Abstract We performed a new melanoma GWAS meta-analysis, roughly doubling the sample size to ∼70,000 cases compared to the most recent study by Landi and colleagues. In the new meta-analysis, we identify 116 independent risk loci, replicating 52/54 loci previously reported. Post-GWAS variant-to-gene mapping remains critical to functionally interpret new loci discovered by this analysis. Most loci contain many candidate causal variants in linkage disequilibrium, with very few altering protein-coding sequence, suggesting cis-regulatory function underlying the majority of loci. Quantitative trait locus (QTL) colocalization methods can effectively prioritize target genes at susceptibility loci but many loci remain without assigned targets, perhaps reflecting context-specific variant effects not well-reflected in QTL datasets. To complement QTLs and more comprehensively identify potential causal genes, including those beyond 1 Mb, we applied H3K27ac-HiChIP in human primary melanocytes, the cell type of origin for melanoma. H3K27ac-HiChIP combines a Hi-C approach with a H3K27ac ChIP step to detect enhancer-promoter interactions at the risk loci. Statistically significant chromatin interactions were identified using the FitHiChIP pipeline. We subsequently performed variant-to-gene (V2G) mapping at all genome-wide significant melanoma loci, nominating target genes where fine-mapped variants overlapped or physically interacted with their respective promoters. HiChIP-based V2G mapping approach identified target genes at 94% loci (779 genes nominated at 109/116 loci), outperforming other gene nomination approaches (QTL colocalization, protein-coding variants), which nominated 102 candidate genes at 57% of risk loci (67/116 loci). Among genes identified by melanocyte or melanoma eQTL colocalization, a majority (19 of 32) were also nominated by V2G mapping. Likewise, 3 of 5 splice QTL genes and 21 of 34 meQTL genes overlapped with the V2G gene set. Next, we compared gene sets including the V2G mapping-identified candidates to those identified solely by QTLs and protein-coding variants. The gene set incorporating V2G candidates showed significant enrichment for oncogenic signaling pathways, including WNT/β-catenin signaling (FDR: Pwith V2G gene set= 6.3 × 10-05 vs Pwithout V2G gene set= 0.2), aryl hydrocarbon receptor signaling (P= 1.6 × 10-4 vs 0.2), and signaling by NOTCH1 (P=0.002 vs0.5). Notably, V2G mapping linked risk-associated variants to known cancer drivers (e.g. PIK3CA, NOTCH2, MDM4etc.) at 46% of loci (53/116; nominated 76 cancer drivers), with several located >1Mb away. Strikingly, we detected a highly significant long-range interaction (∼2 Mb) connecting fine-mapped variants near the locus 8q24.21 to cancer driver MYC. Overall, H3K27ac-HiChIP-based V2G mapping greatly improves the interpretation of melanoma susceptibility loci by identifying distant susceptibility genes, highlighting known cancer drivers as potential targets, and revealing that these loci converge on key oncogenic signaling pathways. Citation Format: Rohit Thakur, G J M Shanika R Jayasinghe, Mai Xu, Linh Bui-Raborn, Jianxin Shi, Diptavo Dutta, Phuc H. Hoang, Mathias Seviiri, Christopher I. Amos, Andrew Bakshi, Anne E. Cust, Florence Demenais, David L. Duffy, Lars G. Fritsche, Jiali Han, Nicholas K. Hayward, Kiarash Khosrotehrani, Rajiv Kumar, John F. Thompson, Stuart MacGregor, Miguel Renteria, Diane T. Smelser, Sarah V. Ward, Maria Concetta Fargnoli, Paola Ghiorzo, Alisa M. Goldstein, Chiara Menin, David Millan-Esteban, Eduardo Nagore, Cristina Pellegrini, Susana Puig, Alex Stratigos, David C. Whiteman, Melanoma Meta-Analysis Consortium, Lee E. Whelees, Rebecca I. Hartman, Maria Teresa Landi, Matthew H. Law, Kevin M. Brown. H3K27ac-HiChIP variant-to-gene mapping confirms the importance of cancer drivers and oncogenic signaling pathways in melanoma risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB384.

Abstract A greater understanding of the biology of nevi will provide insights into the etiology of melanoma. Our large-scale meta-analysis of 14 nevus genome-wide association studies (GWAS) includes 85,965 individuals of European ancestry. We identify 29 nevus-associated loci (p < 5 × 10-8), of which 24 have not been previously reported in a GWAS conducted for nevus count alone. We further identify 255 candidate genes for nevus loci, including SIKE1 which is involved in immune response regulation. This is of interest because immune response regulation influences the formation of nevi and melanoma susceptibility. Gene-set enrichment analyses prioritise immune response-related pathways and cancers that do not have a pigmentation component (e.g. breast, prostate, and glioma). This suggests that the biology underlying nevus count captures risk pathways beyond pigmentation that are relevant to melanoma. In sex-specific analyses, we observe higher total-body nevus count in females than in males, however the genetic architecture is largely shared (genetic correlation = 0.863, 95% CI = 0.453 – 1.273), indicating the difference may be influenced by environmental and behavioural factors rather than genetics. A nevus polygenic risk score explains 5% of the variance in nevus count, indicating its potential to enhance melanoma risk prediction.

Germline genetic variation can influence the number of nevi on the skin. However, nevi can be classified in many ways (e.g., by shape, size or evolution); little is known of how genetics influences the density of different nevus morphologies on the body. Here, we explore how germline genetics influences the density of flat, raised and atypical nevi, and identify which classification correlates most with genetic risk of melanoma. We estimate heritability and perform genome-wide association analysis of flat, raised, and atypical nevus count within the Brisbane Twin Nevus Study (N = 3862). We compare the effect estimates of genetic loci associated with each morphology. We also assess how well each morphology correlates with the genetic risk of melanoma using a polygenic risk score of melanoma. Heritability estimates revealed both unique and shared genetic effects between morphologies. We identified two loci near IRF4 and MC1R showing opposing effects on flat and raised nevus counts. Variation in raised nevus count showed the strongest correlation with melanoma risk using polygenic risk scores. Although some genetic effects are shared between nevus morphologies, they appear to be genetically distinct phenotypes. Given the opposing effects of IRF4 and MC1R on flat and raised nevus counts, future studies of nevus count should consider, where possible, analysing each nevus morphology separately as well as combined.

BACKGROUND:The lifetime risk of cutaneous melanoma in Australia is the highest in the world. The most important melanoma risk factor is the number of acquired cutaneous melanocytic naevi (AMN) on a person, the majority of these forming in adolescence. Childhood exposure to ultraviolet radiation (UVR) is a strong determinant of naevus count. OBJECTIVES:To examine childhood AMN and its risk factors over a 25-year period in the high-UVR environment of South-East Queensland. METHODS:The Brisbane Twin Nevus Study recorded sun behaviours and counted naevi on annual new samples of 12-year-old twins (and siblings nearest in age) from 1992 to 2016. Participants were re-examined 2 years later, and a subset was seen 20 years after the initial exam. RESULTS:Among 3957 participants (158 per year), we saw an approximate halving of naevus counts over the 25-year period, and examined multiple explanations for this trend. As this trend was seen for both large (> 5 mm) and smaller naevi, we inferred errors in counting were unlikely. There was an increase in the number of participants reporting non-European ancestry, but this explained only a small proportion of the change in naevus count. CONCLUSIONS:We propose that, in Queensland through the 1990s and 2000s, children's sun exposure has been altered by changes in behaviour. Looking at studies counting naevi in populations at different latitudes, we estimate the observed fall in naevus counts would be consistent with a 11.7% fall in average annual UVR dose (clear sky erythemal spectrum, from 1503 kJ m-2 to 1327 kJ m-2). Based on published risk prediction equations, the fall in mean naevus number over time should lead to a fourfold drop in lifetime melanoma risk for those born after 2000 compared with those born in the 1980s.

ImportanceIt is unknown whether germline genetic factors influence in situ melanoma risk differently than invasive melanoma risk.ObjectiveTo determine whether differences in risk of in situ melanoma and invasive melanoma are heritable.Design, Setting, and ParticipantsThree genome-wide association study meta-analyses were conducted of in situ melanoma vs controls, invasive melanoma vs controls, and in situ vs invasive melanoma (case-case) using 4 population-based genetic cohorts: the UK Biobank, the FinnGen cohort, the QSkin Sun and Health Study, and the Queensland Study of Melanoma: Environmental and Genetic Associations (Q-MEGA). Melanoma status was determined using International Statistical Classification of Diseases and Related Health Problems codes from cancer registry data. Data were collected from 1987 to 2022, and data were analyzed from September 2022 to June 2023.ExposureIn situ and invasive cutaneous melanoma.Main Outcomes and MeasuresTo test whether in situ and invasive melanoma have independent heritable components, genetic effect estimates were calculated for single-nucleotide variants (SNV; formerly single-nucleotide polymorphisms) throughout the genome for each melanoma. Then, SNV-based heritability was estimated, the genetic correlation between melanoma subtypes was assessed, and polygenic risk scores (PRS) were generated for in situ vs invasive status in Q-MEGA participants.ResultsA total of 6 genome-wide significant loci associated with in situ melanoma and 18 loci with invasive melanoma were identified. A strong genetic correlation (genetic r = 0.96; 95% CI, 0.76-1.15) was observed between the 2 classifications. Notably, loci near IRF4, KLF4, and HULC had significantly larger effects for in situ melanoma compared with invasive melanoma, while MC1R had a significantly larger effect on invasive melanoma compared with in situ melanoma. Heritability estimates were consistent for both, with in situ melanoma heritability of 6.7% (95% CI, 4.1-9.3) and invasive melanoma heritability of 4.9% (95% CI, 2.8-7.2). Finally, a PRS, derived from comparing invasive melanoma with in situ melanoma genetic risk, was on average significantly higher in participants with invasive melanoma (odds ratio per 1-SD increase in PRS, 1.43; 95% CI, 1.16-1.77).Conclusions and RelevanceThere is much shared genetic architecture between in situ melanoma and invasive melanoma. Despite indistinguishable heritability estimates between the melanoma classifications, PRS suggest germline genetics may influence whether a person gets in situ melanoma or invasive melanoma. PRS could potentially help stratify populations based on invasive melanoma risk, informing future screening programs without exacerbating the current burden of melanoma overdiagnosis.

STUDY QUESTION:Which genetic factors regulate female propensity for giving birth to spontaneous dizygotic (DZ) twins? SUMMARY ANSWER:We identified four new loci, GNRH1, FSHR, ZFPM1, and IPO8, in addition to previously identified loci, FSHB and SMAD3. WHAT IS KNOWN ALREADY:The propensity to give birth to DZ twins runs in families. Earlier, we reported that FSHB and SMAD3 as associated with DZ twinning and female fertility measures. STUDY DESIGN, SIZE, DURATION:We conducted a genome-wide association meta-analysis (GWAMA) of mothers of spontaneous dizygotic (DZ) twins (8265 cases, 264 567 controls) and of independent DZ twin offspring (26 252 cases, 417 433 controls). PARTICIPANTS/MATERIALS, SETTING, METHODS:Over 700 000 mothers of DZ twins, twin individuals and singletons from large cohorts in Australia/New Zealand, Europe, and the USA were carefully screened to exclude twins born after use of ARTs. Genetic association analyses by cohort were followed by meta-analysis, phenome wide association studies (PheWAS), in silico and in vivo annotations, and Zebrafish functional validation. MAIN RESULTS AND THE ROLE OF CHANCE:This study enlarges the sample size considerably from previous efforts, finding four genome-wide significant loci, including two novel signals and a further two novel genes that are implicated by gene level enrichment analyses. The novel loci, GNRH1 and FSHR, have well-established roles in female reproduction whereas ZFPM1 and IPO8 have not previously been implicated in female fertility. We found significant genetic correlations with multiple aspects of female reproduction and body size as well as evidence for significant selection against DZ twinning during human evolution. The 26 top single nucleotide polymorphisms (SNPs) from our GWAMA in European-origin participants weakly predicted the crude twinning rates in 47 non-European populations (r = 0.23 between risk score and population prevalence, s.e. 0.11, 1-tail P = 0.058) indicating that genome-wide association studies (GWAS) are needed in African and Asian populations to explore the causes of their respectively high and low DZ twinning rates. In vivo functional tests in zebrafish for IPO8 validated its essential role in female, but not male, fertility. In most regions, risk SNPs linked to known expression quantitative trait loci (eQTLs). Top SNPs were associated with in vivo reproductive hormone levels with the top pathways including hormone ligand binding receptors and the ovulation cycle. LARGE SCALE DATA:The full DZT GWAS summary statistics will made available after publication through the GWAS catalog (https://www.ebi.ac.uk/gwas/). LIMITATIONS, REASONS FOR CAUTION:Our study only included European ancestry cohorts. Inclusion of data from Africa (with the highest twining rate) and Asia (with the lowest rate) would illuminate further the biology of twinning and female fertility. WIDER IMPLICATIONS OF THE FINDINGS:About one in 40 babies born in the world is a twin and there is much speculation on why twinning runs in families. We hope our results will inform investigations of ovarian response in new and existing ARTs and the causes of female infertility. STUDY FUNDING/COMPETING INTEREST(S):Support for the Netherlands Twin Register came from the Netherlands Organization for Scientific Research (NWO) and The Netherlands Organization for Health Research and Development (ZonMW) grants, 904-61-193, 480-04-004, 400-05-717, Addiction-31160008, 911-09-032, Biobanking and Biomolecular Resources Research Infrastructure (BBMRI.NL, 184.021.007), Royal Netherlands Academy of Science Professor Award (PAH/6635) to DIB, European Research Council (ERC-230374), Rutgers University Cell and DNA Repository (NIMH U24 MH068457-06), the Avera Institute, Sioux Falls, South Dakota (USA) and the National Institutes of Health (NIH R01 HD042157-01A1) and the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health and Grand Opportunity grants 1RC2 MH089951. The QIMR Berghofer Medical Research Institute (QIMR) study was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485, 552498, 1050208, 1075175). L.Y. is funded by Australian Research Council (Grant number DE200100425). The Minnesota Center for Twin and Family Research (MCTFR) was supported in part by USPHS Grants from the National Institute on Alcohol Abuse and Alcoholism (AA09367 and AA11886) and the National Institute on Drug Abuse (DA05147, DA13240, and DA024417). The Women's Genome Health Study (WGHS) was funded by the National Heart, Lung, and Blood Institute (HL043851 and HL080467) and the National Cancer Institute (CA047988 and UM1CA182913), with support for genotyping provided by Amgen. Data collection in the Finnish Twin Registry has been supported by the Wellcome Trust Sanger Institute, the Broad Institute, ENGAGE-European Network for Genetic and Genomic Epidemiology, FP7-HEALTH-F4-2007, grant agreement number 201413, National Institute of Alcohol Abuse and Alcoholism (grants AA-12502, AA-00145, AA-09203, AA15416, and K02AA018755) and the Academy of Finland (grants 100499, 205585, 118555, 141054, 264146, 308248, 312073 and 336823 to J. Kaprio). TwinsUK is funded by the Wellcome Trust, Medical Research Council, Versus Arthritis, European Union Horizon 2020, Chronic Disease Research Foundation (CDRF), Zoe Ltd and the National Institute for Health Research (NIHR) Clinical Research Network (CRN) and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. For NESDA, funding was obtained from the Netherlands Organization for Scientific Research (Geestkracht program grant 10000-1002), the Center for Medical Systems Biology (CSMB, NVVO Genomics), Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NL), VU University's Institutes for Health and Care Research (EMGO+) and Neuroscience Campus Amsterdam, University Medical Center Groningen, Leiden University Medical Center, National Institutes of Health (NIH, ROI D0042157-01A, MH081802, Grand Opportunity grants 1 RC2 Ml-1089951 and IRC2 MH089995). Part of the genotyping and analyses were funded by the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health. Computing was supported by BiG Grid, the Dutch e-Science Grid, which is financially supported by NWO. Work in the Del Bene lab was supported by the Programme Investissements d'Avenir IHU FOReSIGHT (ANR-18-IAHU-01). C.R. was supported by an EU Horizon 2020 Marie Skłodowska-Curie Action fellowship (H2020-MSCA-IF-2014 #661527). H.S. and K.S. are employees of deCODE Genetics/Amgen. The other authors declare no competing financial interests. TRIAL REGISTRATION NUMBER:N/A.

BACKGROUND:Nodular melanoma (NM) is a challenge to diagnose early due to its rapid growth and more atypical clinical presentation, making it the largest contributor to melanoma mortality. OBJECTIVES:Our study aim was to perform a rare-variant allele (RVA) analysis of whole-exome sequencing of patients with NM and non-NM (minor allele frequency ≤ 1% non-Finnish European) for a set of 500 candidate genes potentially implicated in melanoma. METHODS:This study recruited 131 participants with NM and 194 with non-NM from South-east Queensland and patients with NM from Victoria to perform a comparative analysis of possible genetic differences or similarities between the two melanoma cohorts. RESULTS:Phenotypic analysis revealed that a majority of patients diagnosed with NM were older males with a higher frequency of fair skin and red hair than is seen in the general population. The distribution of common melanoma polygenic risk scores was similar in patients with NM and non-NM, with over 28% in the highest quantile of scores. There was also a similar frequency of carriage of familial/high-penetrant melanoma gene and loss-of-function variants. We identified 39 genes by filtering 500 candidate genes based on the greatest frequency in NM compared with non-NM cases. The genes with RVAs of greatest frequency in NM included PTCH1, ARID2 and GHR. Rare variants in the SMO gene, which interacts with PTCH1 as ligand and receptor, were also identified, providing evidence that the Hedgehog pathway may contribute to NM risk. There was a cumulative effect in carrying multiple rare variants in the NM-associated genes. A 14.8-fold increased ratio for NM compared with non-NM was seen when two RVAs of the 39 genes were carried by a patient. CONCLUSIONS:This study highlights the importance of considering frequency of RVA to identify those at risk of NM in addition to known high penetrance genes.

IntroductionMarine environments offer a wealth of opportunities to improve understanding and treatment options for cancers, through insights into a range of fields from drug discovery to mechanistic insights. By applying One Health principles the knowledge obtained can benefit both human and animal populations, including marine species suffering from cancer. One such species is green sea turtles (Chelonia mydas), which are under threat from fibropapillomatosis (FP), an epizootic tumor disease (animal epidemic) that continues to spread and increase in prevalence globally. In order to effectively address this epizootic, a more thorough understanding is required of the prevalence of the disease and the approaches to treating afflicted turtles.MethodsTo identify knowledge gaps and assess future needs, we conducted a survey of sea turtle FP experts. The survey consisted of 47 questions designed to assess general perceptions of FP, the areas where more information is needed, local FP trends, the disease status, and mitigation needs, and was voluntarily completed by 44 experts across a broad geographic range.ResultsOver 70% of respondents both recognized FP as a cancerous panzootic disease, and reported that FP is increasing in prevalence. They report several factors contributing to this increase. Nearly all of the respondents reported that FP research, patient treatment and rehabilitation required more funding in their area, and reported inadequate facilities and capacity for dealing with FP patients. Treatment approaches varied: just over 70% of the medical experts that responded surgically remove FP tumors, either using laser or scalpel. Just under half of respondents use anti-cancer drugs in their treatment of FP. Internal tumors were reported as justification for euthanasia by 61.5% of respondents, and 30.8% reported severe external tumors to be sufficient grounds for euthanasia. Most medical respondents (93.3%) routinely perform necropsy on deceased or euthanized FP-afflicted turtles. Over 80% of respondents considered large-scale multidisciplinary collaboration ‘extremely important’ for advancing the field of FP research.DiscussionThe survey responses provide a valuable insight into the current status of FP in sea turtles, FP treatment, rehabilitation and research, and help to identify critical FP-related areas most in need of attention.

Towards the 10th anniversary of the Nagoya Protocol, it is time to embrace key technology developments and adapt existing red tape for genomic monitoring.

Importance:It is unknown whether germline genetic factors influence in situ melanoma risk differently than invasive melanoma risk. Objective:To determine whether differences in risk of in situ melanoma and invasive melanoma are heritable. Design, Setting, and Participants:Three genome-wide association study meta-analyses were conducted of in situ melanoma vs controls, invasive melanoma vs controls, and in situ vs invasive melanoma (case-case) using 4 population-based genetic cohorts: the UK Biobank, the FinnGen cohort, the QSkin Sun and Health Study, and the Queensland Study of Melanoma: Environmental and Genetic Associations (Q-MEGA). Melanoma status was determined using International Statistical Classification of Diseases and Related Health Problems codes from cancer registry data. Data were collected from 1987 to 2022, and data were analyzed from September 2022 to June 2023. Exposure:In situ and invasive cutaneous melanoma. Main Outcomes and Measures:To test whether in situ and invasive melanoma have independent heritable components, genetic effect estimates were calculated for single-nucleotide variants (SNV; formerly single-nucleotide polymorphisms) throughout the genome for each melanoma. Then, SNV-based heritability was estimated, the genetic correlation between melanoma subtypes was assessed, and polygenic risk scores (PRS) were generated for in situ vs invasive status in Q-MEGA participants. Results:A total of 6 genome-wide significant loci associated with in situ melanoma and 18 loci with invasive melanoma were identified. A strong genetic correlation (genetic r = 0.96; 95% CI, 0.76-1.15) was observed between the 2 classifications. Notably, loci near IRF4, KLF4, and HULC had significantly larger effects for in situ melanoma compared with invasive melanoma, while MC1R had a significantly larger effect on invasive melanoma compared with in situ melanoma. Heritability estimates were consistent for both, with in situ melanoma heritability of 6.7% (95% CI, 4.1-9.3) and invasive melanoma heritability of 4.9% (95% CI, 2.8-7.2). Finally, a PRS, derived from comparing invasive melanoma with in situ melanoma genetic risk, was on average significantly higher in participants with invasive melanoma (odds ratio per 1-SD increase in PRS, 1.43; 95% CI, 1.16-1.77). Conclusions and Relevance:There is much shared genetic architecture between in situ melanoma and invasive melanoma. Despite indistinguishable heritability estimates between the melanoma classifications, PRS suggest germline genetics may influence whether a person gets in situ melanoma or invasive melanoma. PRS could potentially help stratify populations based on invasive melanoma risk, informing future screening programs without exacerbating the current burden of melanoma overdiagnosis.

As global temperatures rise, species populations and biodiversity decline, and infectious diseases emerge all at unprecedented rates, it is more vital than ever to accurately understand the current state of natural habitats. While traditional methods including direct sampling and observation have their merits, newer technologies offer additional sampling capabilities. Here, we assess the feasibility of a single assay: shotgun long-read sequencing, to monitor species environmental DNA (eDNA) from across the tree of life, from viruses to complex multicellular organisms, across a river system (mountain tributary to sea). We conducted eDNA sampling and shotgun long-read sampling from water taken from the Avoca River watercourse, Co. Wicklow, Ireland, from a mountain tributary through to the sea, and comparative nearby beach sand sampling. We report that shotgun long-read sequencing and metagenomic analysis have utility for the detection and quantification of organismal DNA present in eDNA samples, from across the tree of life, from microbes (including DNA viruses) to mammals. With this single assay we were able to simultaneously quantify differences in eDNA abundance for a broad range of biodiversity and pathogens across sites and sample types. This included human, wildlife, plant and microbial pathogens and parasites with health, agricultural and economic importance. Additionally, the generated eDNA genomic data enabled population genetic applications even from natural complex community settings, as demonstrated here for blue mussels (Mytilus edulis). The results demonstrate that Oxford Nanopore sequencing provides a quantitative approach for river biodiversity, pollution and environmental health monitoring. This method is more cost-effective and requires less laboratory preparation time or molecular expertise (barcode/primer design) than alternative biodiversity eDNA approaches (e.g. metabarcoding or qPCR). Shotgun analysis approaches will also benefit from the continual expansion of reference genome databases, for environmental, evolutionary and medical reasons, among others. Computation, cloud and artificial intelligence (AI) tools (such as the cloud-based analyses utilized here) can analyze shotgun sequencing eDNA data in a matter of hours and can be used by conservation practitioners, environmentalists and public health personnel without the need for coding or in-depth bioinformatics skills. The proven citizen/community scientist applicability and ease of eDNA sampling may further revolutionize and democratize biodiversity research, conservation surveillance, and environmental health monitoring. Long-read shotgun sequencing of eDNA offers the means to assess whole ecosystems, and the ecological, trophic, and host-pathogen interactions occurring within them. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Approximately 2-20% of cutaneous melanomas (CMs) are diagnosed as amelanotic/hypopigmented melanoma (AHM) and represent a challenge for early diagnosis. OBJECTIVES:To investigate loss-of-function mutations in key pigmentation genes in matched germline and AHM, as well as pigmented melanoma (PM), tumour DNA samples. METHODS:Analysis of clinical and histopathological characteristics - together with whole-exome sequencing data of 34 fresh frozen primary CMs, graded according to the amount of pigmentation present - was performed. Together with germline and somatic variant analysis, 30 samples had previously been analysed for copy number aberration (CNA) changes. This study focused on germline and somatic variants in the coding region of 16 genes known to be associated with albinism/hypopigmentation or variation in human pigmentation in all samples. Chromosomal regions encompassing these 16 genes were examined for DNA copy loss or gain. RESULTS:The finding that red hair-related MC1R and TYR R402Q loss-of-activity gene variant alleles and genotypes are associated with AHM was confirmed. Germline AHM-related gene variants were enriched in 70% (n = 7/10) of patients with AHM vs. 8% (n = 2/24) of those with PM. This surprisingly high frequency of rare germline variants in people with AHM constitutes the 'first hit' and confirms that those with AHM are more likely to be albinism allele carriers than individuals with PM. Next, in CNA analysis of each tumour sample, 50% (n = 4/8) of AHM samples with a pigmentation gene variant had loss of heterozygosity (LOH) in the region containing the corresponding gene and 25% (n = 2/8) had LOH in chromosomal regions of two AHM-related genes. CONCLUSIONS:This study proposes that the likely molecular mechanism for the development of amelanogenesis in AHM is carriage of an albinism/hypopigmentation allele followed by LOH of the corresponding gene in the tumour.

PDF - 219K, Table S1. CAS (Chemical Abstracts Service) numbers, molecular formulas, structures and references for each of the compounds used in the study.

PDF - 98K, Table S2. Flow Cytometry full details in accordance with the MIFlowCyt Standard.

Background Population-wide screening for melanoma is not cost-effective, but genetic characterization could facilitate risk stratification and targeted screening. Common Melanocortin-1 receptor (MC1R) red hair colour (RHC) variants and Microphthalmia-associated transcription factor ( MITF) E318K separately confer moderate melanoma susceptibility, but their interactive effects are relatively unexplored. Objectives To evaluate whether MC1R genotypes differentially affect melanoma risk in MITF E318K(+) vs. E318K(-) individuals. Materials and methods Melanoma status (affected or unaffected) and genotype data (MC1R and MITF E318K) were collated from research cohorts (five Australian and two European). In addition, RHC genotypes from E318K(+) individuals with and without melanoma were extracted from databases (The Cancer Genome Atlas and Medical Genome Research Bank, respectively). chi(2) and logistic regression were used to evaluate RHC allele and genotype frequencies within E318K(+/-) cohorts depending on melanoma status. Replication analysis was conducted on 200 000 general-population exomes (UK Biobank). Results The cohort comprised 1165 MITF E318K(-) and 322 E318K(+) individuals. In E318K(-) cases MC1R R and r alleles increased melanoma risk relative to wild type (wt), P < 0.001 for both. Similarly, each MC1R RHC genotype (R/R, R/r, R/wt, r/r and r/wt) increased melanoma risk relative to wt/wt (P < 0.001 for all). In E318K(+) cases, R alleles increased melanoma risk relative to the wt allele [odds ratio (OR) 2.04 (95% confidence interval 1.67-2.49); P = 0.01], while the r allele risk was comparable with the wt allele [OR 0.78 ( 0.54-1.14) vs. 1.00, respectively]. E318K(+) cases with the r/r genotype had a lower but not significant melanoma risk relative to wt/wt [OR 0.52 (0.20-1.38)]. Within the E318K(+) cohort, R genotypes (R/R, R/r and R/wt) conferred a significantly higher risk compared with non-R genotypes (r/r, r/wt and wt/ wt) (P < 0.001). UK Biobank data supported our findings that r did not increase melanoma risk in E318K(+) individuals. Conclusions RHC alleles/genotypes modify melanoma risk differently in MITF E318K(-) and E318K(+) individuals. Specifically, although all RHC alleles increase risk relative to wt in E318K(-) individuals, only MC1R R increases melanoma risk in E318K(+) individuals. Importantly, in the E318K(+) cohort the MC1R r allele risk is comparable with wt. These findings could inform counselling and management for MITF E318K(+) individuals.

Female fertility is a complex trait with age-specific changes in spontaneous dizygotic (DZ) twinning and fertility. To elucidate factors regulating female fertility and infertility, we conducted a genome-wide association study (GWAS) on mothers of spontaneous DZ twins (MoDZT) versus controls (3273 cases, 24,009 controls). This is a follow-up study to the Australia/New Zealand (ANZ) component of that previously reported (Mbarek et al., 2016), with a sample size almost twice that of the entire discovery sample meta-analysed in the previous article (and five times the ANZ contribution to that), resulting from newly available additional genotyping and representing a significant increase in power. We compare analyses with and without male controls and show unequivocally that it is better to include male controls who have been screened for recent family history, than to use only female controls. Results from the SNP based GWAS identified four genomewide significant signals, including one novel region, ZFPM1 (Zinc Finger Protein, FOG Family Member 1), on chromosome 16. Previous signals near FSHB (Follicle Stimulating Hormone beta subunit) and SMAD3 (SMAD Family Member 3) were also replicated (Mbarek et al., 2016). We also ran the GWAS with a dominance model that identified a further locus ADRB2 on chr 5. These results have been contributed to the International Twinning Genetics Consortium for inclusion in the next GWAS meta-analysis (Mbarek et al., in press).

Environmental DNA (eDNA) sampling is a relatively new technique that has been employed in biodiversity surveys around the world. Environmental DNA can be an effective, non-invasive method of identifying the presence of target host species. Furthermore, advances in DNA deep sequencing technologies are enabling in-depth information to be recovered from eDNA. Here we report the development of a bobcat (Lynx rufus) species-specific probe-based qPCR assay for eDNA studies, which was validated on wild samples and those from a bobcat housed at the Jacksonville Zoo in Florida. Furthermore, we show that long-read shotgun sequencing of eDNA extracted from pawprint soil samples, using an Oxford Nanopore Technologies MinION, could successfully detect bobcat DNA, including enabling correct species level identification and phylogenetic placement within regionally distanced bobcats. This was all achieved without utilizing any DNA enrichment approaches. The long read shotgun sequencing simultaneously recovered genetic information from microbes known to be part of the bobcat microbiome, indicating that potential health-status-related microbial information can be obtained alongside wildlife eDNA. This study revealed that non-targeted (no metabarcoding or enrichment) long-read shotgun sequencing of eDNA samples can be sufficient for species identification, phylogenetic analyses, and population genetics applications. This paves the way for the rapid deployment of eDNA approaches for any species of interest, without requiring laborious development of targeted approaches, thereby increasing the ease and utility of eDNA research for endangered species conservation and management.