Molecular analysis of a deletion hotspot in the NRXN1 region reveals the involvement of short inverted repeats in deletion CNVs

Abstract

NRXN1 microdeletions occur at a relatively high frequency and confer increased risk for neurodevelopmental and neurobehavioral abnormalities. The mechanism that makes NRXN1 a deletion hotspot is unknown. Here, we identified deletions of the NRXN1 region in affected cohorts, confirming a strong association with the autism spectrum and other neurodevelopmental disorders. Interestingly, deletions in both affected and control individuals were clustered in the 5' portion of NRXN1 and its immediate upstream region. To explore the mechanism of deletion, we mapped and analyzed the breakpoints of 32 deletions. At the deletion breakpoints, frequent microhomology (68.8%, 2-19 bp) suggested predominant mechanisms of DNA replication error and/or microhomology-mediated end-joining. Long terminal repeat (LTR) elements, unique non-B-DNA structures, and MEME-defined sequence motifs were significantly enriched, but Alu and LINE sequences were not. Importantly, small-size inverted repeats (minus self chains, minus sequence motifs, and partial complementary sequences) were significantly overrepresented in the vicinity of NRXN1 region deletion breakpoints, suggesting that, although they are not interrupted by the deletion process, such inverted repeats can predispose a region to genomic instability by mediating single-strand DNA looping via the annealing of partially reverse complementary strands and the promoting of DNA replication fork stalling and DNA replication error. Our observations highlight the potential importance of inverted repeats of variable sizes in generating a rearrangement hotspot in which individual breakpoints are not recurrent. Mechanisms that involve short inverted repeats in initiating deletion may also apply to other deletion hotspots in the human genome.

Figure 1

NRXN1 Deletions Identified from Individuals with Neurodevelopmental Disorders, Schizophrenia Cohorts, and the Database of Genomic Variants Controls Displayed as UCSC Genome Browser Custom Tracks The RefSeq Genes track shows the 1.3 Mb NRXN1 region encompassing the longest transcript and 200 kb upstream (chr2: 49990147–chr2: 51317000), and the black arrow indicates the genomic midpoint of this region. Red bars indicate 37 deletions identified from individuals with neurodevelopmental disorders (36 from this affected cohort and one from a literature-reported ASD individual). From our affected cohort, 26 NRXN1 deletions were mapped at the nucleotide level. Grey bars indicate five deletions identified from control individuals with exact breakpoint mapping. The purple and green lines indicate NRXN1 region plus and minus self chains, respectively. The relative positions of 66 NRXN1 region deletions reported in the literature for individuals with schizophrenia are shown as maroon bars. Deletions in control individuals reported in the DGV are shown in the bottom panel. Three independent deletion breakpoints (blue arrow, affected individuals 1 and 34 and 1KG-4) were close to the same minus self chain. The yellow arrow indicates an identical deletion in affected individuals 19 and 20. The green arrows indicate minus self chains in close association with NRXN1 breakpoints.