Disease-Causing 7.4 kb Cis-Regulatory Deletion Disrupting Conserved Non-Coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening

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.

FOXL2copy number changes in the molecular pathogenesis of BPES: unique cohort of 17 deletions

“…Although it was not the primary goal of the study to pinpoint the exact location of the breakpoints, we are able to demonstrate their scattered location. This indicates the absence of rearrangement hotspots, which is in line with previous findings (Beysen, et al, 2005;D'haene, et al, 2009;Shaw and Lupski, 2004). These nonrecurrent rearrangement events are suggestive of nonhomologous end joining (NHEJ), alternative NHEJ (alt-NHEJ), or Fork Stalling and Template Switching (FoSTeS) as possible underlying mechanisms (Lee, et al, 2007;Shaw and Lupski, 2004;Vissers, et al, 2009).…”

“…using MLPA), as gene deletions are present in 10% of this group . If negative, copy number screening targeting the FOXL2 region (e;g. using qPCR or array CGH) is recommended as a third step, to exclude the presence of extragenic copy number changes affecting the long-range genetic control of FOXL2, which have been reported in 4% of the cases (Beysen, et al 2005;D'haene, et al 2009). BPES-like: Genome wide microarray-based copy number screening is recommended in this group, as copy number changes can be detected in a relatively large proportion of these patients (33%) (Gijsbers, et al 2008).…”

“…The single-exon gene FOXL2 (MIM# 605597), encoding a forkhead transcription factor, was identified as the disease causing gene for both types of BPES (Crisponi, et al, 2001). Using a combined mutation detection approach, it is possible to identify the underlying genetic defect in a major proportion (88%) of typical BPES patients (Beysen, et al, 2005;D'haene, et al, 2009). Overall, intragenic FOXL2 mutations represent 81% of the total group of genetic defects found in BPES.…”

Reference-assisted chromosome assembly

“…One of the most important advantages of having whole genome sequences is the capacity to understand the evolutionary history of genome organization and structural variation caused by chromosome rearrangements (4)(5)(6)(7). However, the number of animal genomes sequenced by next generation sequencing (NGS) is rapidly outpacing the number of genomes with physical or genetic maps for anchoring the assemblies to chromosomes, which is necessary for elucidating the biological consequences of chromosome rearrangements and for shedding new light on the molecular signatures of human variation and disease mechanisms (8)(9)(10). More large-scale genome sequencing projects are planned using NGS technologies, including the Genome 10K (G10K) and the 5K Insect Genome (i5K) initiatives (11,12).…”

Circadian changes in long noncoding RNAs in the pineal gland

“…Current research points to a role in cellular regulation through interaction with proteins and DNA (4)(5)(6). The abundance of many lncRNAs changes gradually during development, and many are involved in epigenetic processes impacting gene expression (7)(8)(9)(10)(11)(12) [e.g., chromosome inactivation by XIST (13), imprinting by Kcnq1ot1 (14), development and cancer by HOTAIR (15,16), cancer by PCA3 (17), and disease by PISTR1 (18)]. From this information, it is generally thought that lncRNAs control long-term processes; however, it is also clear that they may play roles in more dynamic processes, including signaling (19)(20)(21)(22)(23).…”