Lung cancer cohort used in the study

Abstract Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue–specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer–target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification. Significance: Mapping enhancer–target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis.

Gene set enrichment analysis on C6 oncogenic dataset from MsigDB

Genomic co-ordinates of lung specific enhancers (hg19)

Supplementary Figures S1-S7

Number of enhancers associated with genes and the number of mutations in the lower right quadrant of figure 4c Overlap with Msigdb C2 dataset

Enhancer target gene prediction results

Epigenomics data from lung cell and tissue type used for Enhancer definition

Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification.

Mutation frequency analysis at enhancer cores

CDH13 enhancer regression: Generalised linear model

Inherited retinal disease (IRD) is a major global cause of blindness caused by mutations in a wide spectrum of genes essential to the retinal structure, maintenance and function. Current clinical diagnostic strategies in the UK are focused on targeted gene panel testing either by enrichment or virtually. Whole exome and genome sequencing (WES and WGS) have been used in rare disease genetic discovery now for a decade and are being integrated into many research pipelines and diagnostic strategies exemplified by the Genomics England 100,000 genomes project.Here, we describe the current approaches to genetic and genomic analysis in IRD, the shortfalls and advantages of gene panel testing, WES and WGS in the context of single nucleotide, structural and copy number variants in coding, non-coding and intractable genomic regions.Looking ahead, the missing heritability in IRD may be consequent on a number of factors: new genes, ignored or undetectable variants, new diseases for known genes, etc. Improved detection of genomic variation afforded by WGS paired with expanded variant databases, advances in variant interpretation, developing our understanding of the effect of non-coding variation using multiomics and integrating deep phenotyping and genomic data into machine learning tools will be the driving forces in better diagnosis of rare disease and discovery of novel causes of disease in the post-genomic era.

North Carolina macular dystrophy (NCMD) and Progressive bifocal chorioretinal atrophy (PBCRA) are a rare set of dominantly inherited disorders that affect central vision from birth. Two linked loci had been previously identified at 5p21 and 6q16. Whole-genome sequencing (WGS) analysis of our cohort of 92 affected individuals has identified 3 novel causative structural variants (SVs) on 5p21 in 13 NCMD families, and 2 novel noncoding single nucleotide variants on 6q16 upstream of the promoter of PRDM13 in three PBCRA families. Combined, the rearrangements in 5p21 have a shared duplicated region of 39kb, located in a gene desert downstream of IRX1 and upstream of ADAMTS16. DNAse-seq data publicly available for human fetal retina identified active open chromatin sites at both loci at restricted times during retinal development. To dissect the molecular mechanism of the identified variants, skin-derived fibroblasts lines from selected patients were established; CRISPR-CAS9 technology was used to recreate the patient variants in a mouse model; and candidate genes expression profile was assessed in three stages of human fetal retina tissue. Additionally, chromosome conformation capture technology was performed in patient fibroblasts and in mouse developing tissue to characterise the genomic landscape of the locus and assess the effect of structural variants. Genome-wide analysis of chromosome occupancy was also performed for the architectural protein CTCF and for histone modifications relevant for identification of cis-acting elements. Expression studies showed altered expression of IRX genes and ADAMTS16 in mutant lines. Together with evidence from human retinal tissue immunohistochemistry and in situ expression data, these genes are suggested to be miss-expressed and/or ectopically expressed during macular development, with involvement of PRDM13, FGF8 and FGF10, which were also found miss-expressed in mutant lines . This work provides novel insight into the gene regulation landscape involved in human macular development and prognostic information for affected families.

The autosomal dominant progressive bifocal chorioretinal atrophy (PBCRA) disease locus has been mapped to chromosome 6q14-16.2 that overlaps the North Carolina macular dystrophy (NCMD) locus MCDR1. NCMD is a nonprogressive developmental macular dystrophy, in which variants upstream of PRDM13 have been implicated. Whole genome sequencing was performed to interrogate structural variants (SVs) and single nucleotide variants (SNVs) in eight individuals, six affected individuals from two families with PBCRA, and two individuals from an additional family with a related developmental macular dystrophy. A SNV (chr6:100,046,804T>C), located 7.8 kb upstream of the PRDM13 gene, was shared by all PBCRA-affected individuals in the disease locus. Haplotype analysis suggested that the variant arose independently in the two families. The two affected individuals from Family 3 were screened for rare variants in the PBCRA and NCMD loci. This revealed a de novo variant in the proband, 21 bp from the first SNV (chr6:100,046,783A>C). This study expands the noncoding variant spectrum upstream of PRDM13 and suggests altered spatio-temporal expression of PRDM13 as a candidate disease mechanism in the phenotypically distinct but related conditions, NCMD and PBCRA.

BACKGROUND:Developmental macular disorders are a heterogeneous group of rare retinal conditions that can cause significant visual impairment from childhood. Among these disorders, autosomal dominant North Carolina macular dystrophy (NCMD) has been mapped to 6q16 (MCDR1) with recent support for a non-coding disease mechanism of PRDM13. A second locus on 5p15-5p13 (MCDR3) has been implicated in a similar phenotype, but the disease-causing mechanism still remains unknown. METHODS:Two families affected by a dominant developmental macular disorder that closely resembles NCMD in association with digit abnormalities were included in the study. Family members with available DNA were genotyped using the Affymetrix GeneChip Human Mapping 250K Sty array. A parametric multipoint linkage analysis assuming a fully penetrant dominant model was performed using MERLIN. Haplotype sharing analysis was carried out using the non-parametric Homozygosity Haplotype method. Whole-exome sequencing was conducted on selected affected individuals. RESULTS:Linkage analysis excluded MCDR1 from the candidate regions (LOD < -2). There was suggestive linkage (LOD = 2.7) at two loci, including 9p24.1 and 5p15.32 that overlapped with MCDR3. The haplotype sharing analysis in one of the families revealed a 5 cM shared IBD segment at 5p15.32 (p value = 0.004). Whole-exome sequencing did not provide conclusive evidence for disease-causing alleles. CONCLUSIONS:These findings do not exclude that this phenotype may be allelic with NCMD MCDR3 at 5p15 and leave the possibility of a non-coding disease mechanism, in keeping with recent findings on 6q16. Further studies, including whole-genome sequencing, may help elucidate the underlying genetic cause of this phenotype and shed light on macular development and function.

AbstractAutosomal dominant North Carolina macular dystrophy (NCMD) is believed to represent a failure of macular development. The disorder has been assigned by linkage to two loci, MCDR1 on chromosome 6q16 and MCDR3 on chromosome 5p15-p13. Recently, noncoding variants upstream ofPRDM13and a large duplication includingIRX1have been identified. However, the underlying disease-causing mechanism remains uncertain. Through a combination of sequencing studies, we report two novel overlapping duplications at the MCDR3 locus, in a gene desert downstream ofIRX1and upstream ofADAMTS16.One duplication of 43 kb was identified in nine NCMD families (with evidence for a shared ancestral haplotype), and another one of 45 kb was found in a single family. The MCDR3 locus is thus refined to a shared region of 39 kb that contains DNAse hypersensitive sites active at a restricted time window during retinal development. Publicly available data confirmed expression ofIRX1andADAMTS16in human fetal retina, withIRX1preferentially expressed in fetal macula. These findings represent a major advance in our understanding of the molecular genetics of NCMD at the MCDR3 locus and provide insights into the genetic pathways involved in human macular development.Abbreviations listaCGHarray comparative genomic hybridizationCNVCopy number variantIBDIdentical-by-descentiPSCInduced pluripotent stem cellDHSDNase hypersensitive siteHHHomozygosity HaplotypeMCDRMacular dystrophy regionNCMDNorth Carolina macular dystrophyPCRPolymerase chain reactionRCHHRegion with a Conserved Homozygosity HaplotypeSNPSingle-nucleotide polymorphismSNVSingle nucleotide variantSVStructural variantWGSWhole-genome sequencing