Genomic copy number variation (CNV) in a part of human chromosome 1 emerged recently in the human lineage by repeated segmental duplications and rearrangements. CNVs here have been associated with autism, schizophrenia and other neurodevelopmental disorders in multiple genome-wide association studies. However, the gene(s) in this interval that contribute to these phenotypes have not been established. We recently identified a family of 3 genes, ?NOTCH2NLA?, -?B and -?C that reside in this locus, are highly expressed during early brain development and are capable of promoting cortical neuron stem cell maintenance and proliferation. In this proposal ?we test the hypothesis that NOTCH2NL genes are required for normal human brain development and alterations in their gene dosage contribute the neurological phenotypes observed in patients with 1q21.1 distal deletions and duplications. This will be accomplished in Aim 1 by ?resequencing this genomic interval ?in diverse human population samples using new long genomic fragment sequencing technologies that will enable us ?to identify the NOTCH2NL alleles present in the human population ?and structural variation present in this highly repetitive genomic region?. This information will inform ?subsequent sequence analysis of 14 samples harboring pathogenic 1q21.1 CNV events that may implicate specific NOTCH2NL loci and variants in these disorders. This information will be used to develop ?high throughput, cost-effective assays to identify the specific NOTCH2NL alleles present in large genomic DNA collections from patients with 1q21.1-associated neurological disorders.
In Aim 2, we will ?test the activity of the various NOTCH2NL alleles identified in 2 ways. First, examine the ability of each NOTCH2NL allele ?to promote NOTCH signaling using reporter based assays?. Second, we will test the activity of specific NOTCH2NL alleles ?to promote normal cortical organoid formation using isogenic CRISPR engineered pluripotent stem cell lines that differ only by the specific NOTCH2NL alleles present. The ability of specific alleles to rescue defects in the balance of neural stem cells and cortical neurons associated with loss of NOTCH2NL will be measured by single-cell RNA Sequencing, bulk gene expression measurements and histology. Finally, in Aim 3 we will test whether ?heterozygous, large-scale 1q21.1 deletions similar to those observed in patients have similar or more severe defects in early brain development as assayed by our human pluripotent stem cell cerebral cortex organoid assay using the analysis methods described for Aim 2. We will then ?test the ability of NOTCH2NL and other genes in locus to rescue any defects observed. The experiments outlined here will improve our understanding of the specific biological defects resulting from 1q21.1 CNVs that ultimately lead to complex neurological disorders. These methods can be applied to other repetitive genomic loci implicated in neurodevelopmental diseases.
Understanding the molecular and cellular defects that underlie complex diseases like autism and schizophrenia are a valuable first step towards developing approaches to prevent or ameliorate their associated pathological phenotypes. Genomic copy number variation of a segment of human chromosome 1 (1q21.1 distal deletion/duplication interval) has been associated with multiple neurodevelopmental disorders including autism and schizophrenia in multiple studies but, since this region is derived from recent segmental duplication events, it has been difficult to dissect the role the many human-specific genes in this genomic locus play in normal development and disease. Here we use new genome sequencing technologies and human pluripotent stem cell-derived cerebral cortex tissue organoids to characterize human variation at this locus and the functional role of these variants in human brain development.
