DNA methylation profiling to assess pathogenicity of BRCA1 unclassified variants in breast cancer

Germline pathogenic mutations in BRCA1 increase risk of developing breast cancer. Screening for mutations in BRCA1 frequently identifies sequence variants of unknown pathogenicity and recent work has aimed to develop methods for determining pathogenicity. We previously observed that tumor DNA methylation can differentiate BRCA1-mutated from BRCA1-wild type tumors. We hypothesized that we could predict pathogenicity of variants based on DNA methylation profiles of tumors that had arisen in carriers of unclassified variants. We selected 150 FFPE breast tumor DNA samples [47 BRCA1 pathogenic mutation carriers, 65 BRCAx (BRCA1-wild type), 38 BRCA1 test variants] and analyzed a subset (n=54) using the Illumina 450K methylation platform, using the remaining samples for bisulphite pyrosequencing validation. Three validated markers (BACH2, C8orf31, and LOC654342) were combined with sequence bioinformatics in a model to predict pathogenicity of 27 variants (independent test set). Predictions were compared with standard multifactorial likelihood analysis. Prediction was consistent for c.5194-12G>A (IVS 19-12 G>A) (P>0.99); 13 variants were considered not pathogenic or likely not pathogenic using both approaches. We conclude that tumor DNA methylation data alone has potential to be used in prediction of BRCA1 variant pathogenicity but is not independent of estrogen receptor status and grade, which are used in current multifactorial models to predict pathogenicity

Understanding the current and future usage of donor human milk in hospitals: An online survey of UK neonatal units

The use of donor human milk (DHM) where there is a shortfall of maternal milk can benefit both infant and maternal outcomes but DHM supply is not always assured. This study aimed to understand current DHM usage in UK neonatal units and potential future demand to inform service planning. An online survey was disseminated to all UK neonatal units using Smart Survey or by telephone between February and April 2022 after development alongside neonatal unit teams. Surveys were completed by 55.4% of units (108/195) from all 13 Operational Delivery Networks. Only four units reported not using DHM, and another two units only if infants are transferred on DHM feeds. There was marked diversity in DHM implementation and usage and unit protocols varied greatly. Five of six units with their own milk bank had needed to source milk from an external milk bank in the last year. Ninety units (84.9%) considered DHM was sometimes (n = 35) or always (n = 55) supportive of maternal breastfeeding, and three units (2.9%) responded that DHM was rarely supportive of breastfeeding. Usage was predicted to increase by 37 units (34.9%), and this drive was principally a result of parental preference, clinical trials and improved evidence. These findings support the assumption that UK hospital DHM demand will increase after updated recommendations from the World Health Organization (WHO) and the British Association of Perinatal Medicine. These data will assist service delivery planning, underpinned by an ongoing programme of implementation science and training development, to ensure future equity of access to DHM nationally

Human milk from tandem feeding dyads does not differ in metabolite and metataxonomic features when compared to single nursling dyads under six months of age

Given the long-term advantages of exclusive breastfeeding to infants and their mothers, there is both an individual and public health benefit to its promotion and support. Data on the composition of human milk over the course of a full period of lactation for a single nursling is sparse, but data on human milk composition during tandem feeding (feeding children of different ages from different pregnancies) is almost entirely absent. This leaves an important knowledge gap that potentially endangers the ability of parents to make a fully informed choice on infant feeding. We compared the metataxonomic and metabolite fingerprints of human milk samples from 15 tandem feeding dyads to that collected from ten exclusively breastfeeding single nursling dyads where the nursling is under six months of age. Uniquely, our cohort also included three tandem feeding nursling dyads where each child showed a preferential side for feeding—allowing a direct comparison between human milk compositions for different aged nurslings. Across our analysis of volume, total fat, estimation of total microbial load, metabolite fingerprinting, and metataxonomics, we showed no statistically significant differences between tandem feeding and single nursling dyads. This included comparisons of preferential side nurslings of different ages. Together, our findings support the practice of tandem feeding of nurslings, even when feeding an infant under six months

Differences in smoking associated DNA methylation patterns in South Asians and Europeans

This is a freely-available open access publication. Please cite the published version which is available via the DOI link in this record.Background DNA methylation is strongly associated with smoking status at multiple sites across the genome. Studies have largely been restricted to European origin individuals yet the greatest increase in smoking is occurring in low income countries, such as the Indian subcontinent. We determined whether there are differences between South Asians and Europeans in smoking related loci, and if a smoking score, combining all smoking related DNA methylation scores, could differentiate smokers from non-smokers. Results Illumina HM450k BeadChip arrays were performed on 192 samples from the Southall And Brent REvisited (SABRE) cohort. Differential methylation in smokers was identified in 29 individual CpG sites at 18 unique loci. Interaction between smoking status and ethnic group was identified at the AHRR locus. Ethnic differences in DNA methylation were identified in non-smokers at two further loci, 6p21.33 and GNG12. With the exception of GFI1 and MYO1G these differences were largely unaffected by adjustment for cell composition. A smoking score based on methylation profile was constructed. Current smokers were identified with 100% sensitivity and 97% specificity in Europeans and with 80% sensitivity and 95% specificity in South Asians. Conclusions Differences in ethnic groups were identified in both single CpG sites and combined smoking score. The smoking score is a valuable tool for identification of true current smoking behaviour. Explanations for ethnic differences in DNA methylation in association with smoking may provide valuable clues to disease pathways.Wellcome Trust Enhancement grantMedical Research CouncilDiabetes UKthe British Heart Foundatio

Establishing a generalized polyepigenetic biomarker for tobacco smoking

Large-scale epigenome-wide association meta-analyses have identified multiple 'signatures'' of smoking. Drawing on these findings, we describe the construction of a polyepigenetic DNA methylation score that indexes smoking behavior and that can be utilized for multiple purposes in population health research. To validate the score, we use data from two birth cohort studies: The Dunedin Longitudinal Study, followed to age-38 years, and the Environmental Risk Study, followed to age-18 years. Longitudinal data show that changes in DNA methylation accumulate with increased exposure to tobacco smoking and attenuate with quitting. Data from twins discordant for smoking behavior show that smoking influences DNA methylation independently of genetic and environmental risk factors. Physiological data show that changes in DNA methylation track smoking-related changes in lung function and gum health over time. Moreover, DNA methylation changes predict corresponding changes in gene expression in pathways related to inflammation, immune response, and cellular trafficking. Finally, we present prospective data about the link between adverse childhood experiences (ACEs) and epigenetic modifications; these findings document the importance of controlling for smoking-related DNA methylation changes when studying biological embedding of stress in life-course research. We introduce the polyepigenetic DNA methylation score as a tool both for discovery and theory-guided research in epigenetic epidemiology.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.The Dunedin Longitudinal Study is funded by the New Zealand Health Research Council, the New Zealand Ministry of Business, Innovation, and Employment, the National Institute on Aging (AG032282), and the Medical Research Council (MR/P005918/1). The E-Risk Study is funded by the Medical Research Council (G1002190) and the National Institute of Child Health and Human Development (HD077482). Additional support was provided by a Distinguished Investigator Award from the American Asthma Foundation to Dr. Mill, and by the Jacobs Foundation and the Avielle Foundation. Dr. Arseneault is the Mental Health Leadership Fellow for the U.K. Economic and Social Research Council. Dr. Belsky is a Jacobs Foundation Fellow. This work used a high-performance computing facility partially supported by grant 2016-IDG-1013 (“HARDAC + : Reproducible HPC for Next-generation Genomics”) from the North Carolina Biotechnology Center. Illumina DNA methylation data are accessible from the Gene Expression Omnibus (accession code: GSE105018).pre-print, post-print, publisher's PD

DNA methylation levels are highly correlated between pooled samples and averaged values when analysed using the Infinium HumanMethylation450 BeadChip array.

BACKGROUND: DNA methylation is a heritable and stable epigenetic mark implicated in complex human traits. Epigenome-wide association studies (EWAS) using array-based technology are becoming widely used to identify differentially methylated sites associated with complex diseases. EWAS studies require large sample sizes to detect small effects, which increases project costs. In the present study we propose to pool DNA samples in methylation array studies as an affordable and accurate alternative to individual samples studies, in order to reduce economic costs or when low amounts of DNA are available. For this study, 20 individual DNA samples and 4 pooled DNA samples were analysed using the Illumina Infinium HumanMethylation450 BeadChip array to evaluate the efficiency of the pooling approach in EWAS studies. Statistical power calculations were also performed to discover the minimum sample size needed for the pooling strategy in EWAS. RESULTS: A total of 485,577 CpG sites across the whole genome were assessed. Comparison of methylation levels of all CpG sites between individual samples and their related pooled samples revealed highly significant correlations (rho > 0.99, p-val  0.98, p-val < 10(-16)). Also, it was calculated that n = 43 is the minimum sample size required to achieve a 95 % statistical power and a 10(-06) significance level in EWAS, when using a DNA pool strategy. CONCLUSIONS: DNA pooling strategies seems to accurately provide estimations of averaged DNA methylation state using array based EWAS studies. This type of approach can be applied to the assessment of disease phenotypes, reducing the amount of DNA required and the cost of large-scale epigenetic analyses