Spatial Enrichment and Genomic Analysis Reveal Association between NOMO1 and Amyotrophic Lateral Sclerosis

Introduction

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease in which motor neurons in the brain and spinal cord gradually degrade. Although the genetic susceptibility of sporadic ALS patients remains uncertain, TDP-43 protein pathology is a defining characteristic in the majority of ALS cases (>97%). Over 25 genes have been reported to be associated with ALS, but known mutations cannot explain approximately 90% of sporadic ALS cases. Existing studies indicate that the complexity of brain tissue structure underpins cell interactions, biological functions, and pathology. Understanding cell types and regional vulnerability is crucial for precision medicine.

Research Background and Objectives

This study aims to assess the vulnerability of ALS-related genes in different brain regions and uncover the genetic links between region-specific genes and ALS risk. The research team developed a tool called Spatiale, based on entropy-weighted differential gene expression matrices, to identify the spatial enrichment of gene sets in spatial transcriptomics (ST). By benchmarking Spatiale against other enrichment tools such as Multi-modal Intersection Analysis (MIA), the team analyzed the spatial enrichment of ALS-related genes in human motor cortex and dorsolateral prefrontal cortex (DLPFC). Additionally, the study used Cell2Location to estimate the abundance of cell types within ALS-related cortical layers, performed burden analysis of rare loss-of-function (LoF) variants in ALS patients and controls through whole-genome sequencing (WGS), and conducted differential gene expression analysis in the TargetALS RNA-Seq dataset.

Workflow

Development and Evaluation of the Spatiale Tool

The research team developed the Spatiale tool, which estimates differential gene expression matrices using an entropy-weighted method and calculates the enrichment significance of predefined gene sets within spatial clusters (regions). The principle of the Spatiale model is that the greater the deviation between the expression of spatially specific genes and the average expression level in the region, the stronger their spatial specificity. To validate the performance of Spatiale, the team first assessed the spatial enrichment of regional cell types in mouse brain tissue and the human DLPFC. The results showed that Spatiale could more accurately and specifically identify regional cell types within the expected spatial regions.

Spatial Transcriptomics Analysis of the Human Motor Cortex

Analysts performed ST on post-mortem motor cortex samples from two neurologically normal subjects and one ALS patient. UMAP clustering revealed 20 cell types within the motor cortex, including excitatory and inhibitory neurons as well as non-neuronal cell types. Spatial clustering and HE staining confirmed the consistency of gray and white matter regions. The team used Spatiale and MIA to analyze the spatial enrichment of three regional cell types (l2/3, l3/5, and oligo) in the aforementioned samples and found that Spatiale produced fewer false positives.

Spatial Enrichment of ALS-Related Genes in the Motor Cortex

Using Spatiale, spatial enrichment analysis of three human motor cortices and two DLPFC tissues revealed that 260 ALS-related genes were significantly enriched in the fifth layer (L5) of the motor cortex and DLPFC. Furthermore, heatmap analysis showed that NeFL (Neurofilament Light Chain) had the highest weighted differential expression among differentially expressed genes in the L5 cortex, consistent with the high expression regions of upper motor neurons and L5 excitatory neurons. In addition, Cell2Location analysis indicated a higher proportion of upper motor neurons and L5 excitatory neurons in ALS-related regions of the motor cortex.

Relationship Between NOMO1 Gene Mutation and ALS Risk

To evaluate the genetic link between other L5-related genes and ALS risk, WGS was performed, and the burden of rare loss-of-function variants was analyzed in 6,814 ALS patients and 3,324 controls for 1,050 common L5-related genes. The results indicated that the functional loss burden of the NOMO1 gene was significantly higher compared to the control group. RNA-Seq data analysis also showed downregulation of NOMO1 expression in ALS patients, suggesting that NOMO1 might increase ALS risk through a loss-of-function mechanism based on single case comparisons and case-control analyses.

Conclusion

By developing the new spatial enrichment tool Spatiale and integrating genomic data, this study expands the understanding of regional vulnerability in ALS. Spatiale demonstrated high specificity and accuracy by evaluating the specificity of marker genes for various cell types through weighted differential gene expression matrices, achieving excellent spatial enrichment performance in multiple human and mouse brain tissues. Furthermore, genetic analysis of L5 motor cortex-related genes provided evidence for NOMO1 as a novel ALS-related gene and revealed a potential loss-of-function mechanism for NOMO1 downregulation in ALS. This comprehensive ST and genomic analysis approach offers new perspectives and methodologies for further exploration of regional vulnerability in ALS.