Heavy Chain Fibrillary Glomerulonephritis: A Case Report

Case Report

Heavy Chain Fibrillary Glomerulonephritis: A Case Report Samih H. Nasr,* Christophe Sirac,* Frank Bridoux, Vincent Javaugue, Sebastien Bender, Alexia Rinsant, Sihem Kaaki, Emilie Pinault, Surendra Dasari, Mariam P. Alexander, Samar M. Said, Jonathan J. Hogan, Angela Dispenzieri, Guy Touchard, Ellen D. McPhail, and Nelson Leung Heavy chain amyloidosis and heavy chain deposition disease are the only known kidney diseases caused by the deposition of truncated immunoglobulin heavy chains. Fibrillary glomerulonephritis typically results from deposition of DNAJB9 (DnaJ heat shock protein family [Hsp40] member B9) and polytypic immunoglobulin G (IgG). We describe a patient with monoclonal gammopathy (IgG with λ light chain) who developed DNAJB9-negative fibrillary glomerulonephritis leading to end-stage kidney disease, with recurrence in 2 kidney allografts. Pre- and postmortem examination showed glomerular deposition of Congo red–negative fibrillar material that was determined to be immunoglobulin heavy chain. We propose the term “heavy chain fibrillary glomerulonephritis” to describe this lesion, which appears to be a rare kidney complication of monoclonal gammopathy. The diagnosis should be suspected when the kidney biopsy shows fibrillary glomerulonephritis with negative staining for immunoglobulin light chains and DNAJB9; the diagnosis can be confirmed using immunochemical and molecular studies.

Introduction Fibrillary glomerulonephritis (GN) is a rare disease encountered in 0.5% to 1% of native kidney biopsies. Patients typically present with hematuria, proteinuria, and decreased kidney function. Most cases are idiopathic, but a minority are associated with autoimmune diseases, hepatitis C virus infection, solid malignancy, or monoclonal gammopathy.1 Prognosis is poor because about half the patients reach end-stage kidney disease within 4 years. Diagnosis requires glomerular deposition of fibrils that are randomly oriented, lack hollow centers, measure <30 nm in thickness, are Congo red negative, and stain with antisera for immunoglobulins using immunofluorescence.1 Recently, a novel tissue biomarker for fibrillary GN, DNAJB9 (DnaJ heat shock protein family [Hsp40] member B9), was identified, having sensitivity of 98% and specificity > 99%.2-4 Glomerular deposits are typically polytypic, staining for immunoglobulin G (IgG) and κ and λ light chains, although in 4% to 11% of fibrillary GN cases, the deposits are monotypic (ie, stain with IgG and 1 light chain), with or without associated circulating monoclonal protein. Furthermore, rare cases of fibrillary GN in which the deposits stained for IgG only (without κ or λ light chain) have been reported, including one with concurrent light chain deposition disease and another with heavy chain deposition disease (HCDD), but the immunochemical and molecular characteristics of the immunoglobulins were not analyzed.1,5,6 We report a case of fibrillary GN in which the deposits are composed of a γ1 heavy chain with a complete variable region (VH) deletion without codeposition of κ or λ chain or DNAJB9. Case Report A 50-year-old man presented with peripheral edema and heavy proteinuria. Kidney biopsy revealed fibrillary GN AJKD Vol XX | Iss XX | Month 2019

Complete author and article information provided before references. *S.H.N. and C.S. contributed equally to this work. Am J Kidney Dis. XX(XX):15. Published online Month X, XXXX. doi: 10.1053/ j.ajkd.2019.01.032

© 2019 by the National Kidney Foundation, Inc.

and bone marrow biopsy showed 7% to 10% monoclonal plasma cells expressing IgG and λ light chain. He became dialysis dependent after 1 year despite multiple therapies, including melphalan, prednisone, dexamethasone, and interferon alfa. After 3 years of hemodialysis, he received a cadaveric kidney transplant at the Mayo Clinic that failed 2 years later from biopsy-proven recurrent fibrillary GN despite therapy with prednisone, rituximab, and subsequently thalidomide. Repeat bone marrow biopsy showed 10% to 20% monoclonal plasma cells expressing IgG and λ light chain. A living donor kidney transplant 5 years after the first transplant also failed after 3 years due to biopsyproven recurrent fibrillary GN. The patient remained on dialysis for another 4 years until his death from sepsis secondary to suppurative peritonitis and colon perforation. Comorbid conditions included hypertension, coronary artery disease, and recurrent episodes of inflammatory oligoarthritis. At the time of presentation, laboratory evaluation revealed the following results: serum albumin, 3.2 g/dL; proteinuria with protein excretion of 3.8 g/d; hematuria; hemoglobin, 10.5 g/dL; and C3 and C4 levels within the reference ranges. Serologic tests for hepatitis C virus and human immunodeficiency virus (HIV), testing for cryoglobulin, bone survey for lytic lesions, and fat, gastrointestinal, and endomyocardial biopsies for amyloid were all negative. Detailed methods for further investigations are provided in Item S1. Serum protein electrophoresis with immunofixation detected a small monoclonal IgG fragment and a small monoclonal IgG λ light chain (IgGλ) that was unquantifiable. Urine protein electrophoresis with immunofixation detected a small monoclonal IgG fragment (97 mg/24 h) and monoclonal λ light chain. Peak serum λ free light 1

Case Report chain level was 41.5 mg/dL with a depressed κ:λ ratio of 0.11. Bone marrow biopsies (n = 6) showed a λ light chain–restricted plasma cell population representing 5% to 40% of cellularity and were negative for amyloid. Standard pathologic analysis of the kidney biopsies (native and 2 allografts with recurrent fibrillary GN) and autopsy showed similar glomerular findings on light microscopy, immunofluorescence, and electron microscopy. On light microscopy, mesangial areas were expanded by weakly periodic acid–Schiff–positive, silver-negative, and Congo red–negative immune material, associated with variable degrees of mesangial hypercellularity (Fig 1A and B). Similar material segmentally thickened glomerular basement membranes (GBMs; Fig 1B). Immunofluorescence performed on frozen tissue of all biopsy specimens and on pronase-digested paraffin tissue on allograft biopsy specimens and autopsy tissue showed bright smudgy mesangial segmental GBMs and focal vascular staining for IgG (Fig 1C) with negative staining for IgA, C1q, and κ and λ light chain; moderate staining for C3; and trace staining for IgM. Glomerular deposits stained brightly for IgG1 subclass (Fig 1D), weakly for IgG4, and negative for

IgG2 and IgG3. They stained for second heavy chain constant domain (CH2) but not for CH1. On electron microscopy, there was mesangial expansion by randomly oriented fibrils that measured 13 nm in thickness (range, 9-16 nm; Figs 1E and F and S1). Similar fibrils segmentally involved the GBM (Fig 1F). There was no linear staining of tubular basement membranes using immunofluorescence or punctate deposits along the GBM and tubular basement membrane using electron microscopy. Postmortem examination did not show evidence of amyloidosis (with negative Congo red stain on sections of native kidneys, transplanted kidney, heart, lungs, liver, large bowel, and bone marrow; negative serum amyloid P immunostaining done on a section of transplanted kidney), or evidence of extrarenal involvement by fibrillary GN (no extracellular deposits seen in the heart or other organs, negative pronase immunofixation staining for IgG on sections of heart, liver, and lungs). Using liquid chromatography–tandem mass spectrometry, glomerular deposits contained high numbers of spectra for γ1 heavy chain (n = 220), apolipoprotein E, and apolipoprotein A4, with few spectra for γ4 heavy

Figure 1. Kidney biopsy findings. (A) The glomerulus shows global mesangial expansion (large arrows) and mild mesangial hypercellularity (small arrow) (periodic acid–Schiff; original magnification, ×400). (B) Another glomerulus shows global mesangial expansion by silver-negative material associated with mild mesangial hypercellularity (large arrows). Similar material segmentally involves and thickens the glomerular basement membrane (small arrow) (silver stain; original magnification, ×600). (C) Low-power view shows diffuse and global smudgy glomerular (large arrows) and focal vascular (small arrow) staining for immunoglobulin G (IgG; immunofluorescence image; original magnification, ×100). Staining for IgA and κ and λ light chain was negative. (D) Glomerular deposits stain brightly for IgG1 (arrows) (immunofluorescence image; original magnification, ×200). (E) The figure shows a mesangial area expanded by randomly oriented nonbranching fibrils (arrows) (electron microscopy; original magnification, ×25,000). (F) Fibrils segmentally extend to the glomerular basement membrane (arrows) without forming spicular projections into the urinary space (electron microscopy; original magnification, ×50,000).

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Case Report chain (n = 5). There were only few spectra for SAP (below our clinical threshold for amyloidosis). No spectra for DNAJB9 or κ or λ light chain were detected. The glomerular negativity for DNAJB9 and SAP was confirmed using immunohistochemistry. Polymerase chain reaction using a leader VH1-specific primer that was performed on complementary DNA prepared from bone marrow samples revealed a truncated heavy chain sequence. No other truncated heavy chain was detected. The size of the polymerase chain reaction product corresponded to a deletion of w400 base pairs compared to full-length heavy chain. The corresponding sequences showed a complete absence of the VDJ

(variable-diversity-joining) exon, but the presence of the entire CH1 domain from a γ1 heavy chain (IGHG1*03). The VH1 leader exon (from the VH1-45*01 germline gene) was found to be directly spliced to the γ1 CH1 exon, leading to an in-frame VDJ-less heavy chain (Fig 2A and B) with a theoretical isoelectric point of 7.2. We did not obtain any sequence corresponding to a CH1-deleted γ1 chain in our analysis. Immunofixation with antibodies to γ, γ1, γ2, γ3, γ4, κ, λ, and γ CH1 and CH2 revealed an isolated monoclonal γ1 chain that stained for CH2 but not CH1, similar to immunofluorescence results on the kidney biopsy (Fig S2). Western blot showed a truncated γ1 with an apparent

A

H

L

VDJ

WT γ1

B

CH1

CH2

CH3

C

FGN HC γ1

75 kDa

-------Leader----------------------------------------------------------------------------atcctcttcttggtggcagcagtcacagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggc I L F L V A A V T A S T K G P S V F P L A P S S K S T S G G

37 kDa

50 kDa 25 kDa

-----------------------------------------------------------------------------------------acagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcac T A A L G C L V K D Y F P E P V T V S W N S G A L T S G V H

-------------------------------------------CH1-------------------------------------------accttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctac T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y

HC FGN

Control

---------------------------------------------------------------------------Hinge---------atctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccg I C N V N H K P S N T K V D K R V E P K S C D K T H T C P P -----------------------------------------------------------------------------------------tgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgag C P A P E L L G G P S V F L F P P K P K D T L M I S R T P E

-----------------------------------------------------------------------------------------gtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaag V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K ------------------------------------------CH2--------------------------------------------acaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggag T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E -----------------------------------------------------------------------------------------tacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtg Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V -------------------------------------------CH3-------------------------------------------tacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgcc Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A

-----------------------------------------------gtggagtgggagagcaatgggcagccggagaacaactacaagaccaca V E W E S N G Q P E N N Y K T T

Figure 2. Molecular and Western blot analysis findings. (A) Schematic representation of the fibrillary glomerulonephritis (FGN) heavy chain (HC) as compared to a normal full-length γ1 HC. (B) Sequence of the HC from the patient with FGN. Nucleotide sequences are indicated together with the deduced amino acid sequence. Immunoglobulin domains are separated by vertical lines. Bold underlining indicates the peptides in the constant domain 1 (CH1) detected using serum mass spectrometry (2 different peptides). Of note, the leader (L) and CH3 sequences are not complete due to the design of the primers. (C) Western blot on denatured serum from the patient with FGN and a control serum was probed using a polyclonal anti-human γ antibody. Molecular weights are indicated on the left. A truncated HC of about 30 to 35 kDa is prominent in serum from the patient with FGN. Abbreviations: H, hinge; VDJ, variablediversity-joining domain; WT, wild type.

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Supplementary Material Supplementary File (PDF) Figure S1: High-power electron microscopic image shows mesangial randomly oriented fibrils. Figure S2: Immunoblotting electrophoresis.

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Item S1: Detailed methods. Table S1: Proteomic analysis of serum. Table S2: Pathologic characteristics of kidney lesions caused by deposition of truncated immunoglobulin heavy chains.

Article Information Authors’ Full Names and Academic Degrees: Samih H. Nasr, MD, Christophe Sirac, PhD, Frank Bridoux, MD, PhD, Vincent Javaugue, MD, Sebastien Bender, MS, Alexia Rinsant, MS, Sihem Kaaki, MS, Emilie Pinault, MS, Surendra Dasari, PhD, Mariam P. Alexander, MD, Samar M. Said, MD, Jonathan J. Hogan, MD, Angela Dispenzieri, MD, Guy Touchard, MD, PhD, Ellen D. McPhail, MD, and Nelson Leung, MD. Authors’ Affiliations: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN (SHN, MPA, SMS, EDM); Department of Immunology, National Center for Scientific Research, Joint Research Unit 7276, University of Limoges, Centre de r ef erence de l’amylose AL et des autres maladies par d epots d’immunoglobuline monoclonale, Limoges (CS, VJ, SB); Department of Nephrology, Dialysis and Renal Transplantation, University Hospital of Poitiers, Centre de r ef erence de l’amylose AL et des autres maladies par d epots d’immunoglobuline monoclonale, Poitiers (FB, VJ, GT); Department of Pathology, University Hospital of Poitiers, Poitiers, France (AR, SK); University of Limoges, BISCEm Mass Spectrometry Platform, F-87000 Limoges, France (EP); Department of Health Sciences Research, Mayo Clinic, Rochester, MN (SD); Division of Nephrology, Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA (JJH); and Division of Hematology, Mayo Clinic, Rochester, MN (AD, NL). ˇ

Discussion Histologic findings using routine light microscopy, immunofluorescence, and electron microscopy in the current case fulfil the established diagnostic criteria for fibrillary GN. However, this case is atypical because glomerular deposits did not stain for DNAJB9 or κ or λ light chain. The kidney deposits were consistently Congo red negative and there was no clinical or histologic evidence of extrarenal amyloidosis pre- or postmortem, arguing against heavy chain amyloidosis.7 HCDD was excluded due to the lack of tubular basement membrane involvement and the fibrillar organization of glomerular deposits (as opposed to the punctate-powdery deposits that would be expected; Table S2).8 The molecular findings in this case are distinct from HCDD and heavy chain amyloidosis. HCDD consistently shows deletion of CH1 (with or without CH2 deletion) and an intact VH domain and deposits generally have a high isoelectric point.9-11 Likewise, most reported cases of heavy chain amyloidosis are composed of VH or a fragment of it with deletion of CH1 (with or without deletion of CH2).12-14 In contrast, heavy chain sequencing in our patient showed a complete VH deletion with intact CH domains and a neutral isoelectric point. VH deletions (most commonly associated with complete deletion of CH1) are the hallmark of γ heavy chain disease,15 but organ deposition does not occur in this condition. Therefore, the constellation of clinicopathologic and molecular characteristics in this case do not conform to any of the previously described conditions associated with structurally abnormal immunoglobulin heavy chains. We propose the term “heavy chain fibrillary GN” to describe this lesion, which appears to be a rare kidney complication of monoclonal gammopathy. Intriguingly, the VH-deleted γ1 in glomerular deposits and serum in this patient did not stain for CH1 despite the presence of an intact CH1 based on sequence analysis and serum proteomic studies. Although we cannot fully explain this finding, we speculate that the absence of VH domain may have resulted in a protein conformational change that impeded the binding of antibodies to the CH1 domain. Such conformational change could also be related to the propensity of the truncated heavy chain to aggregate into a fibrillary structure.

We recommend screening for autoimmune diseases, solid malignancy, paraproteinemia, and hepatitis C virus in all patients with fibrillary GN. The heavy chain variant should be suspected histologically when glomerular deposits stain with IgG but not with κ, λ, or DNAJB9. Investigation in suspected cases should include serum and urine protein electrophoresis/immunofixation, serum free light chain assay, and bone marrow biopsy. Confirmation of diagnosis requires demonstration of a truncated immunoglobulin heavy chain using serum Western blotting and/or bone marrow heavy chain sequencing. In HCDD, bortezomib-based therapies improve kidney prognosis, and serum free light chain assay can be used to monitor hematologic response.9 Whether this also applies to heavy chain fibrillary GN requires further studies.

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molecular weight of about 30 to 35 kDa (expected for a VH deletion; Fig 2C). Proteomic studies conducted on the corresponding band revealed a high number of spectra for a γ1 heavy chain without detectable VH domains (Table S1). Peptides corresponding to the CH1 domain were detected in our analysis with a confidence >99% (peptides are indicated on Figure 2B). As a control, peptides containing VH domain sequences were readily detected in the fragment corresponding to the full-length heavy chain (about 50-55 kDa).

Address for Correspondence: Samih H. Nasr, MD, Mayo Clinic, 200 First St SW, Hilton 10-20, Rochester, MN 55905 (e-mail: [email protected]) or Christophe Sirac, PhD, Centre National de la recherche Scientifique UMR CNRS 7276/INSERM U1262, 2, Rue du Dr. Marcland 87000, Limoges, France (e-mail: christophe. [email protected]).

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Case Report Support: This work was supported in part by funding from Department of Laboratory Medicine and Pathology at the Mayo Clinic (Dr Nasr) and a grant from Ligue contre le cancer Limousin (Dr Sirac).

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Financial Disclosure: The authors declare that they have no relevant financial interests. Peer Review: Received September 21, 2018. Evaluated by 3 external peer reviewers and a pathologist, with direct editorial input from an Associate Editor, who served as Acting Editor-inChief. Accepted in revised form January 21, 2019. The involvement of an Acting Editor-in-Chief was to comply with AJKD’s procedures for potential conflicts of interest for editors, described in the Information for Authors & Journal Policies.

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disease in a patient with monoclonal gammopathy. Ann Clin Lab Sci. 2007;37(4):370-374. Nasr SH, Said SM, Valeri AM, et al. The diagnosis and characteristics of renal heavy-chain and heavy/light-chain amyloidosis and their comparison with renal light-chain amyloidosis. Kidney Int. 2013;83(3):463-470. Nasr SH, Valeri AM, Cornell LD, et al. Renal monoclonal immunoglobulin deposition disease: a report of 64 patients from a single institution. Clin J Am Soc Nephrol. 2012;7(2): 231-239. Bridoux F, Javaugue V, Bender S, et al. Unravelling the immunopathological mechanisms of heavy chain deposition disease with implications for clinical management. Kidney Int. 2017;91(2):423-434. Moulin B, Deret S, Mariette X, et al. Nodular glomerulosclerosis with deposition of monoclonal immunoglobulin heavy chains lacking C(H)1. J Am Soc Nephrol. 1999;10(3): 519-528. Aucouturier P, Khamlichi AA, Touchard G, et al. Brief report: heavy-chain deposition disease. N Engl J Med. 1993;329(19): 1389-1393. Eulitz M, Weiss DT, Solomon A. Immunoglobulin heavy-chainassociated amyloidosis. Proc Natl Acad Sci U S A. 1990;87(17):6542-6546. Solomon A, Weiss DT, Murphy C. Primary amyloidosis associated with a novel heavy-chain fragment (AH amyloidosis). Am J Hematol. 1994;45(2):171-176. Yazaki M, Fushimi T, Tokuda T, et al. A patient with severe renal amyloidosis associated with an immunoglobulin gamma-heavy chain fragment. Am J Kidney Dis. 2004;43(5): e23-e28. Cogne M, Silvain C, Khamlichi AA, Preud’homme JL. Structurally abnormal immunoglobulins in human immunoproliferative disorders. Blood. 1992;79(9):2181-2195.

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