Inter-chromosomal insertions at Xq27.1 associated with retinal dystrophy induce dysregulation of LINC00632 and CDR1as/ciRS-7.

In two unrelated families with X-linked inherited retinal dystrophy, identification of the causative variants was elusive. Interrogation of the next-generation sequencing (NGS) data revealed a "dark" intergenic region on Xq27.1 with poor coverage. Long-range PCR and DNA walking across this region revealed different inter-chromosomal insertions into the human-specific palindrome on Xq27.1: a 58 kb insertion of 9p24.3 [der(X)dir ins(X;9)(q27.1;p24.3)] in family 1 and a 169 kb insertion of 3p14.2 [der(X)inv ins(X;3)(q27.1;p14.2)] in family 2. To explore the functional consequence of these structural variants in genomic and cellular contexts, induced pluripotent stem cells were derived from affected and control fibroblasts and differentiated to retinal organoids (ROs) and retinal pigment epithelium. Transcriptional dysregulation was evaluated using RNA sequencing (RNA-seq) and RT-qPCR. A downstream long non-coding RNA, LINC00632 (Xq27.1), was upregulated in ROs from both families compared to control samples. In contrast, the circular RNA CDR1as/ciRS-7 (circular RNA sponge for miR-7), spliced from linear LINC00632, was downregulated. To investigate this tissue-specific dysregulation, we interrogated the landscape of the locus using Hi-C and cleavage under targets and tagmentation sequencing (CUT&Tag). This revealed active retinal enhancers within the insertions within a topologically associated domain that also contained the upstream promoter of LINC00632, permitting ectopic contact. Furthermore, CDR1as/ciRS-7 acts as a "sponge" for miR-7, and target genes of miR-7 were also dysregulated in ROs derived from both families. We describe a new genomic mechanism for retinal dystrophy, and our data support a convergent tissue-specific mechanism of altered regulation of LINC00632 and CDR1as/ciRS-7 as a consequence of the insertions within the palindrome on Xq27.1.