Abstract
Neutrophil granulocytes are critical mediators of innate immunity and tissue regeneration. Rare diseases of neutrophil granulocytes may affect their differentiation and/or functions. However, there are very few validated diagnostic tests assessing the functions of neutrophil granulocytes in these diseases. Here, we set out to probe omics analysis as a novel diagnostic platform for patients with defective differentiation and function of neutrophil granulocytes. We analyzed highly purified neutrophil granulocytes from 68 healthy individuals and 16 patients with rare monogenic diseases. Cells were isolated from fresh venous blood (purity >99%) and used to create a spectral library covering almost 8000 proteins using strong cation exchange fractionation. Patient neutrophil samples were then analyzed by data-independent acquisition proteomics, quantifying 4154 proteins in each sample. Neutrophils with mutations in the neutrophil elastase gene ELANE showed large proteome changes that suggest these mutations may affect maturation of neutrophil granulocytes and initiate misfolded protein response and cellular stress mechanisms. In contrast, only few proteins changed in patients with leukocyte adhesion deficiency (LAD) and chronic granulomatous disease (CGD). Strikingly, neutrophil transcriptome analysis showed no correlation with its proteome. In case of two patients with undetermined genetic causes, proteome analysis guided the targeted genetic diagnostics and uncovered the underlying genomic mutations. Data-independent acquisition proteomics may help to define novel pathomechanisms in neutrophil diseases and provide a clinically useful diagnostic dimension.
Highlights
14/16 patients had a genetically confirmed molecular diagnosis of neutrophil deficiency (CGD: n ϭ 5, Severe congenital neutropenia (SCN): n ϭ 6, leukocyte adhesion deficiency (LAD): n ϭ 3), in two patients routine genetic workup by exome sequencing did not yield a specific defect in any gene known to cause congenital neutropenia or chronic granulomatous disease (CGD)
Proteome Analysis Guiding Molecular Diagnosis—Two patients in our cohort had clinical signs and symptoms of neutrophil granulocyte deficiency (one patient with CGD confirmed by negative nitro blue tetrazolium (NBT) test and one patient with congenital neutropenia associated with albinism) but exome sequencing analyses did not yield a molecular diagnosis
We looked at neutrophil proteome changes in patients with monogenic diseases
Summary
Data-independent acquisition proteomics was used to study proteome changes of naive human neutrophils in rare monogenic diseases affecting their functions. Data-independent acquisition proteomics analysis of naive neutrophils from patients with rare monogenic diseases. Author’s Choice los Proteome Analysis of Human Neutrophil Granulocytes From Patients With Monogenic Disease Using Data-independent Acquisition*□S. Data-independent acquisition proteomics may help to define novel pathomechanisms in neutrophil diseases and provide a clinically useful diagnostic dimension. To capture the complexities of diseases and to increase the diagnostic yield, additional omics technologies and data integration may be useful This has recently been shown for strategies associating genome and RNA-sequencing in rare neuromuscular diseases [8, 9]. We here systematically analyze proteome changes in neutrophils from patients with different monogenic diseases and demonstrate the usefulness of nextgeneration proteomics for guiding clinical genetic diagnostics
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