Abstract
To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5′ to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular.
Highlights
Many recent studies have provided insights into the biological relevance of the non protein-coding portion of the human genome, previously referred to as junk DNA
One of them is the ENCODE pilot study, which has revealed that the number of functional genomic elements is much higher than previously anticipated, and that the vast majority of elements regulating gene expression are contained in the non-protein coding portion of the genome
We developed a combined strategy consisting of microarray based comparative genome hybridization, high-resolution quantitative PCR and sequencing of coding sequences (CNCs) located in the smallest region of deletion overlap (SRO) 59 to FOXL2
Summary
Many recent studies have provided insights into the biological relevance of the non protein-coding portion of the human genome, previously referred to as junk DNA. One of them is the ENCODE pilot study, which has revealed that the number of functional genomic elements is much higher than previously anticipated, and that the vast majority of elements regulating gene expression are contained in the non-protein coding portion of the genome. It shed light on the pervasively transcribed nature of the human genome [1]. The contribution of disrupted potentially regulatory CNCs to human genetic disease is most likely underestimated, as
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