CIViCdb 2022: evolution of an open-access cancer variant interpretation knowledgebase.

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC's functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing >3200 variants in >470 genes from >3100 publications.

Ulcerated CRTC1::TRIM11 cutaneous tumor with metastases.

CRTC1::TRIM11 cutaneous tumor (CTCT) is a rare skin tumor of uncertain differentiation. In the 49 reported cases, only four cases showed regional or distant metastasis, but follow-up remains limited. Herein, we present a case of metastatic CTCT with ulceration, a histological feature that has not been previously described. A 75-year-old male with a 2-month history of toe ulceration underwent a shave biopsy, which showed a dermal nodular neoplasm that was immunoreactive for SOX10 and S100, negative for Melan-A, and was initially diagnosed as melanoma. Upon pathology review at our institution, the tumor was composed of intersecting fascicles and nests of epithelioid and spindle cells. Additional immunohistochemistry revealed immunoreactivity of the tumor for MiTF and NTRK and negativity for HMB-45 and PRAME. Next-generation sequencing identified CRTC1::TRIM11 fusion, leading to a revised diagnosis of CTCT. The patient proceeded to a toe amputation and sentinel lymph node (SLN) biopsy 5 months after the shave biopsy. The amputation showed residual CTCT and a focus on lymphovascular invasion. The SLN revealed multifocal subcapsular metastases. The patient was started on adjuvant nivolumab and showed biopsy-proven recurrence in the right inguinal lymph nodes and imaging findings suspicious for pulmonary metastases 8 months after the excision. In summary, we present a case of CTCT with ulceration and lymphovascular invasion. We also provide additional evidence that a subset of CTCT behaves aggressively. The optimal surgical and medical treatments are unknown.

Whole-genome sequencing uncovers the structural and transcriptomic landscape of hexaploid wheat/Ambylopyrum muticum introgression lines.

Wheat is a globally vital crop, but its limited genetic variation creates a challenge for breeders aiming to maintain or accelerate agricultural improvements over time. Introducing novel genes and alleles from wheat's wild relatives into the wheat breeding pool via introgression lines is an important component of overcoming this low variation but is constrained by poor genomic resolution and limited understanding of the genomic impact of introgression breeding programmes. By sequencing 17 hexaploid wheat/Ambylopyrum muticum introgression lines and the parent lines, we have precisely pinpointed the borders of introgressed segments, most of which occur within genes. We report a genome assembly and annotation of Am. muticum that has facilitated the identification of Am. muticum resistance genes commonly introgressed in lines resistant to stripe rust. Our analysis has identified an abundance of structural disruption and homoeologous pairing across the introgression lines, likely caused by the suppressed Ph1 locus. mRNAseq analysis of six of these introgression lines revealed that novel introgressed genes are rarely expressed and those that directly replace a wheat orthologue have a tendency towards downregulation, with no discernible compensation in the expression of homoeologous copies. This study explores the genomic impact of introgression breeding and provides a schematic that can be followed to characterize introgression lines and identify segments and candidate genes underlying the phenotype. This will facilitate more effective utilization of introgression pre-breeding material in wheat breeding programmes.

Estimation of wound surface area: an evaluation of medical professionals' assessment in a plastic surgery unit.

The percentage of skin graft take following the resurfacing of burn injuries is routinely calculated in a subjective fashion, in order to make management decisions. Given the gravity of decisions made based on this clinical assessment of graft check, it is notable that limited research has been carried out on this subject. No standardised subjective assessment tools exist to measure surface area of graft take in a manner similar to that of Wallace's Rule of Nines or Lund and Browder. This study set out to examine the accuracy of visual assessments of graft take within the multidisciplinary team regularly making assessments of newly grafted burn wounds. A total of 15 digitally drawn images were used to assess 36 staff members' estimations of percentage of surface area. The results showed a wide variation in estimation in all staff types, including senior burns surgeons, who were found to underestimate surface area by as much as 30%. The British Burns Association has removed 'healing time' as an outcome measurement from its guidance, as it recognises how hard it is to make a standardised assessment of wound healing. This study demonstrates the difficulty in subjectively assessing surface area and makes some suggestions for further research and clinical applications of technology to aid assessment.

Clinical reporting for personalized cancer genomics requires extensive access to subscription-only literature.

Objective: Medical care for cancer is increasingly directed by genomic laboratory testing for alterations in the tumor genome that are significant for diagnosis, prognosis and therapy. Uniquely in medicine, providers must search the biomedical literature for each patient to determine the clinical significance of these alterations. Access to published scientific literature is frequently subject to high fees, with access limited to institutional subscriptions. We sought to investigate the degree to which the scientific literature is accessible to clinical cancer genomics providers, and the potential role of university and hospital system libraries in information access for cancer care. Methods: We identified 265 journals that were accessed during the interpretation and reporting of clinical test results from 1,842 cancer patients at the University Health Network (Toronto, Canada). We determined the degree of open access for this set of clinically important literature, and for any journals not available through open access we surveyed subscription access at seven academic hospital systems and at their affiliated universities. Results: This study found that nearly half (116/265) of journals have open access mandates that make articles freely available within one year of release. For the remaining subscription access journals, universities provided a uniformly high level of access, but access available through hospital system collections varied widely. Conclusion: This study highlights the importance of different modes of access to the use of the scientific literature in clinical practice and points to challenges that must be overcome as genomic medicine grows in scale and complexity.

Refinement of the Superomedial Pedicle Technique: A New Approach to Breast Reshaping Following Reduction.

INTRODUCTION: We present an alteration of the superomedial pedicle technique in breast reduction to control lateral fullness and create a more natural and contoured breast during reshaping. This approach has been adopted by the senior author (NC) in 79 patients over the past 4 years. METHODS: A wise pattern skin incision is used, and the nipple-areola complex (NAC) is maintained on a de-epithelialized superomedial pedicle. Instead of fully releasing the pedicle from the lateral parenchyma for rotation and inset, a bridge of tissue between the pedicle on its most posterior aspect and the lateral pillar is maintained. Key holding sutures are subsequently placed in Scarpa's fascia for reshaping. RESULTS: We find that with this refinement, the connection with the lateral pillar pulls the lateral parenchyma medially and superiorly when the pedicle is rotated into its new position, adding a natural curve to the side. The superior medial pedicle is still attached in its postero-lateral aspect to the lateral pillar and theoretically, will provide an even more robust vascular supply to the NAC. In our series, three patients developed minor skin healing issues amenable to treatment with dressings. No one suffered from nipple loss or other serious complications, and no dog ear revisions were required. CONCLUSIONS: We present a simple alteration of the superomedial pedicle technique that we believe results in improved breast contouring. Our experience suggests that this simple adaptation is safe, effective, and reproducible. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Standards for the classification of pathogenicity of somatic variants in cancer (oncogenicity): Joint recommendations of Clinical Genome Resource (ClinGen), Cancer Genomics Consortium (CGC), and Variant Interpretation for Cancer Consortium (VICC).

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. Although these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (eg, population frequency, functional, and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation, and, importantly, affects patient care. Therefore, it is essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium, and the Variant Interpretation for Cancer Consortium used a consensus approach to develop a standard operating procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification and has been validated on 94 somatic variants in 10 common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of somatic variants.

Rapid identification of homozygosity and site of wild relative introgressions in wheat through chromosome-specific KASP genotyping assays.

For future food security, it is important that wheat, one of the most widely consumed crops in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is currently being introduced into wheat through introgressions from its wild relatives. For trait discovery, it is necessary that each introgression is homozygous and hence stable. Breeding programmes rely on efficient genotyping platforms for marker-assisted selection (MAS). Recently, single nucleotide polymorphism (SNP)-based markers have been made available on high-throughput Axiom® SNP genotyping arrays. However, these arrays are inflexible in their design and sample numbers, making their use unsuitable for long-term MAS. SNPs can potentially be converted into Kompetitive allele-specific PCR (KASP™) assays that are comparatively cost-effective and efficient for low-density genotyping of introgression lines. However, due to the polyploid nature of wheat, KASP assays for homoeologous SNPs can have difficulty in distinguishing between heterozygous and homozygous hybrid lines in a backcross population. To identify co-dominant SNPs, that can differentiate between heterozygotes and homozygotes, we PCR-amplified and sequenced genomic DNA from potential single-copy regions of the wheat genome and compared them to orthologous copies from different wild relatives. A panel of 620 chromosome-specific KASP assays have been developed that allow rapid detection of wild relative segments and provide information on their homozygosity and site of introgression in the wheat genome. A set of 90 chromosome-nonspecific assays was also produced that can be used for genotyping introgression lines. These multipurpose KASP assays represent a powerful tool for wheat breeders worldwide.

Variation in key leaf photosynthetic traits across wheat wild relatives is accession dependent not species dependent.

The wild relatives of modern wheat represent an underutilized source of genetic and phenotypic diversity and are of interest in breeding owing to their wide adaptation to diverse environments. Leaf photosynthetic traits underpin the rate of production of biomass and yield and have not been systematically explored in the wheat relatives. This paper identifies and quantifies the phenotypic variation in photosynthetic, stomatal, and morphological traits in up to 88 wheat wild relative accessions across five genera. Both steady-state measurements and dynamic responses to step changes in light intensity are assessed. A 2.3-fold variation for flag leaf light and CO2 -saturated rates of photosynthesis Amax was observed. Many accessions showing higher and more variable Amax , maximum rates of carboxylation, electron transport, and Rubisco activity when compared with modern genotypes. Variation in dynamic traits was also significant; with distinct genus-specific trends in rates of induction of nonphotochemical quenching and rate of stomatal opening. We conclude that utilization of wild relatives for improvement of photosynthesis is supported by the existence of a high degree of natural variation in key traits and should consider not only genus-level properties but variation between individual accessions.

Exploiting the genome of Thinopyrum elongatum to expand the gene pool of hexaploid wheat.

KEY MESSAGE: One hundred and thirty four introgressions from Thinopyrum elongatum have been transferred into a wheat background and were characterised using 263 SNP markers. Species within the genus Thinopyrum have been shown to carry genetic variation for a very wide range of traits including biotic and abiotic stresses and quality. Research has shown that one of the species within this genus, Th. elongatum, has a close relationship with the genomes of wheat making it a highly suitable candidate to expand the gene pool of wheat. Homoeologous recombination, in the absence of the Ph1 gene, has been exploited to transfer an estimated 134 introgressions from Th. elongatum into a hexaploid wheat background. The introgressions were detected and characterised using 263 single nucleotide polymorphism markers from a 35 K Axiom® Wheat-Relative Genotyping Array, spread across seven linkage groups and validated using genomic in situ hybridisation. The genetic map had a total length of 187.8 cM and the average chromosome length was 26.8 cM. Comparative analyses of the genetic map of Th. elongatum and the physical map of hexaploid wheat confirmed previous work that indicated good synteny at the macro-level, although Th. elongatum does not contain the 4A/5A/7B translocation found in wheat.

Hyaluronan in skin wound healing: therapeutic applications.

Hyaluronan is a vital constituent in effective skin wound healing. This polysaccharide is ubiquitous throughout the human body and has functional significance for tissue repair and remodelling. The importance of hyaluronan in the proliferative phase of healing is diverse, impacting on cell migration, proliferation, modification of the inflammatory response and on angiogenesis. As such, it holds therapeutic potential for a variety of clinical applications that range from facilitating effective wound healing to burns management and scarring. This overview of the multifaceted roles of hyaluronan considers its current applications to clinical practice in plastic surgery as well as the latest advances in research.

Development and characterisation of interspecific hybrid lines with genome-wide introgressions from Triticum timopheevii in a hexaploid wheat background.

BACKGROUND: Triticum timopheevii (2n = 4x = 28; AtAtGG), is an important source for new genetic variation for wheat improvement with genes for potential disease resistance and salt tolerance. By generating a range of interspecific hybrid lines, T. timopheevii can contribute to wheat's narrow gene-pool and be practically utilised in wheat breeding programmes. Previous studies that have generated such introgression lines between wheat and its wild relatives have been unable to use high-throughput methods to detect the presence of wild relative segments in such lines. RESULTS: A whole genome introgression approach, exploiting homoeologous recombination in the absence of the Ph1 locus, has resulted in the transfer of different chromosome segments from both the At and G genomes of T. timopheevii into wheat. These introgressions have been detected and characterised using single nucleotide polymorphism (SNP) markers present on a high-throughput Axiom® Genotyping Array. The analysis of these interspecific hybrid lines has resulted in the detection of 276 putative unique introgressions from T. timopheevii, thereby allowing the generation of a genetic map of T. timopheevii containing 1582 SNP markers, spread across 14 linkage groups representing each of the seven chromosomes of the At and G genomes of T. timopheevii. The genotyping of the hybrid lines was validated through fluorescence in situ hybridisation (FISH). Comparative analysis of the genetic map of T. timopheevii and the physical map of the hexaploid wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed the presence of inter- and intra-genomic translocations within the At and G genomes of T. timopheevii that have been previously only detected through cytological techniques. CONCLUSIONS: In this work, we report a set of SNP markers present on a high-throughput genotyping array, able to detect the presence of T. timopheevii in a hexaploid wheat background making it a potentially valuable tool for marker assisted selection (MAS) in wheat pre-breeding programs. These valuable resources of high-density molecular markers and wheat-T. timopheevii hybrid lines will greatly enhance the work being undertaken for wheat improvement through wild relative introgressions.

Development and validation of an exome-based SNP marker set for identification of the St, Jr and Jvs genomes of Thinopyrym intermedium in a wheat background.

KEY MESSAGE: Cytogenetic analysis and array-based SNP genotyping of wheat- Th. intermedium introgression lines allowed identification of 634 chromosome-specific SNP markers across all twenty-one chromosomes of Th. intermedium (StJ r J vs , 2 n = 6 x = 42). Thinopyrum intermedium (2n = 6x = 42, StJrJvs) is one of the most promising reservoirs of useful genes including tolerance to abiotic stresses, perenniality and disease resistance not available in the cultivated bread wheat. The transfer of genetic diversity from wild species to wheat offers valuable responses to the effects of climate change. The new array-based single-nucleotide polymorphism (SNP) marker technology provides cheap and easy-to-use molecular markers for marker-assisted selection (MAS) in wheat breeding programmes. Here, we focus on the generation of a new chromosome-specific SNP marker set that can be used to characterize and identify the Th. intermedium chromosomes or chromosome segments transferred into wheat. A progressive investigation of marker development was conducted using 187 various newly developed wheat-Th. intermedium introgression lines and the Axiom® Wheat-Relative Genotyping array. We employed molecular cytogenetic techniques to clarify the genome constitution of the Th. intermedium parental lines and validated 634 chromosome-specific SNPs. Our data confirmed the allohexaploid nature of Th. intermedium and demonstrated that the St genome-specific GISH signal and markers are present at the centromeric regions of chromosomes 1Jvs, 2Jvs, 3Jvs and 7Jvs. The SNP markers presented here will be introduced into current wheat improvement programmes, offering a significant speed-up in wheat breeding and making it possible to deal with the transfer of the full genetic potential of Th. intermedium into wheat.

Topoisomerases facilitate transcription of long genes linked to autism.

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

B chromosomes are associated with redistribution of genetic recombination towards lower recombination chromosomal regions in perennial ryegrass.

Supernumerary 'B' chromosomes are non-essential components of the genome present in a range of plant and animal species-including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT (Diversity Arrays Technology) marker genetic maps for six perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progeny and their parents, we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared with zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, namely pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders.

Characterisation of Thinopyrum bessarabicum chromosomes through genome-wide introgressions into wheat.

KEY MESSAGE: Genome-wide introgressions of Thinopyrum bessarabicum into wheat resulted in 12 recombinant lines. Cytological and molecular techniques allowed mapping of 1150 SNP markers across all seven chromosomes of the J genome. Thinopyrum bessarabicum (2n = 2x = 14, JJ) is an important source for new genetic variation for wheat improvement due to its salinity tolerance and disease resistance. Its practical utilisation in wheat improvement can be facilitated through development of genome-wide introgressions leading to a variety of different wheat-Th . bessarabicum translocation lines. In this study, we report the generation of 12 such wheat-Th . bessarabicum recombinant lines, through two different crossing strategies, which were characterized using sequential single colour and multi-colour genomic in situ hybridization (sc-GISH and mc-GISH), multi-colour fluorescent in situ hybridization (mc-FISH) and single nucleotide polymorphic (SNP) DNA markers. We also detected 13 lines containing different Th. bessarabicum chromosome aberrations through sc-GISH. Through a combination of molecular and cytological analysis of all the 25 lines containing Th. bessarabicum recombinants and chromosome aberrations we were able to physically map 1150 SNP markers onto seven Th. bessarabicum J chromosomes which were divided into 36 segmental blocks. Comparative analysis of the physical map of Th. bessarabicum and the wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed that Th. bessarabicum contains the 4/5 translocation also present in the A genome of wheat. These wheat-Th . bessarabicum recombinant lines and SNP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Thinopyrum species containing the J or a closely-related genome such as Thinopyrum intermedium (JrJrJvsJvsStSt) and Thinopyrum elongatum (EeEe), respectively.

Introgression of Aegilops speltoides segments in Triticum aestivum and the effect of the gametocidal genes.

Background and Aims: Bread wheat (Triticum aestivum) has been through a severe genetic bottleneck as a result of its evolution and domestication. It is therefore essential that new sources of genetic variation are generated and utilized. This study aimed to generate genome-wide introgressed segments from Aegilops speltoides. Introgressions generated from this research will be made available for phenotypic analysis. Methods: Aegilops speltoides was crossed as the male parent to T. aestivum 'Paragon'. The interspecific hybrids were then backcrossed to Paragon. Introgressions were detected and characterized using the Affymetrix Axiom Array and genomic in situ hybridization (GISH). Key Results: Recombination in the gametes of the F1 hybrids was at a level where it was possible to generate a genetic linkage map of Ae. speltoides. This was used to identify 294 wheat/Ae. speltoides introgressions. Introgressions from all seven linkage groups of Ae. speltoides were found, including both large and small segments. Comparative analysis showed that overall macro-synteny is conserved between Ae. speltoides and T. aestivum, but that Ae. speltoides does not contain the 4A/5A/7B translocations present in wheat. Aegilops speltoides has been reported to carry gametocidal genes, i.e. genes that ensure their transmission through the gametes to the next generation. Transmission rates of the seven Ae. speltoides linkage groups introgressed into wheat varied. A 100 % transmission rate of linkage group 2 demonstrates the presence of the gametocidal genes on this chromosome. Conclusions: A high level of recombination occurs between the chromosomes of wheat and Ae. speltoides, leading to the generation of large numbers of introgressions with the potential for exploitation in breeding programmes. Due to the gametocidal genes, all germplasm developed will always contain a segment from Ae. speltoides linkage group 2S, in addition to an introgression from any other linkage group.

A step change in the transfer of interspecific variation into wheat from Amblyopyrum muticum.

Despite some notable successes, only a fraction of the genetic variation available in wild relatives has been utilized to produce superior wheat varieties. This is as a direct result of the lack of availability of suitable high-throughput technologies to detect wheat/wild relative introgressions when they occur. Here, we report on the use of a new SNP array to detect wheat/wild relative introgressions in backcross progenies derived from interspecific hexaploid wheat/Ambylopyrum muticum F1 hybrids. The array enabled the detection and characterization of 218 genomewide wheat/Am. muticum introgressions, that is a significant step change in the generation and detection of introgressions compared to previous work in the field. Furthermore, the frequency of introgressions detected was sufficiently high to enable the construction of seven linkage groups of the Am. muticum genome, thus enabling the syntenic relationship between the wild relative and hexaploid wheat to be determined. The importance of the genetic variation from Am. muticum introduced into wheat for the development of superior varieties is discussed.