Genomic copy number variation (CNV) of part of human chromosome 1 (1q21.1 distal deletion/duplication interval) has been associated with autism spectrum disorder (ASD) in multiple studies (e.g.,^1,2). However, since this region is derived from recent segmental duplication events, most genome analysis tools do not work well, making it challenging to determine what role the many human-specific genes in this interval play in development and disease.

Recent work using new genome sequencing methods and human pluripotent stem cell (PSC)-derived cerebral cortex organoids enabled the identification of three paralogs of NOTCH2NL (NOTCH2NLA, -B and –C) that reside in this locus and are highly expressed during early brain development and capable of promoting cortical neuron stem cell maintenance and proliferation³. Further, all of the samples that were tested from individuals with 1q21.1 deletion/duplication syndrome had copy number alterations in NOTCH2NLA and NOTCH2NLB. These results suggested a hypothesis in which alterations in NOTCH2NL gene dosage contribute to the neurological phenotypes observed in individuals with 1q21.1 distal deletions or duplications.

In Aim 1, the potential presence of focal changes affecting NOTCH2NL copy number will be assessed in large collections of autism families by re-analyzing short-read, whole-genome sequencing (WGS) data from the Simons Simplex Collection and the Autism Genetic Resource Exchange (AGRE), a total of over 4,000 families. Samples with altered CNVs will be subject to targeted linked-read sequencing to determine the NOTCH2NL alleles present and to estimate the focal rearrangement boundaries.

In Aim 2, nanopore-based sequencing of large (>50 kb) DNA fragments and the ability of CRISPR/Cas9 to generate large genomic fragments that are highly enriched for a target region of interest will be used to develop a protocol that enables cost-effective, de novo assembly of the 1q21.1 locus in any human sample from which high molecular weight DNA can be generated.

Overall, findings from this project are expected to determine whether focal changes at the 1q21.1 locus, resulting in altered NOTCH2NL gene dosage, are more widespread among individuals with ASD than has previously been appreciated. In addition, the new sequencing methods developed here will likely be of value for future studies of other complex and recently evolved genomic regions that are known to be altered in autism.

1. Shen Y. et al. Pediatrics 125, e727-735 (2010) PubMed
2. Sanders S.J. et al. Neuron 70, 863-885 (2011) PubMed
3. Fiddes I.T. et al. Cell 173, 1356-1369 (2018) PubMed