Chromosomal structural rearrangements implicate long non-coding RNAs in rare germline disorders

Abstract

In recent years, there has been increased focus on exploring the role the non-protein-coding genome plays in Mendelian disorders. One class of particular interest is long non-coding RNAs (lncRNAs), which has recently been implicated in the regulation of diverse molecular processes. However, because lncRNAs do not encode protein, there is uncertainty regarding what constitutes a pathogenic lncRNA variant, and thus annotating such elements is challenging. The Developmental Genome Anatomy Project (DGAP) and similar projects recruit individuals with apparently balanced chromosomal abnormalities (BCAs) that disrupt or dysregulate genes in order to annotate the human genome. We hypothesized that rearrangements disrupting lncRNAs could be the underlying genetic etiology for the phenotypes of a subset of these individuals. Thus, we assessed 279 cases with BCAs and selected 191 cases with simple BCAs (breakpoints at only two genomic locations) for further analysis of lncRNA disruptions. From these, we identified 66 cases in which the chromosomal rearrangements directly disrupt lncRNAs. In 30 cases, no genes of any other class aside from lncRNAs are directly disrupted, consistent with the hypothesis that lncRNA disruptions could underly the phenotypes of these individuals. Strikingly, the lncRNAs MEF2C-AS1 and ENSG00000257522 are each disrupted in two unrelated cases. Furthermore, we experimentally tested the lncRNAs TBX2-AS1 and MEF2C-AS1 and found that knockdown of these lncRNAs resulted in decreased expression of the neighboring transcription factors TBX2 and MEF2C, respectively. To showcase the power of this genomic approach for annotating lncRNAs, here we focus on clinical reports and genetic analysis of seven individuals with likely developmental etiologies due to lncRNA disruptions.

Fig. 1

The lncRNA TBX2-AS1 is a candidate for an association with hearing loss. (A) Chromosome diagrams depict the translocation between 14q23.3 and 17q23.2 in DGAP353. Above, TADs containing the breakpoints are shown, with the breakpoint positions indicated by vertical orange bars and the edges of the region shown in vertical pink bars. TAD borders were defined in (Dixon et al. 2012). Triangular contact maps display micro-C data indicating chromatin conformation (Krietenstein et al. 2020). H3K4Me1 and H3K27Ac tracks depict enhancer-associated chromatin modifications (ENCODE Project Consortium 2012). The VISTA track shows experimentally validated enhancer elements (Visel et al. 2007). Protein-coding genes are shown in blue and non-coding genes in green, with a single isoform depicted per gene. (B) Expanded view of the genomic region surrounding the 14q23.3 breakpoints in DGAP353. (C) Expanded view of the genomic region surrounding the 17q23.2 breakpoints in DGAP353. The directly disrupted lncRNA TBX2-AS1 is highlighted in red. ENSG00000267131 has been identified as an isoform of TBX2-AS1 by LNCipedia (Volders et al. 2019)

Fig. 2

The lncRNA MEF2C-AS1 is disrupted in multiple individuals with neurological phenotypes, as shown here for DGAP191. (A) Chromosome diagrams depict the translocation between 5q14.3 and 7q21.3 in DGAP191. Above, TADs containing the breakpoints are shown, with the breakpoint positions indicated by vertical orange bars and the edges of the region shown in vertical pink bars. TAD borders were defined in (Dixon et al. 2012). Triangular contact maps display micro-C data indicating chromatin conformation (Krietenstein et al. 2020). H3K4Me1 and H3K27Ac tracks depict enhancer-associated chromatin modifications (ENCODE Project Consortium 2012). The VISTA track shows experimentally validated enhancer elements (Visel et al. 2007). Protein-coding genes are shown in blue and non-coding genes in green, with a single isoform depicted per gene. (B) Expanded view of the genomic region surrounding the 5q14.3 breakpoints in DGAP191. The directly disrupted lncRNA MEF2C-AS1 is highlighted in red. (C) Expanded view of the genomic region surrounding the 7q21.3 breakpoints in DGAP191. The directly disrupted lncRNA ENSG00000285090 is highlighted in red

Fig. 3

The lncRNA MEF2C-AS1 is disrupted in multiple individuals with neurological phenotypes, as shown here for DGAP218. (A) Chromosome diagrams depict the inversion between 5p14.2 and 5q14.3 in DGAP218. Above, TADs containing the breakpoints are shown, with the breakpoint positions indicated by vertical orange bars and the edges of the region shown in vertical pink bars. TAD borders were defined in (Dixon et al. 2012). Triangular contact maps display micro-C data indicating chromatin conformation (Krietenstein et al. 2020). H3K4Me1 and H3K27Ac tracks depict enhancer-associated chromatin modifications (ENCODE Project Consortium 2012). The VISTA track shows experimentally validated enhancer elements (Visel et al. 2007). Protein-coding genes are shown in blue and non-coding genes in green, with a single isoform depicted per gene. (B) Expanded view of the genomic region surrounding the 5p14.2 breakpoints in DGAP218. (C) Expanded view of the genomic region surrounding the 5q14.3 breakpoints in DGAP218. The directly disrupted lncRNA MEF2C-AS1 is highlighted in red

Fig. 4

The lncRNA ENSG00000257522 is disrupted in multiple individuals with microcephaly, as shown here for DGAP245. (A) Chromosome diagrams depict the translocation between 3p22.2 and 14q12 in DGAP245. Above, large regions containing the breakpoints are shown, with the breakpoint positions indicated by vertical orange bars and the edges of the region shown in vertical pink bars. The region shown surrounding 14q12 is a TAD, with its borders previously defined in (Dixon et al. 2012). No TAD was defined surrounding 3p22.2, so instead the region including 1 Mb on either side of the breakpoints is displayed. Triangular contact maps display micro-C data indicating chromatin conformation (Krietenstein et al. 2020). H3K4Me1 and H3K27Ac tracks depict enhancer-associated chromatin modifications (ENCODE Project Consortium 2012). The VISTA track shows experimentally validated enhancer elements (Visel et al. 2007). Protein-coding genes are shown in blue and non-coding genes in green, with a single isoform depicted per gene (B) Expanded view of the genomic region surrounding the 3p22.2 breakpoints in DGAP245. (C) Expanded view of the genomic region surrounding the 14q12 breakpoints in DGAP245. The directly disrupted lncRNAs ENSG00000258028 and ENSG00000257522 are highlighted in red

Fig. 5

The lncRNA ENSG00000257522 is disrupted in multiple individuals with microcephaly, as shown here for NIJ1. (A) Chromosome diagrams depict the translocation between 8q21.13 and 14q12 in NIJ1. Above, TADs containing the breakpoints are shown, with the breakpoint positions indicated by vertical orange bars and the edges of the region shown in vertical pink bars. TAD borders were defined in (Dixon et al. 2012). Triangular contact maps display micro-C data indicating chromatin conformation (Krietenstein et al. 2020). H3K4Me1 and H3K27Ac tracks depict enhancer-associated chromatin modifications (ENCODE Project Consortium 2012). The VISTA track shows experimentally validated enhancer elements (Visel et al. 2007). Protein-coding genes are shown in blue and non-coding genes in green, with a single isoform depicted per gene. (B) Expanded view of the genomic region surrounding the 8q21.13 breakpoints in NIJ1. (C) Expanded view of the genomic region surrounding the 14q12 breakpoints in NIJ1. The directly disrupted lncRNAs ENSG00000258028 and ENSG00000257522 are highlighted in red

Fig. 6

Disruption of the lncRNA ENSG00000255087 was identified in an individual with a neurodevelopmental disorder. (A) Chromosome diagrams depict the translocation between Xp11.4 and 11q24.2 in DGAP148. Above, TADs containing the breakpoints are shown, with the breakpoint positions indicated by vertical orange bars and the edges of the region shown in vertical pink bars. TAD borders were defined in (Dixon et al. 2012). Triangular contact maps display micro-C data indicating chromatin conformation (Krietenstein et al. 2020). H3K4Me1 and H3K27Ac tracks depict enhancer-associated chromatin modifications (ENCODE Project Consortium 2012). The VISTA track shows experimentally validated enhancer elements (Visel et al. 2007). Protein-coding genes are shown in blue and non-coding genes in green, with a single isoform depicted per gene. (B) Expanded view of the genomic region surrounding the Xp11.4 breakpoints in DGAP148. (C) Expanded view of the genomic region surrounding the 11q24.2 breakpoints in DGAP148. The directly disrupted lncRNA ENSG00000255087 is highlighted in red

Fig. 7

The lncRNA SOX2-OT is implicated in an individual with epilepsy and autism spectrum disorder. (A) Chromosome diagrams depict the translocation between 3q26.33 and 9q21.13 in DGAP355. Above, TADs containing the breakpoints are shown, with the breakpoint positions indicated by vertical orange bars and the edges of the region shown in vertical pink bars. TAD borders were defined in (Dixon et al. 2012). Triangular contact maps display micro-C data indicating chromatin conformation (Krietenstein et al. 2020). H3K4Me1 and H3K27Ac tracks depict enhancer-associated chromatin modifications (ENCODE Project Consortium 2012). The VISTA track shows experimentally validated enhancer elements (Visel et al. 2007). Protein-coding genes are shown in blue and non-coding genes in green, with a single isoform depicted per gene. (B) Expanded view of the genomic region surrounding the 3q26.33 breakpoints in DGAP355. The directly disrupted lncRNA SOX2-OT is highlighted in red. (C) Expanded view of the genomic region surrounding the 9q21.13 breakpoints in DGAP355

Fig. 8

Knockdown of the lncRNAs TBX2-AS1 and MEF2C-AS1 results in decreased expression of their neighboring genes. (A) Relative expression of TBX2-AS1 upon transfection with siRNAs targeting TBX2-AS1, as compared to negative control (Neg. Ctrl.) siRNAs. **** p < 0.0001. (B) Relative expression of TBX2 upon transfection with siRNAs targeting TBX2-AS1. * p = 0.0367. (C) Relative expression of MEF2C-AS1 upon transfection with siRNAs targeting MEF2C-AS1. **** p < 0.0001. (D) Relative expression of MEF2C upon transfection with siRNAs targeting MEF2C-AS1. ** p = 0.0013. All experiments were performed in human Lenti-X™ 293T cultures with two separate negative control and targeting siRNAs, with the results from siRNA #1 shown in blue and from siRNA #2 in red. Statistical analyses were performed using unpaired t tests