Language

English

Publication Date

5-22-2025

Journal

Genome Medicine

DOI

10.1186/s13073-025-01467-z

PMID

40400026

PMCID

PMC12096736

PubMedCentral® Posted Date

5-22-2025

PubMedCentral® Full Text Version

Post-print

Abstract

Background: Only half of individuals with suspected rare diseases receive a genetic diagnosis following genomic testing. A genetic diagnosis allows access to appropriate care, restores reproductive confidence and reduces the number of potentially unnecessary interventions. A major barrier is the lack of disease agnostic functional tests suitable for implementation in routine diagnostics that can provide evidence supporting pathogenicity of novel variants, especially those refractory to RNA sequencing.

Methods: Focusing on mitochondrial disease, we describe an untargeted mass-spectrometry based proteomics pipeline that can quantify proteins encoded by > 50% of Mendelian disease genes and > 80% of known mitochondrial disease genes in clinically relevant sample types, including peripheral blood mononuclear cells (PBMCs). In total we profiled > 90 individuals including undiagnosed individuals suspected of mitochondrial disease and a supporting cohort of disease controls harbouring pathogenic variants in nuclear and mitochondrial genes. Proteomics data were benchmarked against pathology accredited respiratory chain enzymology to assess the performance of proteomics as a functional test. Proteomics testing was subsequently applied to individuals with suspected mitochondrial disease, including a critically ill infant with a view toward rapid interpretation of variants identified in ultra-rapid genome sequencing.

Results: Proteomics testing provided evidence to support variant pathogenicity in 83% of individuals in a cohort with confirmed mitochondrial disease, outperforming clinical respiratory chain enzymology. Freely available bioinformatic tools and criteria developed for this study ( https://rdms.app/ ) allow mitochondrial dysfunction to be identified in proteomics data with high confidence. Application of proteomics to undiagnosed individuals led to 6 additional diagnoses, including a mitochondrial phenocopy disorder, highlighting the disease agnostic nature of proteomics. Use of PBMCs as a sample type allowed rapid return of proteomics data supporting pathogenicity of novel variants identified through ultra-rapid genome sequencing in as little as 54 h.

Conclusions: This study provides a framework to support the integration of a single untargeted proteomics test into routine diagnostic practice for the diagnosis of mitochondrial and potentially other rare genetic disorders in clinically actionable timelines, offering a paradigm shift for the functional validation of genetic variants.

Keywords

Humans, Mitochondrial Diseases, Proteomics, Rare Diseases, Genetic Variation, Leukocytes, Mononuclear, Genetic Testing, Female, Male, Proteomics, Genetic diagnostics, Mendelian disease, Ultra-rapid genome sequencing, Variant prioritisation

Published Open-Access

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