Title: Exploration of genomic ‘dark matter’ in childhood cancer development
Funder: Cancer Research UK

Abstract: Background Resolution of genetic ‘dark matter’ represents the final frontier in genomic exploration, which will revolutionise our understanding of cancer development. Genomic ‘dark matter’ is comprised of repetitive DNA sequences that constitute centromeric and telomeric regions, which are unsequencable using conventional short read sequencing. Notably, aberrant centromeric and telomeric function has been associated with multiple mechanisms of chromosomal instability, including aneuploidy and chromothripsis. Many childhood cancers develop from this repertoire of chromosomal instability, some arising through genetic predisposition to disease. Intriguingly, aneuploidy and chromothripsis are often associated with specific chromosomes in childhood cancer, usually on the background of a mutationally quiet genome. Thus, abnormalities within the centromere or telomere of individual chromosomes are likely to initiate or promote chromosomal instability in childhood tumourigenesis. The advent of revolutionary, state-of-the-art sequencing technologies (MinION), capable of resolving ultra-long stretches of DNA, has made it possible to explore these regions for the first time. Aims My hypothesis is that genomic abnormalities are present within the genetic ‘dark matter’, which initiate chromosomal instability within childhood cancers. By developing and utilising state-of-the-art sequencing technologies and novel methods, I will create a reference genome for genetic ‘dark matter’ within specific chromosomes. Moreover, I will explore inter-individual and inter-chromosomal variation to elucidate the mechanisms of chromosomal instability in childhood cancer. Methods Chromosomal-flow-sorting and HLS-CATCH (CRISPR-cas9-Assisted-Targeting-of-Chromosome-Segments) methods will be developed to capture the centromere or telomere of individual chromosomes in patient samples. Ultra-long (100kb) read sequencing will be performed using the MinION. A reference genome will be created from BAC constructs of the haploid cell line, CHM1, to infer inter-individual and inter-chromosomal variation among childhood cancer patients (n=50) with recurrent chromothripsis or aneuploidy. Targeted sequencing will determine the true incidence of genetic variation in large well-defined patient cohorts (n1000). The consequence of key genomic abnormalities on centromere and telomere activity will be explored using immunofluorescence techniques. How the results of this research will be used Exploration of genomic ‘dark matter will transform our understanding of cancer development. The discovery of genetic abnormalities that initiate and promote chromosomal instability in tumourigenesis will provide the opportunity to 1) improve diagnosis and genetic-risk-stratification of patients for treatment, 2) lead to the development of targeted therapies for disease prevention and management, 3) facilitate development of diagnostic assays for early detection of childhood cancer.

Lay abstract: Background Resolution of genetic ‘dark matter’ represents the final frontier in genomic exploration, which will revolutionise our understanding of cancer development. Genomic ‘dark matter’ is comprised of repetitive DNA sequences that constitute centromeric and telomeric regions, which are unsequencable using conventional short read sequencing. Notably, aberrant centromeric and telomeric function has been associated with multiple mechanisms of chromosomal instability, including aneuploidy and chromothripsis. Many childhood cancers develop from this repertoire of chromosomal instability, some arising through genetic predisposition to disease. Intriguingly, aneuploidy and chromothripsis are often associated with specific chromosomes in childhood cancer, usually on the background of a mutationally quiet genome. Thus, abnormalities within the centromere or telomere of individual chromosomes are likely to initiate or promote chromosomal instability in childhood tumourigenesis. The advent of revolutionary, state-of-the-art sequencing technologies (MinION), capable of resolving ultra-long stretches of DNA, has made it possible to explore these regions for the first time. Aims My hypothesis is that genomic abnormalities are present within the genetic ‘dark matter’, which initiate chromosomal instability within childhood cancers. By developing and utilising state-of-the-art sequencing technologies and novel methods, I will create a reference genome for genetic ‘dark matter’ within specific chromosomes. Moreover, I will explore inter-individual and inter-chromosomal variation to elucidate the mechanisms of chromosomal instability in childhood cancer. Methods Chromosomal-flow-sorting and HLS-CATCH (CRISPR-cas9-Assisted-Targeting-of-Chromosome-Segments) methods will be developed to capture the centromere or telomere of individual chromosomes in patient samples. Ultra-long (100kb) read sequencing will be performed using the MinION. A reference genome will be created from BAC constructs of the haploid cell line, CHM1, to infer inter-individual and inter-chromosomal variation among childhood cancer patients (n=50) with recurrent chromothripsis or aneuploidy. Targeted sequencing will determine the true incidence of genetic variation in large well-defined patient cohorts (n1000). The consequence of key genomic abnormalities on centromere and telomere activity will be explored using immunofluorescence techniques. How the results of this research will be used Exploration of genomic ‘dark matter will transform our understanding of cancer development. The discovery of genetic abnormalities that initiate and promote chromosomal instability in tumourigenesis will provide the opportunity to 1) improve diagnosis and genetic-risk-stratification of patients for treatment, 2) lead to the development of targeted therapies for disease prevention and management, 3) facilitate development of diagnostic assays for early detection of childhood cancer.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS