Disruption of a large intergenic noncoding RNA in subjects with neurodevelopmental disabilities

Abstract

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.

Figure 1

Identification of Translocation Breakpoints from DGAP162 by CapBP Methodology A distal, short arm translocation involving chromosome 2 (blue) and chromosome 11 (gray) are shown, with split read sequences from fragments containing the breakpoint junctions provided. The next-gen cytogenetic karyotype is designated: 46,XX,der(2)(11pter–15,825,269::chr2 8,247,757–2qter),der(11)(2pter–8,247,756::chr11 15,825,273–11qter)dn (hg19). Additional details of sequence characteristics available in Chiang et al.

Figure 2

LINC00299 RNA Is Both Expressed and Spliced in Normal Human Lymphocytes An RNA FISH probe targeting the spliced product of LINC00299 in EBV-transformed wild-type human lymphocytes. RNA FISH probe was ligated to an Alexa 488 dye, then hybridized to individual cells on a microscope slide. Individual cells are stained with the DNA dye 4′,6-diamidino-2-phenylindole (DAPI). Green dots in the blue nucleus suggest individual molecules of LINC00299 in each cell. Green stain, probe targeting exons 5–7 of LINC00299; blue stain, DAPI.

Figure 3

Structure of LINC00299 Transcripts in Brain, Expression Level across Human Tissues, and Expression of LINC00299 in Lymphoblastoid Cell Lines Comparing DGAP162 to Three Control Subjects (A) LINC00299 alternative splice transcripts from RNA extracted from wild-type human prefrontal cortex (exons and introns not to scale). Numbers represent exons identified by RefSeq, and unreported exons are unnumbered. Each transcript represents a fully cloned RT-PCR fragment, confirmed by Sanger sequencing. Small bars below the final transcript refer to “pre” and “post” primer sets used for expression studies. (B) Agarose electrophoresis gel showing amplification of LINC00299 transcripts in brain prefrontal cortex via different primers. Forward primers (F, top line) and reverse primers (R, bottom line) are noted by numbers representing which exon the specific primer targeted. Exon numbers correspond to exon numbers noted in (A). (C) Expression level of LINC00299 transcripts in spleen, brain, kidney, and liver. “Pre” and “Post” refer to primers that bind 5′ and 3′, respectively, to the translocation breakpoint identified in DGAP162. (D) Quantification of LINC00299 transcripts in lymphoblastoid cell lines from controls and DGAP162 with a probe targeting exon 6 of LINC00299 (pretranslocation), which amplified both the wild-type and mutant alleles of DGAP 162. (E) Quantification of LINC00299 transcripts in lymphoblastoid cells via primers specific to the wild-type allele (posttranslocation probe). ^∗∗∗p < 0.001. All reactions were run in quadruplicate and ACTB was used as an internal control in all cases. All primer sequences can be found in Table S1. All error bars represent standard error of the mean.

Figure 4

LINC00299 Temporal Expression in Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells mRNA expression levels were quantified using qRT-PCR and normalized using two endogenous controls, ACTB and GAPDH. (A) Quantity mean values represent expression levels of LINC00299 in mRNA from exon 6. (B) Quantity mean values represent expression levels of LINC00299 mRNA from exon 8. All error bars represent standard error of the mean.