Spatial enrichment and genomic analyses reveal the link of NOMO1 with amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a severe motor neuron disease with uncertain genetic predisposition in most sporadic cases. Spatial architecture of cell types and gene expression is the basis of cell-cell interactions, biological function and disease pathology, but is not well investigated in human motor cortex, a key ALS relevant brain region. Recent studies indicated single nucleus transcriptomic features of motor neuron vulnerability in ALS motor cortex. However, it remains largely unclear what is the brain regional vulnerability of ALS-associated genes, and what is the genetic link between region-specific genes and ALS risk. Here, we developed an entropy-weighted differential gene expression matrix-based tool (SpatialE) to identify the spatial enrichment of gene sets in spatial transcriptomics (ST). We benchmarked SpatialE against another enrichment tool (Multimodal Intersection Analysis, MIA) using ST data from both human and mouse brain tissues. To investigate regional vulnerability, we analyzed three human motor cortex and two dorsolateral prefrontal cortex tissues for spatial enrichment of ALS-associated genes. We also used Cell2location to estimate the abundance of cell types in ALS-related cortex layers. To dissect the link of regionally expressed genes and ALS risk, we performed burden analyses of rare loss-of-function (LOF) variants detected by whole-genome sequencing in ALS patients and controls, and then analyzed differential gene expression in the TargetALS RNA-seq dataset. SpatialE showed more accurate and specific spatial enrichment of regional cell type markers than MIA in both mouse brain and human dorsolateral prefrontal cortex. Spatial transcriptomic analyses of human motor cortex showed heterogenous cell types and spatial gene expression profiles. We found that 260 manually curated ALS-associated genes are significantly enriched in layer 5 (L5) motor cortex, with abundant expression of upper motor neurons and L5 excitatory neurons. Burden analyses of rare LOF variants in L5-associated genes nominated NOMO1 as a novel ALS-associated gene in a combined sample set of 6,814 ALS patients and 3,324 controls (P = 0.029). Gene expression analyses in central nervous system tissues revealed down-regulation of NOMO1 in ALS, which is consistent with a LOF disease mechanism. In conclusion, our integrated ST and genomic analyses identified regional brain vulnerability in ALS and the association of a L5 gene (NOMO1) with ALS risk.