identification of germline mutations in unselected patients from Latin-America. Identification of pathogenic/likely pathogenic (P/LP) variants is important for shaping national genetic analysis policies, genetic counseling, and early detection strategies. The present study included 400 women with unselected breast cancer (BC), in whom we analyzed ten genes, using Whole Exome Sequencing (WES), know to confer risk for BC, with the aim of determining the genomic profile of previously unreported P/LP variants in the affected population. Additionally, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to identify Large Genomic Rearrangements (LGRs) in the BRCA1/2 genes. To ascertain the functional impact of a recurrent intronic variant (ATM c.5496 + 2_5496 + 5delTAAG), a minigene assay was conducted. ResultsWe ascertained the frequency of P/LP germline variants in BRCA2 (2.5%), ATM (1.25%), BRCA1 (0.75%), PALB2 (0.50%), CHEK2 (0.50%), BARD1 (0.25%), and RAD51D (0.25%) genes in the population of study. P/LP variants account for 6% of the total population analyzed. No LGRs were detected in our study. We identified 1.75% of recurrent variants in BRCA2 and ATM genes. One of them corresponds to the ATM c.5496 + 2_5496 + 5delTAAG. Functional validation of this variant demonstrated a splicing alteration probably modifying the Pincer domain and subsequent protein structure. ConclusionThis study described for the first time the genomic profile of ten risk genes in Colombian women with unselected BC. Our findings underscore the significance of population-based research, advocating the consideration of molecular testing in all women with cancer.

Structural Requirements for Reverse Transcription by a Diversity-generating Retroelement.

Diversity-generating retroelements (DGRs) create massive protein sequence variation in ecologically diverse microbes. Variation occurs during reverse transcription of a protein-encoding RNA template coupled to misincorporation at adenosines. In the prototypical Bordetella bacteriophage DGR, the template must be surrounded by upstream and downstream RNA segments for cDNA synthesis by the reverse transcriptase bRT and associated protein Avd. The function of the surrounding RNA was unknown. Cryo-EM revealed that this RNA enveloped bRT and lay over barrel-shaped Avd, forming an intimate ribonucleoprotein (RNP). An abundance of essential interactions between RNA structural elements and bRT-Avd precisely positioned an RNA homoduplex for initiation of cDNA synthesis by cis -priming. Our results explain how the surrounding RNA primes cDNA synthesis, promotes processivity, terminates polymerization, and strictly limits mutagenesis to select proteins through mechanisms that are likely conserved in DGRs from distant taxa.

Abstract IA020: Functional studies of genetic variation using precision genome editing

Abstract Tumor genomes often harbor a complex spectrum of single nucleotide mutations, small indels, and large chromosome rearrangements that can perturb coding and non-coding regions of the genome in ways that remain poorly understood. The mutational processes responsible for the genesis of these events are also not static; instead, they continue to operate throughout disease evolution and further diversify the genetic and phenotypic landscape of cancer cells. Mounting evidence suggests that tumor genotype can be an important determinant of disease progression and therapy response, such as by modulating the sensitivity or resistance of cancer cells to mutant-specific drugs. These types of observations have motivated efforts to treat cancers with genome-informed therapies, highlighting the need to understand how different genetic variants precisely affect gene function and overall tumor phenotypes. Variant-function maps that provide a mechanistic understanding of the biology driven by cancer-associated mutations are needed to design these types of treatment strategies. Precision genome editing technologies like base and prime editing are uniquely suited to tackle this problem. Nevertheless, deploying these methods for systematic variant-function studies and disease modeling in vivo has not been straightforward due to lack of robust and scalable platforms capable of assessing editing efficiency and precision, particularly at endogenous loci. With this goal in mind, we previously developed and applied high-throughput base editing ‘sensor’ approaches that link endogenous genome editing outcomes with synthetic DNA-based readouts and cellular fitness measurements. Using these approaches, we showed that several uncharacterized mutant p53 alleles drive cancer cell proliferation and in vivo tumor development. Building upon this work, we recently developed new prime editing guide RNA design tools and sensor-based approaches that similarly couple quantitative editing outcomes to cellular fitness, allowing us to significantly expand the breadth of cancer-associated mutations that can be interrogated using these precision genome editing technologies. In this talk, I will describe ongoing work using base and prime editing sensor libraries to probe the biological impact of thousands of functionally-distinct genetic variants in diverse types of protein-coding genes and families to learn how these influence various cancer phenotypes. I will also discuss how the implementation of these modular technologies will allow researchers to functionally disentangle the impact of endogenous and exogenous mutational processes on functional selection and tumor evolution with close to single base pair resolution. This generalizable precision genome editing framework will facilitate the functional interrogation of genetic variants across diverse biological contexts, providing much needed insight into cancer variant-function relationships that could be leveraged to develop more precise cancer treatment paradigms, including synthetic lethal therapies that exploit tumor genotype. Citation Format: Francisco J. Sánchez-Rivera, Samuel I. Gould, Alexandra N. Wuest, Kexin Dong, Grace A. Johnson, Alvin Hsu, Varun K. Narendra, Ondine Atwa, Stuart S. Levine, David R. Liu. Functional studies of genetic variation using precision genome editing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA020.

Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

Kagomerization of transition metal monolayers induced by two-dimensional hexagonal boron nitride

The kagome lattice is an exciting solid state physics platform for the emergence of nontrivial quantum states driven by electronic correlations: topological effects, unconventional superconductivity, charge and spin density waves, and unusual magnetic states such as quantum spin liquids. While kagome lattices have been realized in complex multi-atomic bulk compounds, here we demonstrate from first-principles a process that we dub kagomerization, in which we fabricate a two-dimensional kagome lattice in monolayers of transition metals utilizing an hexagonal boron nitride (h-BN) overlayer. Surprisingly, h-BN induces a large rearrangement of the transition metal atoms supported on a fcc(111) heavy-metal surface. This reconstruction is found to be rather generic for this type of heterostructures and has a profound impact on the underlying magnetic properties, ultimately stabilizing various topological magnetic solitons such as skyrmions and bimerons. Our findings call for a reconsideration of h-BN as merely a passive capping layer, showing its potential for not only reconstructing the atomic structure of the underlying material, e.g. through the kagomerization of magnetic films, but also enabling electronic and magnetic phases that are highly sought for the next generation of device technologies.

Leveraging genomic instability scores (GIS) to investigate BRCA variants of unknown significance (VUS) in ovarian cancer: An analysis of 14,513 patients in the Myriad Collaborative Research Registry (MCRR).

e17549 Background: In ovarian cancer, use of homologous recombination deficiency (HRD) and BRCA mutation ( BRCAm) status is essential in guiding PARP inhibitor (PARPi) therapy, with the greatest survival benefit in patients harboring BRCAm pathogenic variants (PVs). In other indications, PARPi are only approved for patients with BRCAm or homologous recombination repair mutations (HRRm). Genomic instability scores (GIS), an HRD proxy, are also predictive of PARPi benefit in patients without BRCAm or HRRm, suggesting that other DNA alterations without definitive classification, such as BRCA VUS or variants of Special Interpretation (SI), may be contributing to HRD. Here, we evaluated the association of BRCA VUS and SI variants that have complex clinical interpretation with reduced penetrance. Methods: Retrospective analysis was conducted using de-identified genetic testing data from the MCRR. Eligible patients with ovarian, fallopian tube or primary peritoneal cancer were tested with the MyChoice CDx HRD test between 2016 and 2023, with HRD+ defined as GIS≥42 or presence of BRCAm PV. Patient BRCAm and HRRm status were defined using the most clinically significant variant observed in BRCA1/2 and an HRR 13-gene set, respectively. GIS distributions were compared using Kolmogorov-Smirnov tests and decomposed using mixture models. Results: Overall, 31.9% (4,628/14,513) of patients were HRD+ with 9.3% (N=1,344) having BRCAm PVs. An additional 3.7% (N=538) had a BRCA VUS [N=83 uncharacterized large rearrangement (LR), N=455 non-LR]. The distributions of GIS in both non-LR and LR VUS were significantly different from the distributions in BRCAm PVs and BRCA wildtype tumors (ps &lt; 0.001), likely representing a spectrum of pathogenic and benign variants. Mixture models estimated that 28.8% [95% confidence interval (CI) 21.9, 35.8] of non-LR VUS and 55.8% (95% CI 40.0, 70.4) of LR VUS exhibited GIS similar to BRCAm PVs. Of the 15 non-LR VUS observed more than once, two were observed only in tumors with low GIS and are therefore presumed benign. BRCA SI variants (N=6) were observed in 16 patients and had a distribution of GIS significantly different from BRCA wildtype tumors (p &lt; 0.001), but not significantly different from BRCAm tumors (p = 0.1). Two SI variants had high GIS in multiple patients and are therefore presumed pathogenic. One SI variant had low GIS in two patients and is therefore presumed benign. Conclusions: The BRCA VUS GIS distribution spanned across the BRCAm PV and BRCA wildtype GIS distributions, which suggests that BRCA VUS represent a mix of benign variants and PVs. In these patients, GIS is able to correctly classify HRD status. Additional data are needed to further characterize these uncertain BRCA variants and the appropriate therapeutic and preventive strategies required for these patients.