Background and hypothesis Hundreds of protein-coding genes still have unknown functions and no biological pathway annotations. Mechanistic studies typically investigate well-known genes, despite growing evidence of associations between disease and some understudied genes. We hypothesized that examining these overlooked genes lacking pathway annotation could lead to new insights on chronic kidney disease (CKD) pathogenesis.

Methods Human protein-coding genes (N=19,258) from HGNC were annotated with pathway membership using a comprehensive collection of pathway databases (including but not limited to GO, KEGG, MSigDB, Reactome, WikiPathways) to reveal ‘pathway-orphan genes’— genes that are not members of any biological pathways. Expression and enrichment of pathway-orphan genes in healthy kidneys were established using GTEx data. Kidney-expressed pathway-orphan genes were tested for differential case-control expression using publicly available CKD datasets with kidney tissue RNA-seq transcriptomics profiling (GSE98422, GSE142025, GSE175759, GSE197307, Levin et al., 2020).

Results A total of 286 genes in the human genome currently lack any biological pathway membership and are identified as pathway-orphan genes. We have determined 97 of these pathway-orphan genes are expressed in healthy kidneys, with several showing kidney-specific enrichment. Furthermore, 34 pathway-orphan genes show significant modulation of expression (FDR < 0.05) in CKD kidney, out of which 10 robustly demonstrate concordant directionality of change in more than one RNA-seq study. Through interrogating multiple lines of evidence, we showed how possible physiological functions of the pathway-orphan genes in kidney health and disease can be inferred.

Conclusion A substantial number of kidney-expressed genes remain ‘pathway-orphan’ while displaying clear signals of kidney relevance, such as enrichment of expression in the kidney, significant modulation in CKD, and genetic associations with kidney function. Directing mechanistic studies into this overlooked gene group might broaden our biological understanding of kidney physiology and highlight novel disease drivers.

DA, SG, and AR are AstraZeneca employees. DA is an industrial PhD student at AstraZeneca.

This research was supported by the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 860977 titled TrainCKDis.

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