Biomedical Research Council

Abstract Asian populations are currently underrepresented in human genetics research. Here we present whole-genome sequencing data of 4,810 Singaporeans from three diverse ethnic groups: 2,780 Chinese, 903 Malays, and 1,127 Indians. Despite a medium depth of 13.7×, we achieved essentially perfect (>99.8%) sensitivity and accuracy for detecting common variants and good sensitivity (>89%) for detecting extremely rare variants with <0.1% allele frequency. We found 89.2 million single-nucleotide polymorphisms (SNPs) and 9.1 million small insertions and deletions (INDELs), more than half of which have not been cataloged in dbSNP. In particular, we found 126 common deleterious mutations (MAF>0.01) that were absent in the existing public databases, highlighting the importance of local population reference for genetic diagnosis. We describe fine-scale genetic structure of Singapore populations and their relationship to worldwide populations from the 1000 Genomes Project. In addition to revealing noticeable amounts of admixture among three Singapore populations and a Malay-related novel ancestry component that has not been captured by the 1000 Genomes Project, our analysis also identified some fine-scale features of genetic structure consistent with two waves of prehistoric migration from south China to Southeast Asia. Finally, we demonstrate that our data can substantially improve genotype imputation not only for Singapore populations, but also for populations across Asia and Oceania. These results highlight the genetic diversity in Singapore and the potential impacts of our data as a resource to empower human genetics discovery in a broad geographic region.

e16024 Background: Anti-PD-1 immune checkpoint inhibitors have recently been approved in the treatment of advanced gastric cancer. Epstein-Barr virus associated gastric cancer (EBVaGC) has traditionally been associated with high expression of PD-L1 and immune cell infiltration. Correlations between PD-L1 and other immune-related gene (IRG) expression in EBVaGC has not been previously described. We conducted a study of transcriptomic expression of PD-L1 and other IRGs related to intra-tumoral cytolytic activity, cytokines and immune checkpoints in EBVaGC. Methods: EBVaGC samples were obtained from gastric cancer patients undergoing primary tumor resection at Samsung Medical Centre, South Korea, from 1996 to 2011 who consented to research use of their tissue samples. For controls, EBV-negative samples from the previously reported Asian Cancer Research Group (EBVnegACRG) cohort were used. Transcriptomic expression was measured using the NanoString platform on formalin-fixed paraffin embedded gastric cancer samples. EBV status was determined using EBV-encoded RNA in situ hybridization. Samples with PD-L1 expression greater than the 35th percentile were defined as PD-L1high and the remaining as PD-L1low. Disease-free survival (DFS) was calculated from the time of surgery to the time of disease progression or death. Results: We identified 72 cases of EBVaGC and 193 EBV-negative ACRG samples as controls. EBVaGC showed higher expression of all queried IRGs compared to EBVnegACRG samples (p < 0.01). EBVaGC had significantly higher PD-L1 expression (p < 0.001) compared to EBVnegACRG samples. PD-L1low EBVaGC samples (n = 25, 35%) had consistently decreased expression of all other IRGs including markers of cytolytic activity such as CD8A, GZMA and PRF1 and other immune checkpoints such as PD-1 (p < 0.001). PD-L1low EBVaGC samples were also associated with worse DFS (HR = 4.78, p = 0.039) compared to PD-L1high EBVaGC samples. Conclusions: A substantial proportion of EBVaGC do not express high levels of PD-L1 and other IRGs. EBVaGCs with lower transcriptomic expression of PD-L1 tend to have a poorer prognosis through low expression of other IRGs.

Granulation tissue formation requires a robust angiogenic response. As granulation tissue develops, collagen fibers are deposited and compacted. Forces generated in the wake of this process drive wound contraction to reduce the wound area. In diabetics, both angiogenesis and wound contraction are diminished leading to impaired wound healing. To emulate this pathology and to address it pharmacologically, we developed a wound healing model in the diabetic Zucker fatty rat and tested a topical proangiogenic strategy combining antifungal agent ciclopirox olamine (CPX) and lysophospholipid sphingosine-1-phosphate (S1P) to promote diabetic wound closure. In vitro, we demonstrated that CPX + S1P up-regulates a crucial driver of angiogenesis, hypoxia-inducible factor-1, in endothelial cells. Injection of CPX + S1P into subcutaneously implanted sponges in experimental rats showed, in an additive manner, a fivefold increased endothelial infiltration and lectin-perfused vessel length. We developed a splinted diabetic rodent model to achieve low wound contraction rates that are characteristic for the healing mode of diabetic ulcers in humans. We discovered specific dorsal sites that allowed for incremental full-thickness excisional wound depths from 1 mm (superficial) to 3 mm (deep). This enabled us to bring down wound contraction from 51% in superficial wounds to 8% in deep wounds. While the effects of topical gel treatment of CPX + S1P were masked by the rodent-characteristic dominant contraction in superficial wounds, they became clearly evident in deep diabetic wounds. Here, a fivefold increase of functional large vessels resulted in accelerated granulation tissue formulation, accompanied by a 40% increase of compacted thick collagen fibers. This was associated with substantially reduced matrix metalloproteinase-3 and -13 expression. These findings translated into a fivefold increase in granulation-driven contraction, promoting diabetic wound closure. With CPX and S1P analogues already in clinical use, their combination presents itself as an attractive proangiogenic treatment to be repurposed for diabetic wound healing.

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.