Novel Type of Renal Amyloidosis Derived from Apolipoprotein-CII

BRIEF COMMUNICATION www.jasn.org

† Samih H. Nasr,* Surendra Dasari, Linda Hasadsri,* Jason D. Theis,* Julie A. Vrana,* ‡ † Morie A. Gertz, Prasuna Muppa,* Michael T. Zimmermann, Karen L. Grogg,* ‡ Angela Dispenzieri,* Sanjeev Sethi,* W. Edward Highsmith Jr.,* Giampaolo Merlini,§ ‡ Nelson Leung, and Paul J. Kurtin*

Departments of *Laboratory Medicine and Pathology, †Health Sciences Research, and ‡Internal Medicine, Mayo Clinic, Rochester, Minnesota; and §Istituto di Ricovero e Cura a Carattere Scientiﬁco Policlinico San Matteo and Department of BRIEF COMMUNICATION Molecular Medicine, University of Pavia, Pavia, Italy

ABSTRACT Amyloidosis is characterized by extracellular deposition of misfolded proteins as chains by immunofluorescence (IF). A fat insoluble fibrils. Most renal amyloidosis cases are Ig light chain, AA, or leukocyte aspirate was negative for amyloid. Serum- chemotactic factor 2 amyloidosis, but rare hereditary forms can also involve the free k/l light chain ratio was 3.9. Cardiac kidneys. Here, we describe the case of a 61-year-old woman who presented with biomarkers were within normal range. nephrotic syndrome and renal impairment. Examination of the renal biopsy spec- Imaging studies revealed a normal liver imen revealed amyloidosis with predominant involvement of glomeruli and medul- andspleenonultrasound,anegative lary interstitium. Proteomic analysis of Congo red–positive deposits detected large bone survey, and a negative positron amounts of the Apo-CII protein. DNA sequencing of the APOC2 gene in the patient emission tomography scan, other than and one of her children detected a heterozygous c.206A→T transition, causing an metatarsal uptake. The paraffinblock E69V missense mutation. We also detected the mutant peptide in the proband’s was subsequently sent for amyloid typing renal amyloid deposits. Using proteomics, we identified seven additional elderly by proteomics (laser microdissection patients with Apo-CII–rich amyloid deposits, all of whom had kidney involvement [LMD], liquid chromatography [LC], and histologically exhibited nodular glomerular involvement. Although prior in vitro and tandem mass spectrometry [MS/MS]; studies have shown that Apo-CII can form amyloid fibrils and that certain mutations LMD-LC-MS/MS). The patient was hos- in this protein promote amyloid fibrillogenesis, there are no reports of this type of pitalized, due to peripheral edema, 2 weeks amyloidosis in humans. We propose that this study reveals a new form of hereditary before her presentation to our hospital. amyloidosis (AApoCII) that is derived from the Apo-CII protein and appears to man- Our workup revealed a hemoglobin of ifest in the elderly and preferentially affect the kidneys. 11 g/dl, serum albumin of 2.6 g/dl, serum creatinine of 2.8 mg/dl, and 3.3 g/d of J Am Soc Nephrol 28: 439–445, 2017. doi: 10.1681/ASN.2015111228 proteinuria. Serum IFX revealed a small IgG-l M-protein whereas urine IFX was negative. Serum cholesterol, triglycerides, HDL, LDL, and non-HDL cholesterol Amyloidosis refers to a spectrum of rare of cases of renal amyloidosis are cur- diseases that are characterized by abnor- rently unclassified.9 mal extracellular deposition of misfolded A 61-year-old white female with a Received November 13, 2015. Accepted April 9, proteins in the form of insoluble fibrils. history of hypertension and hypothyroid- 2016.

A total of 31 amyloid precursor proteins ism was recently diagnosed with renal S.H.N. and S.D. contributed equally to this work. have been identified so far.1 Renal in- amyloidosis. Four months before, she was fi Published online ahead of print. Publication date volvement is common in AL, AA, and noted to have renal insuf ciency (serum available at www.jasn.org. ALect2 amyloidosis. However, it may creatinine of 2.6 mg/dl) and nephrotic- alsooccurinrarehereditaryformsof range proteinuria. Serum and urine im- Correspondence: Prof.PaulJ.Kurtin,MayoClinic, Laboratory Medicine and Pathology, 200 First Street 2 3 4 5 6 fi AFib, ATTR, AGel, ALys, AApoAI, muno xation(IFX)showedamonoclonal SW, Rochester, MN 55906. Email: kurtin.paul@ AApoAII,7 and AApoAIV8 amyloid- proteinthatcouldnotbequantified. A mayo.edu oses. Even with the availability of pro- renal biopsy showed amyloid deposits Copyright © 2017 by the American Society of teomics for amyloid typing, about 2% that were negative for Ig heavy and light Nephrology

J Am Soc Nephrol 28: 439–445, 2017 ISSN : 1046-6673/2802-439 439 BRIEF COMMUNICATION www.jasn.org were normal. There was no history of re- DNA sequencing of all three exons of On the basis of our experience with this nal disease in the patient’s parents, six sib- the APOC2 gene and found a heterozy- patient, we searched our amyloidosis data lings, or two children (son aged 22 years; gous c.206A→Talterationinexon3, archive for cases that had abundant Apo- daughter aged 23 years). which results in a glutamate to valine CII in the deposits. This archive contains The renal biopsy showed extensive substitution at codon 69 (E69V) (Figure clinical and proteomic data for a total of glomerular and medullary interstitial in- 3A). The same mutation was present in 10,167 amyloidosis cases, distributed be- volvement by acellular eosinophilic mate- the patient’s son (Figure 3B) but not in tween 17 subtypes and 30 different tissues, rial that stained positive with Congo red her daughter (data not shown). We also that were typed in our laboratory between (CR) and exhibited green birefringence detected the mutant peptide in the pa- 2008 and 2015. We identified seven addi- under polarized light (Figure 1). LMD- tient’s renal amyloid deposit (Figure tional cases, and all were initially reported LC-MS/MS performed on the CR-positive 3C), further strengthening the case for as “amyloidosis, indeterminate type” be- amyloid deposits detected Apo-CII and the mutation’s pathogenicity.11 We per- cause they showed both CR positivity and proteins commonly codeposited with formed generalized Born implicit solvent contained proteins commonly deposited amyloids of all types (ApoE, Apo-AIV, molecular dynamics (isMD) simulations with amyloids of all types10 but did not and SAP).10 We did not detect spectral to assess the effect of the mutation on contain any recognized amyloid precursor evidence for other amyloid precursor Apo-CII protein folding. The mutation proteins. Importantly, all of these cases proteins (Patient 1 in Figure 2A). The is located in the linker region and it alters were diagnosed from renal biopsies, sug- Apo-CII protein was also deposited in the native confirmation of the protein gesting that AApoCII primarily affects the entirety of the dissected glomeruli (Figure 3D), which could destabilize the kidneys. Figure 2A shows the Apo-CII ex- (Figure 2B). We performed germline protein and lead to amyloid formation. pression from four AApoCII cases and normal glomeruli from four healthy controls. Apo-CII was exclusively detected in the AApoCII glomeruli along with the ex- pected universal amyloid tissue biomarkers.10 We also wanted to rule out the possibility that Apo-CII can be codeposited in renal amyloid deposits of other types or be present in the glomeruli of patients with nonamyloid glomerular diseases. For this, we utilized normalized MS/MS spectral counts to estimate the relative abundance of Apo-CII protein in the glomerular amyloid deposits of all AApoCII cases (n=8), CR-positive deposits in kidneys of other amyloid subtypes (n=532), glomeruli of patients with nonamyloid glomerular disease (n=49), and normal glomeruli of healthy individ- uals (n=10). As shown in Figure 2C, we detected Apo-CII protein only in amyloidosis cases of AApoCII type. In addition, we performed immunohistochemistry on paraffin sections of three kidney specimens from the AApoCII cases and on seven cases of renal amyloidosis of other types (AL-l, n=2; AL-k, n=1; AA, n=1; AApoAIV, n=3). The glomerular amyloid deposits in the AApoCII cases were all strongly positive for Apo-CII. None of the amyloid deposits in the other Figure 1. Light microscopy of AApoCII renal deposits (patient 1). (A) Hematoxylin and eosin cases stained positively for Apo-CII staining of renal biopsy shows amorphous eosinophilic deposits in the glomeruli. (B) The (Supplemental Figure 1). glomerular deposits are CR-positive. (C) Apple-green birefringence of congophilic amyloid Our total cohort of AApoCII renal deposits using polarized light. (D) Extensive medullary interstitial involvement by CR-positive amyloidosis consisted of five females and amyloid deposits. Original magnification, 3100 in A; 3200 in B and C; 3100 in D. three males, all elderly at diagnosis (median

440 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 439–445, 2017 www.jasn.org BRIEF COMMUNICATION

Figure 2. Proteomic detection of Apo-CII in amyloid deposits from AApoC2 patients and from control patients with other renal diseases. (A) Scaffold display of the amyloid proteome from four patients with AApoCII (Patients 1–4) and from normal glomeruli from four healthy controls (Controls 1–4). Patient 1 represents the index case. Amyloid-related proteins are marked with stars and displayed at the top of the list (APOC2, blue star; other amyloid-associated proteins, yellow stars). The numbers displayed in the boxes in the vertical columns represent the total number of spectra matched to the listed protein. The colors of the boxes represent the probability that the spectra represent the identified protein (only spectra with 95% probability of a match to an identified protein [green boxes] are considered for diagnostic interpretation). In all patient samples there were abundant spectra for APOE, SAP, and APOAIV, providing biochemical evidence for the presence of amyloid in the microdissected sample. In addition, all patient samples contained many spectra corresponding to Apo-CII. The control kidney samples did not contain amyloid-associated proteins or Apo-CII. Structural (actin, vimentin, a-actinin-4) or serum/blood-related proteins (hemoglobin, serum albumin) were also detected in the specimens. (B) Portions of the Apo-CII sequence detected in the index sample are highlighted with bold black letters on yellow background. The first 22 amino acids in the sequence constitute the signal peptide, which is post-translationally clipped. (C) OAT stands for amyloid kidneys of other types (including AL, ALect2, AA, AH, AFib, AGel, AApoAIV, AApoAI, ATTR, and Ab2M). NAGD represents glomeruli of patients with nonamyloid glomerular diseases (dense deposit disease, C3 GN, fibrillary GN, immunotactoid GN, cryoglobulinemic GN, pauci-immune crescentic GN, trans- plant glomerulopathy, diabetic glomerulosclerosis, glomerular thrombotic microangiopathy, and fibronectin glomerulopathy). Spectral counts from each replicate dissection were normalized to account for batch variation and plotted independently. P values were computed using Mann–Whitney rank sum test.

J Am Soc Nephrol 28: 439–445, 2017 Novel Type of Renal Amyloidosis Derived from APOC-II 441 BRIEF COMMUNICATION www.jasn.org

Figure 3. APOC2 gene sequencing, mass spectrometry demonstration of corresponding amino acid sequence variant and molecular modeling of mutant Apo-CII. (A and B) APOC2 gene mutation in proband and kindred. An arrow marks the detected c.206A→T (p.E69V) mutation. (C) MS/MS spectrum of the mutant peptide detected in the CR-positive renal amyloid deposits of the proband. Values along the abscissa represent the mass-to- charge ratio (m/z) of the mutant peptide ions detected by mass spectrometry. The ordinate is the relative intensity of the peaks. The purple line at the top highlights the amino acid sequence of the mutant peptide oriented from left to right in the amino-terminus to the carboxy-terminus direction. The letters represent the individual amino acids (T, threonine; Y, tyrosine; L, leucine; P, proline; A, alanine; V, valine; D, aspartic acid; E, glutamic acid; and K, lysine). This sequence corresponds to the purple peaks in the MS/MS spectrum (b-ion series). The blue line at the top highlights the amino acid sequence of the mutant peptide oriented from left to right in the carboxy-terminus to the amino-terminus direction. This sequence corresponds to the blue peaks in the MS/MS spectrum (y-ion series). Arrows marks the amino acid sequence change corresponding to the genetic mutation. (D) Molecular dynamic trajectories of the wild-type (WT; gray) and mutant (gold) protein were assessed using radius of gyration. Mutant and WT protein conformational representatives with similar radii of gyration were shown. Mutant protein has different conﬁrmation when compared with WT.

442 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 439–445, 2017 www.jasn.org BRIEF COMMUNICATION age 70 years, range 61–86). Patients pre- mutations introduced into lipid and li- moderate; and 3, severe. The following clinical sented with proteinuria with or without poprotein lipase-binding portions of the definitions were used: proteinuria, .150 mg/d; renal insufficiency (Table 1). The median Apo-CII protein can promote amyloid nephrotic-range proteinuria, .3.0 g/d; ne- 24-hour urine protein was 3.4 g/d (range fibrillogenesis.21–23 The E69V mutation phrotic syndrome, nephrotic-range protein- 1.5–9.7). Proteinuria was in the nephrotic discovered in this study is in the linker uria, serum albumin ,3.5 g/dl, and peripheral range in 67% of patients and 43% had full region, and isMD simulations suggest edema; and renal insufficiency, serum creati- nephrotic syndrome. The median serum that the mutation can alter the confor- nine .1.2 mg/dl. creatinine at diagnosis was 1.8 mg/dl mation of the protein. Hence, it is pos- Immunohistochemistry for APOC2 was (range 0.9–2.6) and 57% of patients had sible that the E69V alteration mediates performed on paraffin sections of 11 kidney renal insufficiency (Table 1). Histologi- amyloid fibrillogenesis through destabi- biopsy specimens involved by amyloidosis cally, the glomeruli were affected in all ca- lization of the mutant Apo-CII protein. (AApoCII, n=3; AL-k, n=1; AL-l, n=2; AA, ses. Whereas the index patient showed Alternatively, the E69V mutation might n=1; AApoA4, n=3). Tissue sections were global glomerular involvement (Figure 1, alter the interaction between Apo-CII deparaffinized, subjected to citrate buffer A–C), the remaining seven cases ex- and lipids, sufficiently freeing the Apo-CII antigen retrieval, and incubated with rabbit hibited a distinctive nodular glomerular from the lipids such that amyloid forma- polyclonal anti-APOC2 antibodies (Abcam, involvement in which the mesangium tion can occur. Inc., Cambridge, MA; 1:2500 dilution), fol- was asymmetrically expanded by large lowed by incubation in a biotin-free, polymeric rounded masses of amyloid that en- horseradish peroxidase–linker antibody conju- croached on the capillary spaces (Supple- CONCISE METHODS gate system (Leica Biosystems, Buffalo Grove, mental Figure 2). This pattern can also be IL). Antibody localization was visualized using seen in AA amyloidosis.12 The massive The biopsy processing and diagnosis of renal diaminobenzidine and the sections were coun- obliterative glomerular involvement that amyloidosis in the eight cases was performed terstained with hematoxylin. is characteristic of AFib was not seen in in other institutions. Evaluation of renal bi- We utilized a previously established proteo- any case. In contrast to AL and AA amy- opsies included staining of light-microscopy mics method for typing the amyloid deposits.24 loidosis, vascular involvement was absent sections with hematoxylin and eosin, peri- For each case, CR-stained, formalin-fixed, (six cases) or minimal (two cases). In con- odic acid–Schiff, Masson trichrome, Jones paraffin-embedded tissue sections were ex- trast to ALect2 amyloidosis, cortical inter- methenamine silver, and CR; and electron amined to confirm the presence of amyloid. stitial involvement was absent (six cases) microscopy and standard IF staining for The amyloid type was identified using an or minimal (two cases). Renal medulla IgG, IgM, IgA, C3, C1q, albumin, fibrinogen, LMD-assisted LC-MS/MS method as previ- was sampled in five cases, two of which k,andl.Theparaffinblocksweresubse- ously described.24 Multiple independent showed medullary interstitial involve- quently sent to the Mayo Clinic for amyloid samples (replicates) were analyzed for each pa- ment (Figure 1D, Table 1). Prominent typing by proteomics (LMD-LC-MS/MS). tient. Data for each patient was processed medullary interstitial involvement is also The extent of amyloid deposits in the kidney, using a previously described bioinformatics common in amyloidosis derived from tubular atrophy and interstitial fibrosis, and pipeline and a patient-specific amyloid pro- other apolipoproteins (Apo-AI, Apo-AII, arteriosclerosis was evaluated by reviewing teome profile was created.25 For each patient and Apo-AIV). Electron microscopy theCRandhematoxylinandeosinslides sample, a pathologist reviewed the microdis- showed the typical ultrastructural appear- routinely prepared at the Mayo Clinic for section images to confirm that the amyloid ance of amyloid with randomly oriented specimens received for proteomic analysis. deposits were included in the LC/MS-MS- fibrils that measured 8–14 nm in thick- Glomerular amyloid deposits were scored analyzed tissue. After LC-MS/MS, the pro- ness (Supplemental Figure 3). In all cases, using a semiquantitative scale: 0, absent; 1, am- teomic results, as displayed using Scaffold amyloid deposits were negative for IgG, yloid deposits affecting ,25% of the glomer- software (Proteome Software, Portland, OR), IgA, k,andl,asassessedbyIF. ulus; 2, amyloid deposits affecting 25%–50% were reviewed (Figure 2A). The proteome pro- Apo-CII is a major constituent of chy- of the glomerulus; and 3, amyloid deposits af- file was first scrutinized for the presence of lomicrons, VLDL particles, and HDL fecting .50% of the glomerulus. The extent of proteins that are codeposited with amyloids particles. Apo-CII activates lipoprotein li- amyloid deposition in the cortical and medul- of all types (Apo-E, SAP, and Apo-AIV)10 to pase,13 which hydrolyzes triglycerides into lary interstitium was scored as: 0, absent; 1, verify that the basic biochemical signature of free fatty acids. Homozygous or compound ,25% of parenchyma involved; 2, 25%–50% amyloid was detected. Next, the proteome was heterozygous mutations in the APOC2 gene of parenchyma involved; and 3, .50% of searched for potential amyloid proteins. In all can cause hyperlipoproteinemia type IB,14 parenchyma involved. The extent of amyloid eight cases, previously described canonical characterized by hypertriglyceridemia.15 deposition in tubular basement membranes amyloid proteins were not identified in the In contrast, our index patient did not was scored as: 0, absent; 1, ,25% of renal proteome; rather, abundant spectra corre- have abnormal levels of lipids. Previous tubules involved; 2, 25%–50% of renal tu- sponding to Apo-CII were consistently de- in vitro studies have shown that lipid-free bules involved; and 3, .50% of renal tubules tected across all replicates. This protein was human Apo-CII can also form amyloid involved. The extent of amyloid deposition previously shown to be amyloidogenic in ani- fibrils.16–20 Researchers have shown that in arteries was scored as: 0, absent; 1, mild; 2, mal models and in vitro studies.16,17,19–23 Thus,

J Am Soc Nephrol 28: 439–445, 2017 Novel Type of Renal Amyloidosis Derived from APOC-II 443 BRIEF COMMUNICATION www.jasn.org

b Apo-CII was considered a potential primary amyloid precursor protein in these cases. 1+ No No No No Genetic evaluation for germline mutations Tubular Basement Membrane in the APOC2 gene was performed using a Involvement

b blood sample. All three exons of the APOC2 gene were ampliﬁed using hybrid primers con-

ciency are described – fi 2+ 3+ fi No No No taining 20 22 bases of a gene-speci c sequence Medullary Interstitial and a universal sequencing primer sequence Involvement (19 or 23 bases for the forward and reverse b primers) at the 59 end. Amplified products were sequenced using universal sequencing 1+ 1+ No No No MNS No No No MNS No No MNS No

Cortical primers, ABI BigDye terminators (Applied Interstitial

Involvement Biosystems, Foster City, CA), and capillary b electrophoresis on an ABI 3730 sequencer. Data were analyzed using Mutation Surveyor 1+ 1+ No No No No No No (SoftGenetics, College Station, PA), conﬁg- Vascular

Involvement ured to use corresponding reference se- quences obtained from GenBank. isMD simulations were carried out using No Yes Yes Yes Yes Yes Yes Yes NAMD26 and the CHARMM22 with CMAP27 Nodular

Glomerular ﬁ 28 Involvement force eld. The PDB structure 1SOH was uti- b lized for our initial conformation. Initial mutant conformations were generated using nitions of proteinuria, nephrotic-range proteinuria, and renal insuf 2+ 2+ 3+ 3+ 3+

ﬁ PyMOL (The PyMOL Molecular Graphics Sys-

Glomerular tem, version 1.5.0.3; Schrödinger, LLC, Cam- Involvement bridge, MA). We utilized an interaction cutoff of 15 Å with strength tapering (or switching) beginning at 12 Å, a simulation time step of 1 femtoseconds, conformations recorded every

Arteriosclerosis 2 picoseconds. Each initial conformation was used to generate ﬁve replicates and each was energy-minimized for 20,000 steps, followed Tubular Fibrosis by randomized temperature initialization (at Atrophy/ Degree of Interstitial 10 K; a standard approach that provides variation between the replicates), followed by 0 Moderate Mild 0 Mild Moderate 3+ heating to 300 K over 300 picoseconds via a Globally Sclerotic Langevin thermostat. A further 12 nanosec- Glomeruli, % onds of simulation trajectory was generated and the ﬁnal 10 nanoseconds analyzed. Anal- ysis was carried out using custom scripts, No. of Sampled Glomeruli leveraging the Bio3D R package29 and visual molecular dynamics.30 Full Nephrotic Syndrome

a ACKNOWLEDGMENTS

Protein We would like to acknowledge that Drs. at Biopsy 24-h Urine

a Giovanni Palladini and Laura Obici have contributed to the discovery of this novel type of amyloidosis. Their manuscript on Serum at Biopsy Creatinine AApoCII is currently in preparation. Clinical and pathologic characteristics of the patients with AApoCII

Gender DISCLOSURES yr Scoring criteria for glomerular involvement, vascular involvement, cortical interstitial involvement, medullary interstitial involvement, and tubular basement membrane involvement are described in the Concise Serum creatinine was represented as mg/dl. Measurements of 24-h urine total protein were represented as g/d. Clinical de Methods section. Table 1. Age, 61 F 2.3 6F, female; M,a male; MNS, medulla not sampled; NA, not available. b Yes 9 11 Moderate Marked 6372 M M 2.5 1.8 9.7 2 Yes No 16 13 46 Moderate Moderate 2+ 75 F NA NA Yes 25 686186 F M F 0.5 0.9 2.6 3.2 3.5 NA No No NA 10 8 8 20 50 38 None Mild Mild Moderate Mild Marked 2+ 73 F 1.1 1.5 No 17 12 Mild None in Concise Methods section. None.

444 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 439–445, 2017 www.jasn.org BRIEF COMMUNICATION

REFERENCES diagnosis and typing of systemic amyloid- Fibril Formation by Apolipoprotein C-II. Adv osis in subcutaneous fat aspirates by mass Exp Med Biol 855: 157–174, 2015 spectrometry-based proteomics. Haematologica 21. Legge FS, Treutlein H, Howlett GJ, Yarovsky I: 1. Sipe JD, Benson MD, Buxbaum JN, Ikeda S, 99: 1239–1247, 2014 Molecular dynamics simulations of a fi brillo- Merlini G, Saraiva MJ, Westermark P: No- 11. Dasari S, Theis JD, Vrana JA, Zenka RM, genic peptide derived from apolipoprotein menclature 2014: Amyloid fibril proteins Zimmermann MT, Kocher JP, Highsmith WE C-II. Biophys Chem 130: 102–113, 2007 and clinical classification of the amyloidosis. Jr, Kurtin PJ, Dogan A: Clinical proteome 22. Todorova N, Hung A, Maaser SM, Griffin Amyloid 21: 221–224, 2014 informatics workbench detects pathogenic MD, Karas J, Howlett GJ, Yarovsky I: Effects 2. de Carvalho M, Linke RP, Domingos F, mutations in hereditary amyloidoses. JPro- of mutation on the amyloidogenic propensity Evangelista T, Ducla-Soares JL, Nathrath WB, teome Res 13: 2352–2358, 2014 of apolipoprotein C-II(60-70) peptide. Phys Azevedo-Coutinho C, Lima R, Saraiva MJ: 12. Verine J, Mourad N, Desseaux K, Vanhille P, Chem Chem Phys 12: 14762–14774, 2010 Mutant fibrinogen A-alpha-chain associated Noël LH, Beaufils H, Grateau G, Janin A, Droz 23. Mao Y, Zlatic CO, Griffin MD, Howlett GJ, with hereditary renal amyloidosis and periph- Todorova N, Yarovsky I, Gooley PR: Hydro- eral neuropathy. Amyloid 11: 200–207, 2004 D: Clinical and histological characteristics of gen/Deuterium Exchange and Molecular 3. Haagsma EB, Hawkins PN, Benson MD, renal AA amyloidosis: a retrospective study of Dynamics Analysis of Amyloid Fibrils Formed Lachmann HJ, Bybee A, Hazenberg BP: Famil- 68 cases with a special interest to amyloid- fl by a D69K Charge-Pair Mutant of Human ial amyloidotic polyneuropathy with severe re- associated in ammatory response. Hum Pathol – Apolipoprotein C-II. Biochemistry 54: 4805– nal involvement in association with transthyretin 38: 1798 1809, 2007 4814, 2015 Gly47Glu in Dutch, British and American- 13. Kinnunen PK, Jackson RL, Smith LC, Gotto 24. Vrana JA, Gamez JD, Madden BJ, Theis JD, Finnish families. Amyloid 11: 44–49, 2004 AM Jr, Sparrow JT: Activation of lipoprotein Bergen HR 3rd, Dogan A: Classification of 4. Rowczenio D, Tennent GA, Gilbertson J, lipase by native and synthetic fragments of amyloidosis by laser microdissection and Lachmann HJ, Hutt DF, Bybee A, Hawkins PN, human plasma apolipoprotein C-II. Proc Natl – mass spectrometry-based proteomic analy- Gillmore JD: Clinical characteristics and SAP Acad Sci U S A 74: 4848 4851, 1977 sis in clinical biopsy specimens. Blood 114: scintigraphic findings in 10 patients with AGel 14. Baggio G, Manzato E, Gabelli C, Fellin R, 4957–4959, 2009 amyloidosis. Amyloid 21: 276–281, 2014 Martini S, Enzi GB, Verlato F, Baiocchi MR, 25. Theis JD, Dasari S, Vrana JA, Kurtin PJ, Dogan 5. Sattianayagam PT, Gibbs SD, Rowczenio D, Sprecher DL, Kashyap ML: Apolipoprotein fi A: Shotgun-proteomics-based clinical testing Pinney JH, Wechalekar AD, Gilbertson JA, C-II de ciency syndrome. Clinical features, li- for diagnosis and classification of amyloidosis. Hawkins PN, Lachmann HJ, Gillmore JD: poprotein characterization, lipase activity, and J Mass Spectrom 48: 1067–1077, 2013 Hereditary lysozyme amyloidosis – phenotypic correction of hypertriglyceridemia after apoli- 26. Phillips JC, Braun R, Wang W, Gumbart J, heterogeneity and the role of solid organ poprotein C-II administration in two affected – Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kalé transplantation. JInternMed272: 36–44, 2012 patients. J Clin Invest 77: 520 527, 1986 L, Schulten K: Scalable molecular dynamics with 6. Vigushin DM, Gough J, Allan D, Alguacil A, 15. Breckenridge WC, Little JA, Steiner G, Chow NAMD. JComputChem26: 1781–1802, 2005 Penner B, Pettigrew NM, Quinonez G, A, Poapst M: Hypertriglyceridemia associ- 27. Mackerell AD Jr, Feig M, Brooks CL 3rd: Bernstein K, Booth SE, Booth DR, Soutar, AK, ated with deficiency of apolipoprotein C-II. N Extending the treatment of backbone ener- Hawkins PN, Pepys, MB: Familial nephro- Engl J Med 298: 1265–1273, 1978 getics in protein force fields: limitations of pathic systemic amyloidosis caused by apo- 16. Hatters DM, MacPhee CE, Lawrence LJ, gas-phase quantum mechanics in reproduc- lipoprotein AI variant Arg26. QJMed87: Sawyer WH, Howlett GJ: Human apolipoprotein ing protein conformational distributions in 149–154, 1994 C-II forms twisted amyloid ribbons and closed molecular dynamics simulations. JComput 7. Yazaki M, Liepnieks JJ, Barats MS, Cohen AH, loops. Biochemistry 39: 8276–8283, 2000 Chem 25: 1400–1415, 2004 Benson MD: Hereditary systemic amyloidosis 17. Ryan TM, Teoh CL, Griffin MD, Bailey MF, 28. MacRaild CA, Howlett GJ, Gooley PR: The associated with a new apolipoprotein AII Schuck P, Howlett GJ: Phospholipids enhance structure and interactions of human apoli- stop codon mutation Stop78Arg. Kidney Int nucleation but not elongation of apolipoprotein poprotein C-II in dodecyl phosphocholine. 64: 11–16, 2003 C-II amyloid fibrils. J Mol Biol 399: 731–740, 2010 Biochemistry 43: 8084–8093, 2004 8. Sethi S, Theis JD, Shiller SM, Nast CC, 18. Lindgren M, Hammarström P: Amyloid 29. Grant BJ, Rodrigues AP, ElSawy KM, Harrison D, Rennke HG, Vrana JA, Dogan A: oligomers: spectroscopic characterization of McCammon JA, Caves LS: Bio3d: an R package Medullary amyloidosis associated with apo- amyloidogenic protein states. FEBS J 277: for the comparative analysis of protein struc- lipoprotein A-IV deposition. Kidney Int 81: 1380–1388, 2010 tures. Bioinformatics 22: 2695–2696, 2006 201–206, 2012 19. Teoh CL, Pham CL, Todorova N, Hung A, 30. Humphrey W, Dalke A, Schulten K: VMD: 9. Said SM, Sethi S, Valeri AM, Leung N, Cornell Lincoln CN, Lees E, Lam YH, Binger KJ, visual molecular dynamics. JMolGraph14: LD, Fidler ME, Herrera Hernandez L, Vrana Thomson NH, Radford SE, Smith TA, Müller 27–38, 1996. JA, Theis JD, Quint PS, Dogan A, Nasr SH: SA, Engel A, Griffin MD, Yarovsky I, Gooley Renal amyloidosis: origin and clinicopatho- PR, Howlett GJ: A structural model for apoli- logic correlations of 474 recent cases. Clin J poprotein C-II amyloid fibrils: experimental Am Soc Nephrol 8: 1515–1523, 2013 characterization and molecular dynamics sim- – 10. Vrana JA, Theis JD, Dasari S, Mereuta OM, ulations. J Mol Biol 405: 1246 1266, 2011 This article contains supplemental material online Dispenzieri A, Zeldenrust SR, Gertz MA, 20. Ryan TM, Mok YF, Howlett GJ, GriffinMD: at http://jasn.asnjournals.org/lookup/suppl/doi:10. Kurtin PJ, Grogg KL, Dogan A: Clinical The Role of Lipid in Misfolding and Amyloid 1681/ASN.2015111228/-/DCSupplemental.

J Am Soc Nephrol 28: 439–445, 2017 Novel Type of Renal Amyloidosis Derived from APOC-II 445 Supplementary Figure 1: Renal specimens from four patients stained for APOC2 using immunohistochemistry (APOC2 immunohistochemistry with diaminobenzidine chromagen and hematoxylin counterstain, all X400). The identity of the amyloid in each case was confirmed by mass spectrometry and/or immunofluorescence. (A) Glomerular involvement by AApoCII, patient 3 of Table 1. (B) Glomerular involvement by AL-. (C) Glomerular involvement by AL-. (D) Medullary involvement by AApoAIV. Strong specific staining for APOC2 highlights the nodular amyloid deposits in the specimen from the patient with AApoCII. The amyloid deposits in the AL and AApoAIV cases are negative. Supplementary Figure 2: Renal biopsies from two patients demonstrating nodular glomerular involvement by amyloid. In both examples, the mesangium is asymmetrically expanded by large rounded masses of amyloid that encroach on the capillary spaces. (A: from patient #6 in Table 1, hematoxylin & eosin X200; B: from patient #8 in Table 1, hematoxylin & eosin X400) Supplementary Figure 3: Ultrastructural findings in patient #4 in Table 1. (A) Low power electron microscopic figure shows nodular mesangial expansion by moderately electron dense deposits (original magnificaiton, X5600). (B) On higher magnification, the mesangial deposits are composed of randomly-oriented fibrils typical of amyloid (original magnification, X22000).