Abberrant DNA methylation is one of the hallmarks of cancerogenesis. Our study aims to delineate differential DNA methylation in cirrhosis and hepatic cancerogenesis. Patterns of methylation of 27,578 individual CpG loci in 12 hepatocellular carcinomas (HCCs), 15 cirrhotic controls and 12 normal liver samples were investigated using an array-based technology. A supervised principal component analysis (PCA) revealed 167 hypomethylated loci and 100 hypermethylated loci in cirrhosis and HCC as compared to normal controls. Thus, these loci show a ''cirrhotic'' methylation pattern that is maintained in HCC. In pairwise supervised PCAs between normal liver, cirrhosis and HCC, eight loci were significantly changed in all analyses differentiating the three groups (p < 0.0001). Of these, five loci showed highest methylation levels in HCC and lowest in control tissue (LOC55908, CELSR1, CRMP1, GNRH2, ALOX12 and ANGPTL7), whereas two loci showed the opposite direction of change (SPRR3 and TNFSF15). Genes hypermethylated between normal liver to cirrhosis, which maintain this methylation pattern during the development of HCC, are depleted for CpG islands, high CpG content promoters and polycomb repressive complex 2 (PRC2) targets in embryonic stem cells. In contrast, genes selectively hypermethylated in HCC as compared to nonmalignant samples showed an enrichment of CpG islands, high CpG content promoters and PRC2 target genes (p < 0.0001). Cirrhosis and HCC show distinct patterns of differential methylation with regards to promoter structure, PRC2 targets and CpG islands.Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and globally the third most common cause of cancer mortality. 1,2 Approximately one million new cases are diagnosed globally each year. The highest incidences are observed in sub-Saharan Africa and Eastern Asia, where hepatitis B virus and hepatitis C virus infections are endemic. HCC incidence is rising also due to an increase in incidence of alcoholic cirrhosis and nonalcoholic steatohepatitis. 3,4 As for many other tumors, the development of HCC is a multistep process characterized by the accumulation of genetic and epigenetic alterations leading to the activation of oncogenes and inactivation or loss of tumor suppressor genes. Genomic alterations as part of the somatic evolution of the cancer genome are common in human cancer in general and also in

BackgroundThe search to identify disease-susceptible genes requires access to biological material from numerous well-characterized subjects. Archived residual dried blood spot (DBS) samples, also known as Guthrie cards, from national newborn screening programs may provide a DNA source for entire populations. Combined with clinical information from medical registries, DBS samples could provide a rich source for productive research. However, the amounts of DNA which can be extracted from these precious samples are minute and may be prohibitive for numerous genotypings. Previously, we demonstrated that DBS DNA can be whole-genome amplified and used for reliable genetic analysis on different platforms, including genome-wide scanning arrays. However, it remains unclear whether this approach is workable on a large sample scale. We examined the robustness of using DBS samples for whole-genome amplification following genome-wide scanning, using arrays from Illumina and Affymetrix.ResultsThis study is based on 4,641 DBS samples from the Danish Newborn Screening Biobank, extracted for three separate genome-wide association studies. The amount of amplified DNA was significantly (P < 0.05) affected by the year of storage and storage conditions. Nine (0.2%) DBS samples failed whole-genome amplification. A total of 4,586 (98.8%) samples met our criterion of success of a genetic call-rate above 97%. The three studies used different arrays, with mean genotyping call-rates of 99.385% (Illumina Infinium Human610-Quad), 99.722% (Illumina Infinium HD HumanOmni1-Quad), and 99.206% (Affymetrix Axiom Genome-Wide CEU). We observed a concordance rate of 99.997% in the 38 methodological replications, and 99.999% in the 27 technical replications. Handling variables such as time of storage, storage conditions and type of filter paper were shown too significantly (P < 0.05) affect the genotype call-rates in some of the arrays, although the effect was minimal.ConclusionOur study indicates that archived DBS samples from the Danish Newborn Screening Biobank represent a reliable resource of DNA for whole-genome amplification and subsequent genome-wide association studies. With call-rates equivalent to high quality DNA samples, our results point to new opportunities for using the neonatal biobanks available worldwide in the hunt for genetic components of disease.

Background: Identification of disease susceptible genes requires access to DNA from numerous well-characterised subjects. Archived residual dried blood spot samples from national newborn screening programs may provide DNA from entire populations and medical registries the corresponding clinical information. The amount of DNA available in these samples is however rarely sufficient for reliable genome-wide scans, and whole-genome amplification may thus be necessary. This study assess the quality of DNA obtained from different amplification protocols by evaluating fidelity and robustness of the genotyping of 610,000 single nucleotide polymorphisms, using the Illumina Infinium HD Human610-Quad BeadChip. Whole-genome amplified DNA from 24 neonatal dried blood spot samples stored between 15 to 25 years was tested, and high-quality genomic DNA from 8 of the same individuals was used as reference.

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