MSACL 2018 US Abstract

Topic: Proteomics

Proteomics Identifies DNAJB9 as a Pathogenic Protein in Fibrillary Glomerulonephritis

Surendra Dasari (Presenter)
Mayo Clinic

Bio: Dr. Surendra Dasari is an assistant professor of biomedical informatics at Mayo Clinic. He specializes in developing informatics solutions for proteomics-based diagnostic assays. His informatics solutions are being used to support the amyloid typing assay and MALDI-based monoclonal gammopathy screening assay at the Mayo Clinic. Dr. Dasari also actively works with collaborators in the areas of aging, insulin resistance, molecular response to exercise training, and molecular pathogenesis of anaplastic large cell lymphomas.

Authorship: Surendra Dasari(1), Mariam P. Alexander(2), Jason D. Theis(2), Julie A. Vrana(2), Samih H. Nasr(2), Paul J. Kurtin(2)
(1) Department of Health Sciences Research, Mayo Clinic, Rochester, MN (2) Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN

Short Abstract

Fibrillary glomerulonephritis (FGN) is a rare primary glomerular disease with unknown pathogenesis. A proteomic analysis of FGN glomeruli in patient biopsies detected six-fold overexpression of DNAJB9 protein when compared to amyloid glomeruli. DNAJB9 was not detected in glomeruli of healthy subjects and 19 types of non-FGN glomerular diseases. This highlights 100% sensitivity and 100% specificity of DNAJB9 as an FGN biomarker. Additional experiments showed that DNAJB9 was deposited extracellularly, localized to fibrils, and co-localized with Ig-gamma chains in FGN glomeruli. With this evidence, we propose that DNAJB9 is a strong biomarker for rapid diagnosis of FGN in renal biopsies.

Long Abstract

Introduction

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease seen in 0.5-1% of native kidney biopsies. FGN patients present with proteinuria, hematuria, renal impairment, and hypertension. Most cases are idiopathic, although a third of the cases occur in association with hepatitis C infection, dysproteinemia, or autoimmune diseases. FGN has a poor prognosis with nearly 50% of patients progressing to end-stage renal disease (ESRD) within 4 years despite therapeutic interventions. The pathogenesis of FGN is unknown. Traditionally, an FGN diagnosis relies on integrating ultrastructural, immunofluorescence (IF) and light microscopic (LM) findings: a time consuming and expensive process. Also, the pathologic features of FGN overlap with other glomerular diseases, like immunotactoid and lupus nephritis, creating diagnostic grey zones. To date, there is no unique biomarker for diagnosing FGN. Further, there are no targeted therapies for FGN patients due to lack of information on the pathogenic protein underlying the disease, which is the underlying cause for the poor prognosis of these patients.

Methods

We assembled a large renal biopsy cohort containing 253 cases to study the proteomic characteristics of FGN in search of an FGN-specific diagnostic marker. This cohort contained N=24 FGN cases, N=145 renal amyloidosis cases, N=72 cases of 19 different types of non-FGN glomerular diseases (NFGNGDs), and 12 healthy controls. A 10-micrometer thick serial section was obtained from each case and renal glomeruli were laser microdissected (LMD). Two independent dissections of 60,000 square micrometers were performed and proteins present in the FFPE fragments of each dissection were subjected to shotgun proteomics analysis. Resulting MS/MS data were analyzed using a previously described bioinformatics pipeline[1] to find proteins that uniquely overexpressed in FGN samples when compared to the rest of the cohort.

Results

We detected a more than six-fold overexpression of DNAJB9 protein in FGN glomeruli when compared to amyloid glomeruli (FDR corrected p-value=3.79E-286). DNAJB9 was detected in all FGN cases and it was not detected in glomeruli of healthy subjects and 19 types of NFGNGDs. This highlights 100% sensitivity and 100% specificity of DNAJB9 as an FGN marker. DNAJB9 went from being present at trace, undetectable, levels in normal glomeruli to the fourth most abundant protein in FGN glomeruli. Immunohistochemistry (IHC) of DNAJB9 in FGN cases showed that the protein was deposited extracellularly. Immunoelectron microscopy (IEM) of DNAJB9 showed that the extracellular protein is present in the fibrils. Dual label immunofluorescence (IF) for DNAJB9 and Ig-gamma showed co-localization of DNAJB9 and Ig-gamma, suggesting its role in autoantigenecity (a known feature of FGN). All of these data are suggestive of DNAJB9’s role in FGN pathogenesis.

Conclusions & Discussion

The pathogenic protein in FGN has not been identified since the disease was first defined in 1977. Based on the evidence of this study, we propose that DNAJB9 is a strong biomarker for rapid diagnosis of FGN in renal biopsies. Mass spectrometry can readily distinguish FGN from amyloidosis and other pathologically similar renal diseases. A simple DNAJB9 IHC-based diagnostic test could be used to simplify the current clinical algorithms for diagnosing FGN. Further, this biomarker would be extremely useful for diagnosing FGN in the developing world where instrumentation needed for mass spectrometry or ultrastructural evaluation is either limited or non-existent. Finally, the identification of DNAJB9 as pathogenic protein in FGN would enable the development of targeted therapies for these patients and improve their outcomes.


References & Acknowledgements:

1. Theis JD, Dasari S, Vrana JA, Kurtin PJ, Dogan A. Shotgun-proteomics-based clinical testing for diagnosis and classification of amyloidosis. Journal of mass spectrometry : JMS. 2013;48(10):1067-1077. Prepublished on 2013/10/17 as DOI 10.1002/jms.3264.


Financial Disclosure

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