D25V apolipoprotein C-III variant causes dominant hereditary systemic amyloidosis and confers cardiovascular protective lipoprotein profile

Abstract

Apolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients.

ARTICLE

Received 1 Jun 2015 | Accepted 2 Dec 2015 | Published 21 Jan 2016

DOI: 10.1038/ncomms10353 OPEN

D25V apolipoprotein C-III variant causes dominant hereditary systemic amyloidosis and confers cardiovascular protective lipoprotein prole

Sophie Valleix1,2,3,4,w, Guglielmo Verona5,6, Nomie Jourde-Chiche7, Brigitte Ndelec2,3,4,

P. Patrizia Mangione5,6, Frank Bridoux8, Alain Mang9,10,11,12, Ahmet Dogan13,14, Jean-Michel Goujon15,

Marie Lhomme16, Carolane Dauteuille17, Michle Chabert4,17,18, Riccardo Porcari5, Christopher A. Waudby19, Annalisa Relini20, Philippa J. Talmud21, Oleg Kovrov22, Gunilla Olivecrona22, Monica Stoppini6,John Christodoulou19, Philip N. Hawkins5, Gilles Grateau23, Marc Delpech1, Anatol Kontush17,Julian D. Gillmore5, Athina D. Kalopissis4 & Vittorio Bellotti5,6

Apolipoprotein C-III deciency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufciency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) prole. Amyloid brils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably brillogenic under physiological conditions in vitro producing typical amyloid brils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the rst conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deciency. Thus, brate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients.

1 Universit Paris-Descartes, Sorbonne Paris Cit, Assistance Publique-Hpitaux de Paris, Laboratoire de Biologie et Gntique Molculaire, Hpital Cochin, Paris 75014, France.

2 INSERM, UMR_1163, Institut Imagine, Laboratoire de Gntique Ophtalmologique (LGO), Universit Paris Descartes, Sorbonne Paris Cit, Paris 75015, France. 3 INSERM, U1016, Institut Cochin, Universit Paris Descartes, Sorbonne Paris Cit, Paris 75014, France. 4 Sorbonne Universits, UPMC Univ Paris 06, INSERM, Universit Paris-Descartes, Sorbonne Paris Cit, UMR_S 1138, Centre de Recherche des Cordeliers, Paris 75006, France. 5 Centre for Amyloidosis and Acute Phase Proteins, National Amyloidosis Centre, University College London, London NW3 2PF, UK. 6 Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Via Taramelli 3b, Pavia 27100, Italy. 7 Universit de Marseille, AP-HM, Hpital de la Conception, Marseille 13005, France. 8 Universit de Poitiers, CHU Poitiers, Department of Nephrology and Kidney Transplantation, Centre National de Rfrence Amylose AL et autres maladies par dpts dimmunoglobulines monoclonales, Poitiers 86021, France. 9 Institut de Recherche en Cancrologie de Montpellier (IRCM), Montpellier 34298, France. 10 INSERM, U1194, Montpellier 34298, France. 11 Universit de Montpellier, Montpellier 34090, France. 12 Institut rgional du Cancer de Montpellier, Montpellier 34298, France. 13 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55901, USA. 14 Departments of Laboratory Medicine and Pathology, Memorial Sloan-Kettering Cancer Centre, New York, NY 10065, USA. 15 Universit de Poitiers, CHU Poitiers, Service dAnatomie et Cytologie Pathologiques, Centre National de Rfrence Amylose AL et autres maladies par dpts dimmunoglobulines monoclonales, Poitiers 86021, France. 16 Lipidomic core, ICANalytics, Institute of Cardiometabolism and Nutrition, ICAN, Piti-Salptrire Hospital, F-75013 Paris, France. 17 Sorbonne Universits, UPMC Univ Paris 06, Institute of Cardiometabolism and Nutrition (ICAN), UMR_S 1166, Hpital de la Piti, Paris 75013, France. 18 Ecole Pratique des Hautes Etudes, PSL Research University, Laboratoire de Pharmacologie cellulaire et Molculaire, Paris 75006, France. 19 Institute of Structural and Molecular Biology, University College London and Birkbeck College, University of London, London WC1E 6BT, UK. 20 Department of Physics, University of Genoa, Via Dodecaneso 33, Genoa 16146, Italy. 21 Centre for Cardiovascular Genetics, Institute of Cardiovascular Science, University College London, London WC1E 6JF, UK. 22 Department of Medical Biosciences, Ume University, Ume SE-901 87, Sweden. 23 Hpital Tenon, AP-HP, Service de Mdecine Interne, Centre de rfrence des amyloses dorigine inammatoire et de la vre mditerranenne familiale, Paris 75020, France. w Present address: Laboratoire de Gntique Molculaire, Service de

Gntique molculaire, Hpital Necker-Enfants Malades, Assistance Publique-Hpitaux de Paris, Universit Paris Descartes, Sorbonne Paris Cit, 149 rue de Svres, 75015 Paris, France. Institut National de la Sant et de la Recherche Mdicale, Unit Mixte de Recherche 1163, Institut Imagine, Laboratoire de Gntique Ophtalmologique, Universit Paris Descartes, Sorbonne Paris Cit, 24 Bd du Montparnasse 75015 Paris, France (S.V.). Correspondence and requests for materials should be addressed to S.V. (email: mailto:[email protected]

Web End [email protected] ) or to V.B. (email: mailto:[email protected]

Web End [email protected] ).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10353

There is growing evidence that apolipoprotein C-III (apoC-III), a small exchangeable apolipoprotein carried in the circulation by VLDL and HDL1, is a master

regulator of plasma TG homeostasis and downregulation of its expression has a cardiovasculo-protective effect2. ApoC-III inhibits the activity of lipoprotein lipase (LPL), the enzyme catalysing the rst and rate-limiting step of TG-rich lipoprotein catabolism3, and also impairs uptake of TG-rich lipoprotein-remnants by the low-density lipoprotein (LDL)-receptor (LDL-R), increasing remnant residence time in the circulation4. ApoC-III is a glycosylated apolipoprotein composed of 79 amino acids, synthesized mainly in the liver, and existing in three isoforms, apoC-III0, apoC-III1 and apoC-III2 with, respectively, 0, 1 and 2 residues of sialic acid per molecule, but little is known about sialylation of apoC-III in disease states. Heterozygosity for the rare null allele of APOC3, R19X, has been associated with high levels of plasma HDL cholesterol (HDL-C), low levels of plasma TG and reduced incidence of ischaemic cardiovascular disease (CVD) in the Amish population5. Subsequently, genome-wide association studies also identied the Arg19Stop variant in other population isolates with a strong atheroprotective phenotype6. More importantly, a recent general population-based study conrmed the association of APOC3 deciency with an atheroprotective lipid prole and clinical protection against ischaemic CVD7,8.

In its lipid-bound state, apoC-III is composed of six amphipathic a-helices, the structural motif shared by all exchangeable apolipoproteins that confers their structural stability9. Conversely, apolipoproteins in lipid-free form have lower conformational stability and some, such as native and/or variants of apoA-I (ref. 10), apoA-II (ref. 11) and apoA-IV (ref. 12), are prone to self-assembly in vitro and in vivo as amyloid brils. Recombinant wild-type apoC-III forms insoluble aggregates with cross-b structure in vitro, reminiscent but not typical of amyloid brils13. Amyloid bril formation is associated with a wide variety of diseases, and the hereditary monogenic forms of human amyloidoses, although rare, provide unique insight into mechanisms of protein misfolding and brillogenesis as recently highlighted by the discovery of the amyloidogenic variant of b2-microglobulin14.

Here we report a French family with severe renal amyloidosis and hypotriglyceridemia, both caused by a novel D25V apoC-III variant. Remarkably, this variant induced a favourable cardiovascular lipoprotein prole, even in the context of chronic kidney disease (CKD). The D25V protein is highly brillogenic in its lipid-free state, and forms amyloid brils under physiological conditions in vitro. This study provides new insights into the genetics of apoC-III and into VLDL and HDL metabolism in health and disease, illustrating the increasing importance of apoC-III in diverse biological processes.

ResultsPatients. The proband (III.3 in Fig. 1a) presented at age 51 years with sicca syndrome and hypertension. Two years later, he was diagnosed with CKD; his serum creatinine level was 1.9 mg dl 1 with modest proteinuria (40 mg dl 1). Salivary gland and renal biopsies showed amyloid deposits. A search for an underlying monoclonal gammopathy was negative, and plasma concentrations of serum amyloid A protein (SAA) and C-reactive protein were in the normal range. The proband reached end-stage renal disease (ESRD) aged 56 years and received a cadaveric kidney transplant uneventfully. 123I-labelled serum amyloid P component (SAP) scintigraphy, undertaken just before renal transplantation in the proband III.3 (Fig. 1b) and in affected member IV.3 (Fig. 1c), showed a small total body amyloid load in

the spleen and kidneys, at which point a fat aspirate demonstrated extensive amyloid. Four years after renal transplantation, graft function was normal (serum creatinine 0.88 mg dl 1), without detectable proteinuria. There was a strong family history of amyloidosis (Fig. 1a) with all affected members suffering rst from sicca syndrome and later developing mildly proteinuric CKD. The probands grandmother (patient I.2) and aunt (patientII.1) had suffered from sicca syndrome for many years before developing systemic amyloidosis and had died aged 74 and 80 years, respectively. The probands father (patient II.3) had presented with sicca syndrome aged 63 years, Raynaud phenomenon, high blood pressure and diffuse pruritus. Salivary gland, rectal and bronchial biopsies had all shown amyloid. He reached ESRD aged 64 years. The probands younger sister (patient III.5) had died aged 49 years of staphylococcal infection, shortly after kidney transplantation. She had presented with cardiac amyloidosis proven by biopsy 10 years earlier, associated with CKD and mild proteinuria. She had reached ESRD and started haemodialysis aged 40 years. She had also suffered from sicca syndrome, Raynaud phenomenon, oesophageal reux with oesophagitis, diffuse pruritus and severe hypertension. Her two children (patients IV.3 and IV.4) presented with sicca syndrome at age 23 and 20 years, respectively, and both of them showed amyloid deposits on salivary gland biopsies (Supplementary Fig. 1). Both of them subsequently developed hypertension and stage II CKD. The probands son (IV.1) suffered from sicca syndrome aged 30 years and showed amyloid deposition on salivary gland biopsy (Supplementary Fig. 1), but he had no hypertension and his renal function was normal with a serum creatinine level at 0.73 mg dl 1 and no proteinuria. The probands daughter (IV.2) was clinically asymptomatic, and had no amyloid deposits on salivary gland biopsy.

Amyloid and hypotriglyceridemia in D25V apoC-III carriers. An initial genetic screening for mutations in all genes recognized until now to be linked to hereditary amyloidosis was negative (see Methods for details). Based on the fact that affected patients with amyloidosis also displayed hypotriglyceridemia and increased plasma levels of HDL-C as compared with non-carriers in the family (Table 1), we next screened several candidate genes including the APOC3, APOC1, APOC2, APOC4, APOA4, APOA5 and APOE genes. The unique variant identied was a single-base substitution (c.134A4T) in exon 3 of APOC3, encoding replacement of the negatively charged aspartate residue at position 25 of the mature apoC-III by a valine residue (D25V; Fig. 1d). All D25V-carriers displayed a 3050% decrease in plasma apoC-III concentration (Table 1), and there was no correlation between APOC3 promoter polymorphism genotypes and plasma TG levels among family members (Fig. 1a). Therefore, D25V apoC-III variant was uniquely found in family members affected with amyloidosis, hypotriglyceridemia and reduced apoC-III levels, and not in healthy normotriglycemic family members, demonstrating the co-inheritance of this variant with both the lipidemic and amyloidogenic phenotypic traits.

Histology and LMD/MS-proteomic analysis of amyloid deposits. To establish that apoC-III D25V variant is the causative amyloidogenic protein, we analysed amyloid deposits by immunohistochemical staining, immunogold technology and proteomic methodologies. Amyloid deposits were discovered in a total of 17 diverse disease tissues from 6 different affected family members through three generations (kidney, salivary glands, heart, bowel, fat, and bronchia from III.3; kidney, salivary glands, skin, heart, liver from III.5; salivary glands, kidney, skin, kidney, bowel from II.3; salivary glands from IV.1, IV.3 and IV.4).

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10353 ARTICLE

Exon2: Asp25Val (D25V) p.Asp45Val rs2854117 C-482T*rs2854116 T-455C*

a

d

I

II

III

IV

III.3

1 2

1

2

3

III.1

Congo red Polarized light ApoC-III IF

Salivary glands

Digestive tract Kidney Kidney

III.3

e

f

g

1

2

3

4

5

D25V+/ C C

D25V+/ C C

D25V+/ C T*T C*

T

C*

C*

T

1

2

3

4

D25V+/ C C

T T

D25V/ C C

D25V+/ C C

T T

D25V+/ C C

T T

T

C*

b c

Jones staining ApoC-III immunogold

h

Ant Post Ant Post

Figure 1 | Apolipoprotein C-III amyloidosis in a French family. (a) Pedigree showing three generations of transmission of the ApoC3 D25V variant with systemic amyloidosis and decreased plasma triglyceride levels: APOC3 mutation present; clinical syndrome; ApoC-III amyloid conrmed

histologically; decreased plasma triglyceride levels; no clinical syndrome, no amyloid on histology, absence of APOC3 mutation, no hypotriglyceridemia. APOC3 haplotypes distribution of C-482T and T-455C polymorphisms is shown. D25V / , heterozygous for the apoC-III

amyloidogenic variant; D25V / , absence of apoC-III mutation. (b,c) Serial anterior and posterior planar whole-body 123I- SAP scan. The scans from III.3

in b and IV.3 in c show a congested liver and amyloid in the spleen and kidneys. (d) Partial sequence chromatograms of APOC3 exon 3. (e) Amyloid deposits in salivary glands from proband III.3. (f) Amyloid deposits in the duodenum from proband III.3 predominate in the submucosa around the vessels and around Brenners glands. There are also amyloid deposits grouped in small clods within the intestinal villi. (g) Abundant vascular, moderate glomerular and interstitial amyloid deposits in kidney from proband III.3. For each tissue in eg, left panels show the Congo red staining, scale bar, 100 mm; middle panels show the Congo red-stained section viewed under polarized light, scale bar, 100 mm; right panels show the immunouorescence with anti-apoC-III antibody; scale bar, 100 mm. (h) Left panel: amyloid deposits in the kidney are observed in the mesangium and the glomerular arteriole (arrow) (Jones staining, scale bar, 100 mm); middle and right panels: electron micrograph of amyloid brils on immunogold staining with anti-apoC-III antibody, scale bar, 500 and 200 nm, respectively).

Table 1 | Plasma TG, HDL-C and APOC-III levels.

II.3 III.1 III.3 III.4 III.5 IV.1 IV.2 IV.3 IV.4 TG (g l 1) 0.30 1.80 0.31 1.38 0.44 0.34 0.86 0.35 0.50

HDL-C (g l 1) ND 0.35 0.71 0.57 0.79 0.59 ND 0.69 0.83 ApoC-III (mg l 1) ND 54.0 17.7 ND ND ND 44.2 18.8 34.4

apoC-III, apolipoprotein C-III; HDL-C, high-density lipoprotein-cholesterol; ND, not determined; TG, triglyceride.II.3, III.1, III.3, III.4, III.5, IV.1, IV.2, IV.3, IV.4refers to individuals in Fig. 1a.

All patients with sicca syndrome, the rst clinical symptom, were carriers of D25V (II.3, III.3, III.5, IV.1, IV.3 and IV.4) and had amyloid deposits in salivary gland biopsies. Conversely, clinically non-affected individuals and non-carriers of D25V (III.1 andIV.2) demonstrated no amyloid deposits on salivary gland biospsies. In the proband III.3, all amyloid deposits observed in salivary gland, digestive tract and kidney were Congo red positive and immunoreactive with the anti-apoC-III antibody (Fig. 1eg). Moreover, extensive amyloid deposits were found around salivary acini, in the interstitial matrix and in the walls of blood vessels, and were associated with areas of brosis and massive acinar atrophy (Fig. 1e). Similar pathological ndings in salivary glands

from younger affected family D25V-carriers (individuals IV.1,IV.3 and IV.4) were found and are shown in Supplementary Fig. 1. Therefore, this extensive amyloid accumulation associated with gland atrophy could explain the sicca symptoms of mouth reported by the patients. Renal histology from affected probandIII.3 at an advanced stage of renal insufciency showed diffuse amyloid deposition in the renal cortex that mostly affected the vascular compartment with abundant amyloid deposits in the walls of renal arterioles leading to lumen obliteration (Fig. 1g,h). Deposits were also observed in the glomeruli with prominent mesangial distribution, within the renal interstitium, and in the peritubular basement membranes with tubular atrophy. Amyloid

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10353

a

Heart amyloid proteome

b

Digestive tract amyloid proteome

Probability legend:

Over 95% 8094%

5079%

2049%

019%

Over 95% 8094% 5079%

2049%

019%

Accession number

Probability legend:

Molecular weight

Starred

B S1 S2 S3

Accession number

Molecular weight

# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Identified proteins (294)

69 kDa

109 kDa

15 kDa

16 kDa

36 kDa

34 kDa

ALBU_HUMANAPOE_HUMAN CO6A3_HUMAN VTNC_HUMAN APOA4_HUMAN SAMP_HUMANACTB_HUMAN CASPE_HUMAN APOC3_HUMAN CO6A1_HUMAN S10A7_HUMANHBB_HUMANHBA_HUMANZA2G_HUMANDCD_HUMANG3P_HUMANCYTA_HUMANPGBM_HUMAN CO6A2_HUMANCLUS_HUMANACTA_HUMAN APOA1_HUMAN ENOA_HUMANFILA_HUMANH4_HUMAN APOC3_D45V_HUMAN ANXA2_HUMANTRFL_HUMANCO1A2_HUMANCALL5 HUMAN 16 kDa

129 kDa

Serum albumin Apolipoprotein E

Apolipoprotein A-IV

Apolipoprotein C-III

Apolipoprotein A-I

Apolipoprotein C-III D45V mutation

48 44 29 29 30 31 21 12 21 13

9 19 17

9 6 5 6 14

9

67 71

44 18 33 26 28 18 25 14

7 12 12 14 14 13 13 11

4 8 7 21

7 6 8 8 9 8 5 11

9

36 kDa

36

Collagen alpha-3(VI) chain

Collagen alpha-1(VI) chain

Collagen alpha-2(VI) chain

Collagen alpha-2(I) chain Calmodulin-like protein 5

344 kDa

44

Vitronectin

Serum amyloid P-component Actin, cytoplasmic 1 Caspase-14

Protein S100-A7Haemoglobin subunit betaHaemoglobin subunit alpha Zinc-alpha-2-glycoproteinDermcidinGlyceraldehyde-3-phosphate dehydrogenase Cystatin-ABasement membrane-specific heparan sulfate

ClusterinActin, aortic smooth muscle

Alpha-enolase Filaggrin Histone H4

Annexin A2 Lactotransferrin

54 kDa

21

32

# Identified proteins (314)

C 1 2

45 kDa

25 kDa

30

26

35 19

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Apolipoprotein ESerum amyloid P-component Apolipoprotein C-III D45V mutation Myosin-7

Actin, alpha skeletal muscleDesminCollagen alpha-3(VI)chainMyosin regulatory light chain 2, ventricular/car.. VitronectinMyosin light chain 3

VimentinATP synthase subunit alpha, mitochondrial Myosin-binding protein C, cardiac-type Alpha-actinin-2ATP synthase subunit beta, mitochondrial Isocitrate dehydrogenase [NADP], mitochondrial.. Collagen alpha-2(I) chainTroponin T, cardiac muscleFibrillin-1Creatine kinase S-type, mitochondrial

APOE_HUMAN SAMP_HUMAN APOC3_D45V_HUMAN MYH7_HUMAN ACTS_HUMAN DESM_HUMAN CO6A3_HUMAN MLRV_HUMAN VTNC_HUMAN MYL3_HUMAN VIME_HUMAN ATPA_HUMAN MYPC3_HUMAN ACTN2_HUMAN ATPB_HUMAN IDHP_HUMAN CO1A2_HUMAN TNNT2_HUMAN FBN1_HUMAN KCRS_HUMAN

36 kDa 25 kDa

3 kDa 223 kDa

42 kDa

54 kDa

54 kDa

54 kDa

60 kDa

57 kDa 51 kDa 129 kDa

141 kDa 104 kDa

42 kDa

11

12

28 kDa

14

4 63

14

23

11

22

11

3

11 kDa

13

138

21

31

11 kDa

14

34

8

13

344 kDa

19 kDa

36 kDa 312 kDa

48 kDa

10

11 kDa

7

9

9

9

22

22 kDa

16

9

20

5

2 2

5

11 kDa

20

469 kDa

12

21

109 kDa

12

20

52 kDa

7

3

3

16

42 kDa

10

25

6

7

21

11

31 kDa

8

10

1

1

11

21 22 23 24 25 26 27 28 29 30

4

47 kDa

5

13

11

2 8 9 9 13

64 5

3

435 kDa

8

2

11 kDa

7

3 kDa

10

39 kDa

5

78 kDa

3

c

Signal peptide

1 20

10 90

80

70

30 40 45

45

45

50

60

60

60

99

APOC-III_Human wild type

APOC-III_Human D25V (p.D45V)

Heart

Digestive tract

Figure 2 | Proteomic analysis of amyloid plaques. Proteomic analyses of amyloid plaques from heart (a) and digestive tract (b) biopsy specimens of patient III.3 showing the most abundant proteins found. In both amyloid proteomes, the variant apoC-III protein was identied along with the apolipoprotein E and the serum amyloid P component. (c) ApoC-III tryptic peptide coverage: the two top lines represent the wild-type apoC-III sequence with the20 amino-acid signal peptide and the mutant D25V (p. D45V) apoC-III sequence, respectively, and the two bottom lines represent the apoC-III peptides identied from the amyloid plaques from the heart and digestive tract, respectively. All amyloid plaques contained only the mutated apoC-III peptide with the valine at position 45 (corresponding to position 25 of the mature protein) and not the corresponding apoC-III wild-type peptide with the aspartate residue.

P component co-localized with the above-mentioned deposits, and vascular, glomerular, tubular and interstitial amyloid deposits were positively and specically immunoreactive with apoC-III antibody (Fig. 1g). Electron micrographs of the kidney specimen from III.3 showed numerous non-branched and randomly arranged deposits with a brillar appearance, measuring 710 nm in diameter, typical of amyloid (Fig. 1h). Immunoelectron microscopic analysis subsequently showed that these renal brillar aggregates were specically labelled by gold-conjugated anti-apoC-III antibody (Fig. 1h). Therefore, our histologic analysis, in conjunction with 123I-SAP scintigraphy, clearly established the correlation between the clinical symptoms of D25V-carriers (sicca syndrome and renal failure) and the presence of amyloid in the respective tissues. In all 11 affected tissues from 6 D25V-carriers over three generations, the anti-apoC-III antibody consistently and specically bound to all Congo red-staining amyloid areas, whereas there was no binding of antibodies against the other known amyloid bril proteins. Moreover, the fact that anti-apoC-III antibody directly bound to the brillar aggregates conrms at the ultrastructural level the diagnosis of apoC-III amyloidosis.

In addition to immunouorescence and immunogold staining, amyloid subtyping was also conrmed by laser microdissection/ mass spectrometry (LMD/MS)-based proteomics on three

different tissues: digestive tract and heart from proband III.3, and salivary glands from individual IV.3, with several amyloid deposits microdissected per tissue (Fig. 2a,b and Supplementary Table 1). In all proteomes, only the variant apoC-III containing Valine 25 was invariably found in the diverse amyloid deposits along with SAP and apoE, with no trace of the corresponding wild-type apoC-III peptide (Fig. 2c). In the amyloid proteomes of the digestive tract and salivary glands, wild-type apoA-IV and apoA-I proteins were variably present, whereas both proteins were absent in heart proteome (Fig. 2a,b and Supplementary Table 1). These ndings are in agreement with a number of studies showing the presence of apoA-I and A-IV in various amyloid deposits, irrespective of the nature of the amyloidogenic protein15. The concordance between the amyloid proteomes from different tissues of three D25V-carriers of this family, and the fact that no other amyloid-associated proteins were found in the heart except the mutated apoC-III clearly demonstrated that the mutated apoC-III was the amyloid bril protein. Importantly, neither peptides representing the D25V or the wild-type apoC-III have been identied in the amyloid proteomes from over 5,000 patients with different types of systemic amyloidosis15, clearly indicating that apoC-III is not a protein commonly co-deposited in amyloid aggregates. Therefore, the D25V variant was specically found in this kindred for the rst time,

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10353 ARTICLE

a

HDL2

HDL3

IV.3

100

50

IV.3

III.4

ApoC-III0a

ApoC-III0a

ApoC-III2

100

D25V

D25V D25V

ApoC-III0c

ApoC-III1

ApoC-III2

D25V

% Intensity

% Intensity

ApoCIII0c

ApoC-III1

50

08,700 8,960 9,220

m/z

9,480 9,740 10,000 8,700 8,960 9,220

m/z

9,480 9,740 10,000

0

III.4

ApoC-III1

100

% Intensity

100

% Intensity

ApoC-III0a ApoC-III0c

ApoC-III2 ApoC-III0a

ApoC-III0c

ApoC-III1

ApoC-III2

50

50

0

0

8,700

8,960 9,220

m/z

9,480 9,740 10,000 8,700 8,960 9,220

m/z

9,480 9,740 10,000

b c

ApoC-III1

ApoC-III wild type

ApoC-III D25V

D25V

IV.3

=15.5

9,350 m/z

Peptide sequence Protein identity D25V carrier Non-carrier

Alignment

Apolipoprotein C-III

m/z 2,021.0565 4560 m/z 2,005.0979 4560 m/z 2,515.2668 2137 m/z 1,064.6672 3841

6171

m/z 1,506.8412 7278 m/z 2,153.0186 8199

m/z 1,805.0054

III.4

9,480

Figure 3 | MS characterization of wild-type and D25V apoC-III isoforms in HDL2 and HDL3. (a) MS analysis of apoC-III isoforms from HDL2/HDL3. Representative graphs of apoC-III isoforms from D25V carrier IV.3 and non-carrier III.4 are shown. Three apoC-III isoforms are commonly referred to as apoC-III0, apoC-III1 and apoC-III2, where the index represents the number of sialic acid residues in each isoform. The subscript lower case letter indicates the absence of glycans (apoC-III0a) or the presence of a GalNAc-Gal disaccharide (apoC-III0c), respectively. The arrow points to the peak corresponding to the D25V variant of apoC-III isoforms with a reduced mass of 15,500 Da (apoC-III1: 9,406.11; apoC-III2: 9,708.90) in comparison to the wild-type apoC-III isoforms (apoC-III1: 9,421.61; apoC-III2: 9,714.40). The variant apoC-III1 and apoC-III2 isoforms are detected only in D25V-carriers. (b) Higher magnication of apoC-III1 from the D25V-carrier IV.3 and a non-carrier III.4. (c) Mass spectrometric identication of apoC-III in HDL2/3 using LC-MS/MS sequencing.

The amino-acid sequences obtained using MS/MS are in bold. Specic wild-type or mutated peptides are in red. The observed m/z of peptides (including variable modications) are listed in the table with their position in the apoC-III protein sequence.

and histologic and proteomic analyses were consistent with the genetic ndings.

MS analysis of apoC-III from D25V-HDLs. To determine the relative amounts of wild-type and mutant apoC-III as well as the proportions of apoC-III isoforms in HDL2 and HDL3, MS was carried out in these lipoproteins from hypotriglyceridemic

D25V carriers III.3, IV.3 and IV.4 and normotriglyceridemic non-D25V-carrier III.4. The resulting MS proles showed that D25V-carriers exhibited a double peak for apoC-III1 and apoC-III2 isoforms (only the prole of individual IV.3 is presented), whereas the non-carrier of D25V had a normal prole (Fig. 3a). The measured mass difference of 15.5 Da between the two signals is in agreement with the difference between the

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Table 2 | Relative quantication of apoC-III isoforms in D25V-carriers.

HDL2 HDL3III.4 III.3 IV.3 IV.4 III.4 III.3 IV.3 IV.4 D25V / D25V / D25V / D25V / D25V / D25V / D25V / D25V /

ApoC-III0 8.2 11.8 3.8 3.4 11.2 17.8 5.9 5.9 ApoC-III1 60.3 44.1 46.4 51.4 62.7 45.9 53.7 53.7 ApoC-III2 31.5 44.1 49.8 45.2 26.1 36.3 40.4 40.4 Ratio WT/D25V Apo-CIII1 2.4 2.1 2.4 1.6 1.7 2.0 Ratio WT/D25V Apo-CIII2 1.9 1.6 1.7 2.1 1.6 1.8

apoC-III, apolipoprotein C-III; WT, wild type.

Relative quantication of each apoC-III isoform in three D25V-carriers (III.3, IV.3 and IV.4) in comparison with a healthy non-D25V-carrier (III.4). The ratio of apoC-III isoforms is modied in the D25V-carriers and less D25V variant than wild-type apoC-III is present in HDL2/HDL3.

a

Control

Proband TG TC

VLDL LDL HDL

20

0

20

TG (mg dl1 )

12

8

4

12

8

4

0

VLDL LDL HDL

15

15

10

TC (mg dl1 )

TG (mg dl1 )

10

TC (mg dl1 )

5

5

0

8

0 1 22 36 50 64 78 85 99106 120

15

1 22 36 50 64 78 85 99106 120

15

8

b c

Fraction number Fraction number

[3Hcholesterol efflux from

THP-1 cells (%)]

6543210 HDL3c

Proband Control

Proband Control

Maximal concentration of

conjugated

dienes in LDL (mol l1 )

300 250 150 100

50

0

HDL3c

Total HDL

Total HDL

Figure 4 | Lipoprotein studies. (a) Fast protein liquid chromatography (FPLC) size exclusion chromatogram of lipoproteins of III.3 in comparison to a normolipidemic control subject. In each case 200 ml of serum was analyzed using two GE Healthcare 10/300 GL Superose 6 columns coupled in series, equilibrated and eluted with a buffer containing 0.15 M NaCl, 3 mM EDTA, 0.04% sodium azide, pH 7.4 at 0.4 ml min 1. Triglyceride (TG) and total cholesterol (TC) were determined in each fraction. (b) Cholesterol efux capacity of HDL. THP-1 macrophages were cholesterol-loaded by incubation with [3H]cholesterol-labelled acetylated LDL (1 mCi ml 1) for 24 h as detailed in the Methods. Cellular cholesterol efux to HDL3c and total HDL from III.3 and one normolipidemic control subject (15 mg ml 1 HDL-PL per well) was measured in serum-free medium for a 4 h chase period. The percentage of cholesterol efux was calculated as (medium cpm)/(medium cpm cell cpm) 100%. Specic cholesterol efux was determined by subtracting

nonspecic cholesterol efux occurring in the absence of cholesterol acceptors (HDL). (c) Antioxidative activity of HDL. The antioxidative capacity of HDL3c and total HDL from III.3 and one normolipidemic control subject to inhibit LDL oxidation was assayed at a physiological HDL to LDL ratio. HDL particles were compared on the basis of their total mass. Lipoprotein oxidation was induced by the azo-initiator 2,20-azo-bis-(2-amidinopropane)

hydrochloride (nal concentration 1 mmol l 1). Oxidation was expressed as the concentration of lipid hydroperoxides with conjugated diene structure in LDL.

wild-type aspartate residue and the mutated valine residue(15.9 Da). A higher magnication of MS prole is presented in Fig. 3b. To conrm that the additional peak observed in D25V-carriers corresponds to the apoC-III variant, LC/MS-MS sequencing was performed. As shown in Fig. 3c, both the tryptic peptides containing the mutant Val25 and the wild-type Asp25 of apoC-III isoforms were identied in heterozygous D25V-carriers, whereas individual III.4 exhibited the wild-type apoC-III tryptic peptide on HDL2/HDL3 subfractions.

Relative quantication of each apoC-III isoforms was performed in the three D25V-carriers (III.3, IV.3 and IV.4) and compared with the non-carrier individual III.4. Data, summarized in Table 2, show a twofold reduction of D25V-apoC-III compared with its wild-type counterpart in all three affected patients with amyloidosis. The levels of di-sialylated apoC-III were higher in D25Vcarriers compared with the non-D25V-carrier, with a corresponding decrease in mono- and non-sialylated apoC-III.

Lipoprotein studies. The lipoprotein proles of III.3 and a normolipidemic subject were substantially different. Interestingly, VLDL was nearly absent, LDL was reduced and HDL was massively increased in the probands plasma (Fig. 4a). A detailed analysis of all lipoprotein constituents shows a vefold increase in HDL2 (Table 3) but a lower apoC-III content in the probands HDL2/HDL3 fractions (Supplementary Fig. 2). In addition, HDL particles from III.3 were enriched in esteried and free cholesterol, and in phospholipid, but depleted in TG and protein, a composition typical of hyperalphalipoproteinemic HDL (Table 4). The prole of several lipid classes in the probands HDL was altered (Table 5). Although the proband had CKD, his HDL maintained an elevated cholesterol efux capacity compared with HDL of the normolipidemic control (Fig. 4b), whereas the antioxidative activities of total HDL and small, dense HDL3c were similar between the proband and the normolipidemic control subject (Fig. 4c). The hypotriglyceridemia and

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Table 3 | Lipoprotein composition.

Protein TG TC PL Total (mg dl 1)

(mg dl 1) (%) (mg dl 1) (%) (mg dl 1) (%) (mg dl 1) (%)

ControlVLDL 12.55 (9.3) 87.80 (65.0) 8.55 (6.3) 26.26 (19.4) 135.16 IDL 4.56 (20.6) 8.45 (38.1) 2.99 (13.5) 6.16 (27.8) 22.16 LDL 82.90 (35.2) 13.72 (7.2) 39.13 (20.4) 71.47 (37.3) 207.22 HDL2 22.79 (47.4) 2.86 (5.9) 8.19 (17.0) 14.26 (29.6) 48.09 HDL3 93.15 (63.1) 4.63 (3.1) 13.23 (9.0) 36.70 (24.9) 147.71

Proband (III.3)VLDL 3.55 (11.4) 20.39 (65.7) 1.60 (5.1) 5.51 (17.8) 31.04 IDL 0.42 (4.9) 3.87 (45.9) 1.32 (15.7) 2.82 (33.4) 8.43 LDL 45.80 (31.7) 11.36 (7.9) 30.98 (21.4) 56.53 (39.1) 144.67 HDL2 117.22 (47.5) 5.02 (2.0) 40.19 (16.3) 84.63 (34.3) 247.06 HDL3 62.97 (56.6) 2.79 (2.5) 12.72 (11.4) 32.79 (29.5) 111.27

HDL, high-density lipoprotein; IDL, intermediate density lipoprotein; LDL, low-density lipoprotein; PL, phospholipid; TC, total cholesterol; TG, triglyceride; VLDL, very-low-density lipoprotein. Lipoproteins of the proband and of a control subject were isolated in parallel by sequential ultracentrifugation of serum (see Methods for details). All lipoprotein components were expressed in mg dl 1;

in parentheses are the percentages of components in each lipoprotein class.

Total is the sum of all constituents in each lipoprotein class.

Table 4 | Chemical composition (WT%) of HDL subpopulations.

Subjects HDL2b HDL2a HDL3a HDL3b HDL3c CE (WT%) Proband 29.8 21.1 22.2 27.0 20.1

Control 26.0 23.3 21.6 13.1 12.6 FC (WT%) Proband 7.5 3.1 3.8 6.2 4.2

Control 6.5 4.1 3.3 4.5 1.7 PL (WT%) Proband 31.9 24.4 26.7 31.8 26.6

Control 32.0 25.8 25.6 24.9 16.9 TG (WT%) Proband 2.2 1.4 1.4 2.1 1.9

Control 3.1 2.5 1.9 2.0 1.0 Total protein (WT%) Proband 28.6 49.9 46.0 32.9 47.1

Control 32.4 44.4 47.6 55.4 67.9

CE, cholesteryl ester; FC, free cholesterol; HDL, high-density lipoprotein; PL, phospholipid; TG, triglyceride.

Data are shown as WT% of total HDL mass. The proband corresponds to individual III.3, and the control to one healthy normolipidemic subject.

Table 5 | Phospholipid and sphingolipid composition (WT%) of total HDL.

PC LPC SM Cer PE PI PS PG PA Proband 78.6 0.59 18.1 0.211 1.2 1.1 0.030 0.008 0.013

Control 83.0 0.44 12.9 0.160 1.8 1.7 0.055 0.006 0.006

Cer, ceramide; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin.

Data are shown as WT% of total phospholipids sphingolipids. The proband corresponds to individual III.3, and the control to one healthy normolipidemic subject.

hyperalphalipoproteinemia of apoC-III-D25V carriers confer an atheroprotective prole. This was corroborated by clinical data (detailed imaging of coronary angiography, pulse arterial Doppler ultrasound of the lower limbs and supra-aortic trunks, computed tomography (CT) angiography of the aorta and iliac vessels) establishing the absence of atheroma or vascular wall calcication, whereas carotid intima-media thickness was normal in the pro-band and his sister (III.5). Similarly, there was no record of ischaemic CVD in D25V-patients I.2 or II.1, who died aged 74 and 82 years, respectively.

Analysis of structure, function and aggregation propensity. Predictive analysis of the secondary structure of the apoC-III polypeptide in its lipid-free state revealed that the D25V mutation enhances both its beta-sheet content and aggregation propensity (Fig. 5a,b). Although wild-type and D25V apoC-III initially

showed similar circular dichroic spectra, the mutation appears to cause a transition towards prominent beta-sheet content (Fig. 5c,d) associated with rapid formation of aggregates (Fig. 6a), which were shown to increase the thioavin T uorescence16, the typical signature of amyloid brils (Fig. 6b). Transmission electron microscopy (Fig. 6c) and atomic force microscopy (Fig. 6d) conrmed the presence of brillar structures in the D25V apoC-III aggregated material.

The effect of the mutation on the lipid binding was also carried out using dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles17. Analysis of the binding curves based on the decrease in turbidity at 325 nm showed that the time required to reduce by 50% the initial turbidity of the vesicles was 168.78.1 s for the variant and 114.74.2 s for the wild-type, respectively (Fig. 7a), suggesting that the mutation may cause a less efcient lipid binding similar to other apoC-III variants17. At the equilibrium,

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a b

c

d

3

WT

1,000

500

Aggregation propensity

D25V

0 h0.5 h 4 h6 h

2

1

0

[] (deg cm2 dmol1 )

0

P

5,000

5,000

10,000

15,000

1

2

0 0 20 80

60

40

3 0 20 80

60

40

200 220 240

Wavelength (nm)

200 220 240

Amino-acid position

Relaxation

Wavelength (nm)

Figure 5 | Predicted and experimental beta-aggregation propensity at the site of mutation. (a) Protein b-sheet content was calculated using the GOR secondary structure prediction method53. (b) Aggregation propensity calculated according to the Zyaggregator method54. (c) Change in secondary structure monitored by far-UV CD during aggregation of both wild-type (WT) and (d) D25V apoC-III in PBS, pH 7.4, 37 C at 1,500 r.p.m. (IKA magnetic stirrer). CD spectra of samples (200 ml) at 0, 0.5, 4, 6 h, respectively, were recorded in 1.0 mm path length quartz cell, buffer subtracted and expressed as mean ellipticity per residue (y).

1.5

1.0

0.5

0.0 0 1 2 3 4 5 Time (h)

a b c d

Absorbance 350 nm (a.u.)

D25V

WT

ThT fluorescence (a.u.)

100

50

0 WT D25V

Figure 6 | Amyloid bril formation by D25V apoC-III. (a) Increase in turbidity at 350 nm was used to monitor the aggregation of wild-type (WT) and D25V ApoC-III in PBS, pH 7.4, and 1,500 r.p.m. (IKA magnetic stirrer). The curve shown is representative of three replicates. (b) Thioavin T (ThT) uorescence emission at 480 nm after excitation at 445 nm was performed for each apoC-III isoform at the end of protein aggregation, as shown in Fig. 5a. Bars show the ThTrelative intensity uorescence (arbitrary units, a.u.) of a 10-ml aliquot of protein samples in the presence of 10 mM ThT. Values are shown as means.d. of three independent experiments. (c) Negatively stained transmission electron micrograph of D25V apoC-III brils generated in physiological conditions. Scale bar, 100 nm. (d) Tapping mode atomic force microscopy (AFM) image (amplitude data) of ApoC-III brils. AFM measurements were performed in tapping mode with a Multimode Scanning Probe Microscope driven by a Nanoscope IV controller (Digital Instruments, Bruker), using single-beam uncoated silicon cantilevers (type OMCL-AC160TS, Olympus). The drive frequency was between 290 and 320 kHz, the scan rate was between 0.5 and 0.8 Hz. Scan size is 1.2 mm.

however, the variant was integrated in the vesicles as shown in the native electrophoresis (inset, Fig. 7a). The native electrophoresis revealed the strong propensity of the variant to self-aggregate in its free form when it aggregated and precipitated at the site of deposition (inset, Fig. 7a). Solubility of D25V apoC-III was, however, rescued when the variant was integrated within the vesicles. We have also investigated the effect of the mutation on the well-known inhibition of LPL activity by apoC-III (refs 3,18). The D25V apoC-III variant exhibited the same inhibitory activity when compared with the wild-type (Fig. 7b). It is worth noting that another mutation in position 25 (D25N, designed in silico), with the change in charge but unmodied hydrophobicity, was as efcient as the wild-type in inhibiting LPL activity19.

We nally compared the 3D NMR structure of D25V apoC-III with that of the wild-type counterpart already characterized in the presence of SDS9. Two-dimensional 1H,15N NMR spectra (Fig. 8a) showed small (o0.2 p.p.m., excluding residue 25) amide chemical shift changes in the variant (Fig. 8b,c), which were clustered within approximately one helical turn (3.6 residues) of the mutation site. No chemical shift perturbations were observed elsewhere in the sequence. The hydrodynamic radii of wild-type and D25V apoC-III micellar complexes, recorded by TRACT relaxation interference experiments, were virtually indistinguishable (Fig. 8d). Moreover, there was no difference in the helical conformation of residues 2030 (Fig. 8e).

DiscussionHere we report a previously unappreciated, naturally occurring APOC3 mutation68, which causes low levels of plasma TG and apoC-III and a favourable lipoprotein prole in a French kindred affected with amyloidosis. Concordance between the APOC3 mutation and the presence of amyloid among members of this kindred, coupled with identication of D25V apoC-III within amyloid brils from numerous affected tissues and in vitro demonstration of its amyloidogenicity, establishes that the D25V variant also causes amyloidosis.

The phenotype of this new form of amyloidosis is mainly characterized by onset with sicca syndrome as early as age 20 years, secondary to diffuse salivary gland deposits and progressive renal insufciency. We showed that these clinical symptoms are caused by amyloid deposition in the respective tissues with progressive and widespread amyloid accumulation within different organs. In this novel form of renal amyloidosis, amyloid deposition is characterized by prominent vascular involvement in all compartments of the kidney and ischaemic glomerular lesions that probably account for low-grade proteinuria and severe hypertension. This clinical renal presentation contrasts those described for Leu75Pro apoA1 amyloidosis20 and A-alpha chain amyloidosis21, which typically manifests as a chronic tubulointerstitial nephritis with amyloid deposits restricted to the inner medulla, or as proteinuria/nephrotic syndrome because of massive glomerular amyloid deposition, respectively, ApoC-III

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a b

100

120

+

100

Absorbance 325 nm

80

% LPL activity

80

60

60

40 40 20 20

0 0

0 0

1 2 3 4 5

D25V (t =168.78.1 s)

D25V WT

2 4 6 8 apoC-III (M)

WT (t =114.74.2 s)

200 400 600 800Time (s)

Figure 7 | Functional properties of wild-type and D25V apoC-III in vitro. (a) Lipid binding of ApoC-III was monitored by decrease in turbidity at 325 nm of DMPC vesicles mixed with recombinant wild-type or D25V ApoC-III at a lipid/protein ratio of 2:1 (w/w) over 900 s at 30 C. The binding curves shown are representative of three independent experiments and expressed as percentage of the initial absorbance of the vesicles at 325 nm. The time required to reduce by 50% the initial signal at 325 nm (t50%) is given as means.d. of three replicates. Inset, native gel electrophoresis shows the shift in mobility of both proteins after formation of the vesicles with DMPC. Lanes: wild-type apoC-III alone (1) and with DMPC (2); marker (3); D25V apoC-III alone (4) and with DMPC (5). Marker for electrophoretic mobility is a vefold diluted plasma (see Methods for details) in which the bands corresponding to the major plasma protein components are highlighted: albumin (I), a-1 antitrypsin (II), haptoglobin (III), transferrin (IV), brinogen (V) and g-globulin (VI). The arrow indicates the site of deposition of the samples where clearly the apoC-III variant precipitated. Negative and positive gel polarities indicate the direction of the electrophoretic run. (b) Inhibition of LPL activity was determined keeping constant the incubation time and measuring the free fatty acid release at different concentrations of the two apoC-III isoforms. Data plotted as means.d. of three independent experiments.

should be added to the list of protein variants associated with hereditary renal amyloidosis22, along with apoA-I (ref. 10, apoAII (ref. 11), lysozyme23, brinogen Aa-chain21 and gelsolin24. The diagnosis of apoC-III amyloidosis may be suggested by hypotriglyceridemia, present in all amyloidotic patients from this kindred, a biological marker that is usually absent in patients with other forms of hereditary renal amyloidosis.

Our in vitro studies clearly show that, despite the remarkable differences in folding dynamics and kinetics of aggregation between wild-type and D25V apoC-III in their lipid-free state, their conformation and colloidal stability are similar in the lipid-bound state. Wild-type apoC-III was recently reported to self-aggregate into polymeric ordered structures with a peculiar triangular geometry and a Mbius strip conformation after 3 days of shaking in physiological buffer. Although these structures do not resemble the genuine amyloid brils that we obtained under physiological conditions with the D25V variant, it is worth noting that aggregation of lipid-free wild-type apoC-III was preceded by a gradual structural transition that resembles, in circular dichroism (CD) spectroscopy, the conformation rapidly adopted by the D25V variant. There is no evidence suggesting in vivo amyloidogenicity of wild-type apoC-III and demonstration of aggregation in vitro does not necessarily imply amyloidogenicity in vivo. Importantly, the absence of wild-type apoC-III within the ex vivo amyloid brils from three patients in this kindred suggests that the wild-type protein does not contribute to amyloid deposition even in the presence of abundant D25V brillar seeds, analogous to what we have recently observed in patients with familial b2-microglobulin amyloidosis14.

We hypothesize that the preferential incorporation of D25V apoC-III into amyloid brils is likely the main cause of the imbalance in the ratio of wild-type/D25V apoC-III observed

within HDL particles. It will be worth investigating further in appropriate cellular and/or animal models whether the secretion of the variant may be partially impaired by the intracellular quality control, similar to what we previously described for some of the amyloidogenic variants of apoA-I (ref. 25), in which we found an imbalance in the wild type/variant ratio.

D25V-carriers displayed a reduction in plasma and HDL apoCIII concentration, hypotriglyceridemia associated with a dramatic decrease in the number of VLDL particles, and a concomitant massive increase in the larger HDL2 fraction. We propose that these are indirect effects of the D25V variant because of its very low concentration in plasma (and HDL fractions) because of its preferential association with amyloid brils. The lower plasma apoC-III concentration in D25V-carriers probably results in lack of inhibition of LPL and LDL-R. As a consequence, VLDL-TG hydrolysis proceeds at high rate and is followed by rapid uptake of VLDL-remnants (or intermediate density lipoprotein (IDL) in Fig. 4b), provoking the drastic decreases in VLDL and IDL of D25V-carriers (Fig. 4a,b). It has been well established that surface lipids of VLDL, which are liberated after LPL-mediated VLDL-TG hydrolysis, are transferred to HDL by phospholipid transfer protein, resulting in an increase in HDL size and particle number, as conrmed in mice decient either in LPL26 or in phospholipid transfer protein27 and which lack HDL. Importantly, subjects heterozygous for other loss-of-function APOC3 mutations (K58E28 and R19X5,6) also present with hypotriglyceridemia and increased plasma HDL.

The favourable lipid/lipoprotein prole of D25V-carriers is remarkable in the context of CKD and/or ESRD, pathological conditions predisposing to premature atherosclerosis and major ischaemic CVD events and commonly associated with an unfavourable lipid prole, an elevated plasma apoC-III concentration and dysfunctional HDL carrying increased amounts of apoCIII, serum amyloid A1 and lipoprotein-associated phospholipase A2 (refs 2931). Hypertriglyceridemia, often due to accumulation of VLDL remnants/IDL in the circulation, is an independent CVD risk factor32, and low plasma HDL is a second independent CVD risk factor33. D25V-carriers displaying hypotriglyceridemia and very high HDL2 are obviously lacking in CVD risk factors. Moreover, their HDL is functional, as evidenced by their normal antioxidant activity and cholesterol efux capacity possibly related to their enrichment in sphingomyelin (SM). Furthermore, the lower apoC-III content of HDL may contribute to atheroprotection as HDL particles without apoC-III reduce monocyte adhesion to vascular endothelial cells, whereas apoC-III-rich HDL do not, suggesting that apoC-III may have additional proatherogenic effects on vascular endothelial cells34. Thus, the pathologic amyloidogenic properties of D25V apoC-III unexpectedly confer a favourable cardioprotective lipoprotein prole indirectly, most probably through lowering of plasma apoC-III concentration, extending the evidence for an important antiatherogenic role of APOC3 deciency in ESRD patients.

As reducing the amyloid bril precursor protein concentration is known to slow amyloid formation and improve prognosis among patients with a variety of systemic amyloidoses, and as PPARa agonists (brates) signicantly reduce hepatic APOC3 transcription35,36, we postulate that brate therapy may have therapeutic potential for treatment of this new form of amyloidosis. Because the safety and side-effect proles of brates are well known, it might be reasonable to prescribe brates to the affected individuals from this kindred in an attempt to slow down amyloid formation. If successful, this therapy could represent an alternative to the gene silencing approach currently under evaluation in other systemic amyloidoses37.

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a

b

0.5

106

108

110

112

114

116

118

120

122

124

*

Chemical shift difference (p.p.m.)

WT D25V

G14

G41

0.4

0.3

G46

0.2

T74

T20

0.1

0 0 10 20 30 40 50 60 70 80

S55

S28

Residue

15 N chemical shift (p.p.m.)

S62 T44

c

S48 S10

S7

S33

T22

Q37

D25

H82

R40 V43 H18

V77

K24 S75 29S

M16 T56H83 S49

E81

Chemical shift difference (p.p.m.)

0.45

L27 H84 K51

L9/E32

D59

Q13 M12

V35 W65

F61

L80 E4

K17 F64

N

H86 25V

Q38/K58

H85

D52

C

F47 D5

D66

E63 K60

0

W54 Q31 Y53

V30

K21

d

E2

D45

1

Y15/V57

A39

A36 A78

R (WT) R (WT)

R (D25V) R (D25V)

Amide intensity

Q34 W42

0.8

F11

L50

A19 D68

A3 A6

0.6

A23L8 A26

0.4

0.2

A76

H87

8.8

8.6

8.4

8.2

8.0

7.8

7.6

0

1H chemical shift (p.p.m.)

0 0.1 0.2 0.3 0.4

Relaxation time (s)

e

K21 T22 A23 K24 V25 A26 L27 S28 S29 V30

V25H

1 H chemical shift (p.p.m.)

3.8

4.0

4.2

4.4

K24H

V30H

E63

T20H

K21H

S28H

S28H

S29H

S29H

A23H

T22H

L27H

M12

F61

A26H

H87

T22H

15N

1H

121.37.73

114.37.82

124.08.29

116.78.06

119.17.70

124.58.10

118.18.29

113.47.98

116.57.89

120.17.77

Figure 8 | NMR spectroscopy. (a) 1H,15N-Heteronuclear single quantum coherence (HSQC) spectra of wild-type (WT, blue) and D25V (red) apoC-III. Residue assignments of the variant were based on the Gangabadage9 assignment of the wild-type protein, using a nuclear overhauser effect spectroscopy (NOESY)-HSQC spectrum (120 ms mixing time) to reassign perturbed residues around the mutation site. Large chemical shift changes from the WT are highlighted with arrows. (b) Amide chemical shift differences between WT and D25V apoC-III, calculated as the weighted combination DdNH (DdH2

(DdN/ 5)2)1/2. An asterisk (*) highlights the position of residue 25. (c) Amide chemical shift differences projected onto the WT NMR structure (pdb2jq3) according to the indicated colour scale. Grey areas indicate unassigned residues or proline. (d) TRACT relaxation interference measurements34 of WT and

D25V apoC-III, determined by integration of the amide envelope and tting to single exponential decays. Fitted 15N relaxation ratess.e. were7.20.4 s 1 (WT) and 7.20.2 s 1 (D25V) in the a spin state, and 19.31.0 s 1 (WT) and 18.20.4 s 1 (D25V) in the b spin state, corresponding to hydrodynamic radii valuess.e. of 20.30.6 (WT) and 19.60.2 (D25V). (e) 1H strips from the NOESY-HSQC spectrum of D25V apoC-III. Dashed arrows highlight sequential NOEs from the Ha atom of residue i to the HN atom of residue i 1/i 3, which are diagnostic of a-helical structure.

Methods

Patients. The study was carried out in accordance with the Declaration of Helsinki, and was approved by the University of Marseille Institutional Review

Board. All authors vouch for the completeness and accuracy of the analyses and results. Oral and written informed consents were obtained from all participants, including the transfer of biopsy material outside of France.

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Genetic analysis. Genomic DNAs were extracted from peripheral blood samples, using the QIAamp DNA mini kit (Qiagen). Coding and consensus splice site regions of APOA1, APOA2, TTR, B2M, FGA, LYZ, GSN, SAA, APOE, APOC3, APOC2, APOC1, APOC4, APOA4 and APOA5 genes were amplied by PCR (AmpliTaq Gold; ABI, or Platinium Taq; Invitrogen), according to the manufacturers instructions, in a DNA thermal cycler 9700 (Perkin-Elmer). The primer sequences for APOC3 amplication are listed in Supplementary Table 2. The resulting PCR fragments were puried by QIAquick PCR Purication kit (Qiagen), and sequenced using the Big Dye terminator cycle sequencing kit (DNA sequencing kit; Applied Biosystems) on an automatic genetic analyser (ABI PRISM 3100 genetic analyzer; Applied Biosystems)14. APOC3 polymorphims at positions

455 (rs 2854116), and 482 (rs2854117) in the promoter region were

genotyped by direct sequencing. Mutation nomenclature was based on the APOC3 transcript reference (NCBI RefSeqcDNA accession number NM_000040.1), and nucleotides were numbered according to the cDNA with 1 corresponding to the

A of ATG translation initiation codon according to the Human Genome Variation Society guidelines, http://www.hgvs.org/mutnom//

Web End =http://www.hgvs.org/mutnom// . Therefore, the apoC-III variant is described as p.Asp45Val (with the signal peptide), whereas the mature protein is designed as D25V.

Histology and immunohistochemistry. Amyloid was detected by Congo red staining of formalin-xed wax-embedded biopsy sections14 (kidney, salivary glands, heart, bowel, fat and bronchia from III.3; kidney, salivary glands, skin, heart, liver from III.5; salivary glands, kidney, skin, kidney, bowel from II.3; salivary glands from IV.1, IV.3, and IV.4). Amyloid typing was performed by indirect immunouorescence and by immunoelectron microscopy14. For indirect immunouorescence, 3 mm cryostat sections of frozen tissue were air dried, incubated 1 h with primary goat anti-human ApoC3 polyclonal antibody (Abnova, 50 mg ml 1 in PBS), washed with PBS and stained with uorescent isothiocyanateconjugated donkey anti-goat antibody IgG, F(ab,)2 (Santa Cruz, 20 mg ml 1 in

PBS). For the immunogold labelling procedure, thin sections (80 nm) were cut from the routine (glutaraldehyde-xed, osmicated, Araldite embedded) electron microscopic blocks, collected on nickel grids. Grids were oated on one drop of 5% sodium metaperiodate (Sigma Chemical Co) in distilled water for 30 min. Sections were washed with distilled water three times and then saturated for 1 h with 3% BSA in PBS in order to minimize nonspecic labelling. The grids were then incubated overnight at 4 C with primary goat anti-human ApoC3 polyclonal antibody (Abnova, 100 mg ml 1in PBS).

Sections were washed six times with PBS, incubated for 1 h with a rabbit polyclonal biotin-conjugated anti-goat IgG secondary antibody (Clinisciences, 100 mg ml 1 in PBS) and 1 h with gold-labelled strepatavidin (Zymed Laboratories, 6 mg ml 1 in PBS). Immunolabelled sections were contrasted using uranyl acetate and observed under a JEOL 1010 transmission electron microscope (Jeol Ltd).

In all cases, parallel control negative sections consisting of the absence of the primary apoC-III antibody were performed, demonstrating the specicity of the apoC-III antibody.

LMD and tandem MS-based proteomics. Congo red-stained sections from salivary glands (IV.3), heart and digestive tract (III.3) were examined for the presence of amyloid under uorescence (Leica, B/G/R lter cube). Positive areas were microdissected into 0.5 ml microcentrifuge tube caps containing 10 mM Tris, 1 mM EDTA, 0.002% Zwittergent 316 (Calbiochem) using a Leica DM6000B Microdissection System (Leica). For each case, two to four different micro-dissections (60,000 mm2 each) were collected. Collected tissues were heated at 98 C for 90 min with occasional vortexing. Following 60 min of sonication in a water-bath, samples were digested overnight at 37 C with 1.5 ml of 1 mg ml 1 trypsin (Promega)15. The trypsin generated digests were reduced with dithiothreitol and separated by nanoow liquid chromatography-electrospray tandem mass spectrometry using a ThermoFinnigan LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron) coupled to an Eksigent nanoLC-2D HPLC system (Eksigent). A 0.25-m1 trap (Optimize Technologies) packed with Michrom Magic C-8 was plumbed into a 10-port valve. A 75 mm B15 cm C-18 column was utilized for the

separation utilizing an organic gradient from 6 to 86% in 55 min at 400 nl min 1. The Thermo-Fisher MS/MS raw data les were searched using three different algorithms (Mascot, Sequest and X!Tandem) and the results assigned peptide and protein probability scores. The results were then combined and displayed using Scaffold (Proteome Software). All searches were conducted with variable modications and restricted to full trypsin-generated peptides allowing for two missed cleavages. Peptide mass search tolerances were set to 10 p.p.m. and fragment mass tolerance to 1.00 Da. The human SwissProt database (24 July 2007) was complemented with frequently observed contaminants (porcine trypsin and human keratins). Protein identications below the 90% condence level and those with single peptide identication were not considered

Lipid and lipoprotein analysis. Plasma levels of TGs and HDL-cholesterol were measured in the non-fasting state using standard hospital biochemical assays. Plasma apoC-III concentrations were measured by electro-immunodiffusion. Lipoprotein classes were prepared by sequential ultracentrifugation of sera from the proband III-3 and a control subject run in parallel. Sequential ultracentrifugations

were performed at 100,000g and 10 C to isolate VLDL, IDL and LDL (for 18 h, at densities 1.006, 1.020 and 1.063 g ml 1, respectively) and HDL (for 40 h, at density1.21 g ml 1) in an OPTIMA XL 100 K Beckman ultracentrifuge38. After extensive dialysis of all lipoproteins against PBS containing/0.3 mM EDTA, their protein contents were measured with the Bio-Rad DC Protein assay kit II (Bio-Rad), and their TG, total cholesterol and phospholipid with commercial kits (DiaSys). HDL apolipoproteins were analysed by 15% SDSPAGE under non-reducing conditions to distinguish the apoA-II homodimer (17 kDa) from apo Cs38, and stained with Coomassie Brilliant Blue G 250. Puried human apoA-I and apoA-II, as major HDL apolipoproteins, were run as standards (4 mg per lane) in the gel. For apoC-III detection, proteins were transferred to a nitrocellulose membrane and immunoblotted with rabbit anti-human apoC-III antibody (1 mg ml 1 in PBS,

Abnova) and secondary ECL anti-rabbit IgG-HRP-Linked specic whole antibody (5,000-fold diluted in PBS, GE Healthcare). Puried human apoC-III (4 mg per lane) was added as standard in the western blotting. Protein bands were visualized with ECL Western Blotting Detection Reagent (GE Healthcare). Standard human apoA-I, apoA-II and apoC-III were puried from delipidated HDL by preparative isoelectric focusing with a pH gradient 46.

Fractionation of HDL subpopulations. HDL subpopulations were isolated from plasma (3 ml) by single step, isopycnic non-denaturing density gradient ultracentrifugation in a Beckman SW41 Ti rotor at 40,000 r.p.m. for 44 h in a Beckman XL70 ultracentrifuge at 15 C by a slight modication of the method of Chapman et al.39,40. After centrifugation, each gradient was fractionatedin predened volumes from the meniscus downwards with an Eppendorf precision pipette into 11 fractions corresponding to very-low-density lipoprotein and intermediate-density lipoprotein (VLDL IDL; 800 ml, do1.019 g ml 1),

LDL (5 subfractions, 800 ml each, LDL1, d 1.0191.023 g ml 1;

LDL2, d 1.0231.029 g ml 1; LDL3, d 1.0291.039 g ml 1; LDL4,

d 1.0391.050 g ml 1 and LDL5, d 1.0501.063 g ml 1) and HDL.

Five major HDL subclasses were isolated, that is, large, light HDL2b (1,200 ml,d 1.0631.087 g ml 1) and HDL2a (800 ml, d 1.0881.110 g ml 1), and small,

dense HDL3a (d 1.1101.129 g ml 1), HDL3b (800 ml, d 1.1291.154 g ml 1)

and HDL3c (800 ml, d 1.1541.170 g ml 1). The validity and reproducibility of

this density gradient procedure, which facilitates preparative fractionation of HDL particle subspecies in a non-denaturated, native state, have been extensively documented3941. All HDL subclasses isolated by this procedure are essentially albumin free (o1% of total protein, that is, o0.05 mg dl 1)42. Lipoproteins were extensively dialysed (3 cycles of at least 8 h) against PBS pH 7.4 at 4 C in the dark, stored at 4 C and used within 8 days.

Phosphosphingolipidome analysis. For the phosphosphingolipidome analysis, HDL subpopulations (30 mg phospholipid mass determined using a commercially available assay) were added to 4 ml of cold CHCl3/acidied CH3OH (5:2 (v/v))

containing seven internal lipid standards (4 mg of phosphatidylcholine d9 32:0, 100 ng of phosphatidylinositol (PI) 25:0, 80 ng of phosphatidylethanolamine, 25:0, 80 ng of phosphatidic acid 25:0, 40 ng of phosphatidylserine 25:0, 20 ng, of phosphatidylglycerol 25:0 and 20 ng of ceramide 17:0; Avanti Polar Lipids)43.

A blank and a control sample were extracted in parallel with each batch to ensure for quality control. K4EDTA (200 mM) solution was added (1:5 (v/v)) and the mixture was vortexed and centrifuged. The organic phase was dried under nitrogen and lipids were reconstituted into isopropanol/hexane/water (10:5:2 (v/v)), transferred into LC/MS vials, dried under nitrogen and resuspended in isopropanol/hexane/water (10:5:2 (v/v)). Seven principal phospholipid (PL) subclasses (phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, PI, phosphatidylglycerol, phosphatidylserine and phosphatidic acid) and two principal sphingolipid (SL) subclasses, SM and ceramide, which together comprise 4160 individual molecular lipid species and account for 495% of total plasma PL and SM44 were assayed by LC/MS/MS. Lipids were quantied using a QTrap 4000 mass spectrometer (AB Sciex), an LC20AD HPLC system, and the Analyst 1.5 data acquisition system (AB Sciex). Quantication of PLs and SLs was performed in positive-ion mode, except for PI species that were detected in negative-ion mode. Sample (4 ml) was injected onto a

Symmetry Shield RP8 3.5 mm, 2.1 50 mm2 reverse phase column (Waters

Corporation) using a gradient from 85:15 to 91:9 (v/v) methanol/water containing 5 mM ammonium formate and 0.1% formic acid at a ow rate of0.1 ml min 1 for 30 min. Lipid species were detected using multiple reaction monitoring reecting the head-group fragmentation of each lipid class and quantied using calibration curves specic for the nine individual lipid classes with up to 12 component fatty acid moieties; 23 calibration curves were generated in non-diluted and tenfold diluted matrices to correct for matrix-induced ion suppression effects. More abundant lipid species that displayed a nonlinear response in non-diluted extracts were quantied from a 10- or 100-fold diluted sample. An in-house developed script was used to compile data from the three successive injections.

Cellular cholesterol efux capacity of HDL. The cholesterol efux capacity of HDL was characterized in a human THP-1 monocytic cell system (ATCC)45. In this assay, HDL particles were compared on the basis of their PL concentrations

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because PL was shown to represent the key component determining cholesterol efux capacity of HDL46. Briey, THP-1 monocytes were cultured on 24-well tissue culture plates, grown in RPMI-1640 media with 10% FBS and differentiated into macrophage-like cells with 50 ng ml 1 phorbol 12-myristate 13-acetate for 48 h and 37 C. The cells were washed and loaded for 24 h with [3H]cholesterol-labelled acetylated LDL (1 mCi ml 1) in serum-free RPMI-1640 culture medium supplemented with 50 mM glucose, 2 mM glutamine, 0.2% BSA, 100 mg ml 1 penicillin and 100 mg ml 1 streptomycin (further abbreviated as RGGB) to allow equilibration of cellular cholesterol pools. The labelling medium was removed and human macrophages were then equilibrated in RGGB for an additional 1624 h period. Cellular cholesterol efux to 15 mg ml 1 HDL-PL was assayed in 300 ml serum-free medium for a 4-h chase period. Finally, culture media were harvested and cleared of cellular debris by brief centrifugation. Cell radioactivity was determined by extraction in hexane-isopropanol (3:2), evaporation of the solvent under nitrogen and liquid scintillation counting (Wallac Trilux 1450 Microbeta, Perkin Elmer). The percentage of cholesterol efux was calculated as (medium cpm)/ (medium cpm cell cpm) 100%. Specic cholesterol efux was

determined by subtracting nonspecic cholesterol efux occurring in the absence of cholesterol acceptors.

Antioxidative activity of HDL. Antioxidative activity of HDL (nal concentration of each, 10 mg total mass per dl) was assessed at a physiological HDL to LDL ratio towards reference LDL (d 1.0191.063 g ml 1; nal concentration, 10 mg dl 1

TC) isolated from one healthy normolipidemic control subject42,47,48. In this assay, HDL particles were compared on the basis of their total mass concentrations because both protein and lipid components were shown to contribute to the capacity of HDL to inhibit LDL oxidation43,49,50. HDL subfractions were added to LDL directly before oxidation. Lipoprotein oxidation was induced by an azo-initiator 2,20-azo-bis-(2-amidinopropane) hydrochloride (nal concentration 1 mmol l 1) as model of mild oxidation induced by free radicals in the arterial intima42,47,48. Serum was used as a source of HDL for this assay to ensure intact paraoxonase activity, which is inhibited by EDTA. Thereby, this assay employs mild oxidative conditions and integrates the antioxidative activities of several HDL components, that is, apoA-I, antioxidative enzymes and lipophilic low-molecular-weight antioxidants43,49,50. Accumulation of conjugated dienes was measured as the increment in absorbance at 234 nm. Absorbance kinetics values were corrected for the absorbance of 2,20-azo-bis-(2-amidinopropane) hydrochloride itself run in parallel as a blank. Three consecutive phases were identied, the lag, the propagation and the decomposition phases for each curve and duration of the propagation rate, oxidation rate in the propagation phase and amount of dienes formed at the end of the propagation phase (maximal concentration of dienes) were calculated as markers of antioxidative activity of HDL42,47,48.

MS analysis of apoC-III isoforms from HDL. Twenty micrograms of HDL fraction was dissolved and acidied in 20 ml of 0.1% triuoroacetic acid for 15 min with agitation. The protein was desalted using ZipTips C18 (Millipore) following the manufacturers instructions and eluted with 20 ml of saturated matrix solution of sinapinic acid in 50% acetonitrile and 0.5% (v/v) triuoroacetic acid. The mass spectrometric measurements were performed using a MALDI TOF/TOF 4800 Proteomics Analyzer mass spectrometer51. The MS spectra from m/z 8,000 to 10,000 Da were acquired both in reectron and in linear positive ion modes using 1,000 laser shots. For mass spectrometric identication of ApoC-III in HDL2/3 using LC-MS/MS, 25 mg of HDL fraction was dissolved into 500 mM triethylammonium bicarbonate, 1% SDS. Proteins were reduced with 10 mM dithiothreitol, alkylated with 20 mM iodoacetamide and trypsin digested overnight. The resulting peptides were cleaned up using OMIX C18 100 ml pipette tips (Agilent), and lyophilized before being reconstituted for the LC-MS/MS analysis. The peptides were separated using an Eksigent Ultra nano-LC HPLC system coupled with an AB Sciex Triple TOF 5600 mass spectrometer. The LC separations were performed using a Discovery Bio Wide Pore HPLC column (C18, 3 mm, 100 5 mm2). The mobile phases used were 0.1% formic acid in water (A) and

100% acetonitrile with 0.1% formic acid (B). The gradient elution steps were performed with a ow rate of 5 ml min 1 as follows: 040% B for 106 min, 4080%

for 5 min and then 80% B for an additional 5 min. All data were acquired using Analyst software (AB Sciex) in the data-dependent mode. Peptide proling was performed using a mass range of 3501,600 Da, followed by a MS/MS product ion scan from 100 to 1,500 Da. For each survey MS1 scan (accumulation time of250 ms), MS/MS spectra (accumulation time of 75 ms per MS/MS) were obtained for the 30 most abundant precursor ions. The protein identication was performed with the ProteinPilot Software 4.5.0 (AB Sciex). The MS/MS spectra obtained were searched against the UniProt database (release 2014_03). The search parameters for tryptic cleavage and accuracy are built-in functions of the software. Other data analysis parameters were as follows: sample type: identication; Cys-alkylation: iodoacetamide; digestion: trypsin; instrument: TripleTOF 5600; special factor: biological modications; species: Homo sapiens; search effort: thorough ID. Proteins comprising one or more peptides with a high condence score (95%) and a low false discovery rate (estimated local false discovery rate of 1%) were considered positively identied.

Radiolabelled SAP scintigraphy. Patients III.3 and IV.3 underwent 123I-labelled SAP scintigraphy52 (http://www.amyloidosis.org.uk/diagnosis/sap-scintigraphy-3/

Web End =http://www.amyloidosis.org.uk/diagnosis/sap-scintigraphy-3/). Puried human SAP was labelled with 123I using the N-bromosuccinimide method. Each patient received B100 mg SAP bearing 200 MBq of 123I, corresponding to an effective dose equivalent of B3 mSv. Thyroid uptake was blocked by oral ingestion of potassium iodide for 2 days before and following isotope injection. Anterior and posterior whole-body scintigraphy was performed 24 h after isotope administration using a GE Healthcare-Discovery gamma-camera.

Expression and purication of recombinant ApoC-III. ApoC-III was expressed from a pET23b vector containing the full-length cDNA for human ApoC-III, including the sequence encoding a C-terminal His6-tag preceded by Leu and Glu additional residues17. pET23b/D25V was generated by using a QuikChange site-directed mutagenesis kit (Stratagene). In a 50 ml reaction, 50 ng of plasmid DNA pET23b/ApoC-III was used as template and two synthetic oligonucleotide primers, the forward primer (50-CCAAGACCGCCAAGGtTGCACTGAGCA

GCG-30) and the reverse primer (50-CGCTGCTCAGTGCAaCCTTGGCGGTCT TGG-30) with the A-to-T base change complementary to opposite strands of the vector, were extended by Pfu Turbo DNA polymerase (Stratagene). For PCR, there was an initial cycle of 95 C for 30 s, followed by 12 cycles of 95 C for 30 s, 55 C for 1 min and 68 C for 2 min. After DpnI digestion of the parental deoxyadenosine methylase-methylated template, the synthesized mutated DNA was transformed into E. coli XL1Blue supercompetent cells. Candidate clones were screened by sequencing the ApoC3 insert on an ABI 377 prism DNA sequencer.

Prediction of beta aggregation propensity. The Garnier-Osguthorpe-Robson (GOR) method version IV (ref. 53) was used to predict the beta propensity at the mutation site. The aggregation propensities of wild-type and D25V ApoC-III were predicted using the Zyggregator method54.

Fibrillogenesis. Fibrillogenesis experiments were performed in standard quartz cells stirred at 1,500 r.p.m. (IKA magnetic stirrer) at 37 C using 100 mM ApoC-III isoforms in PBS, pH 7.4. Aggregation was carried out without seeds of preformed brils and the increase in turbidity at 350 nm was monitored. Thioavin T uorescence emission16 was measured at the end-point aggregation. Transmission electron microscopy analysis was performed using protein samples stained with 2% (w/v) uranyl acetate using a CM120 microscope at 80 keV. Atomic force microscopy analysis was carried out on 10 ml of brillar sample incubated on a freshly cleaved mica substrate for 5 min, then rinsed and dried. Conformational modications during aggregation of apoC-III were monitored by far-UV CD.

Lipid-binding properties of apoC-III. Association of recombinant wild-type and D25V apoC-III to DMPC multilamellar vesicles55, at a lipid-to-protein ratio of 2:1 (w/w), was followed by monitoring the decrease in turbidity of DMPC liposomes at 325 nm as a function of time at 30 C (ref. 17) in 0.01 M Tris-HCl buffer pH 8.0 containing 150 mM NaCl, 8.5% KBr, 0.01% NaN3 and 0.01% EDTA using a 1 cm path length quartz cell in a Jasco V-650 spectrophotometer equipped with a Peltier temperature controller. The lipid/protein association was expressed as percentage of the turbidity of DMPC vesicles in the absence of apoC-III; the time required to reduce the initial absorbance at 325 nm by 50% was determined from the curves to compare the lipid-binding properties of the two apoC-III isoforms. Electrophoretic mobility of wild-type and D25V apoC-III with and without DMPC was analysed using 1% medium electroendosmosis agarose gel in 70 mM barbiturate-sodium barbiturate buffer, pH 8.6 containing 2 mM calcium lactate. Samples (3 ml) were applied to the gel including vefold diluted human plasma in 2% bromophenol blue solution used as marker for protein electrophoretic mobility. Electrophoresis was carried out in a 10 C water-thermostated chamber at 20 V cm 1. Gel was then xed for 15 min in a 15% acetic acid solution containing 1% picric acid, dried and stained with Coomassie Blue.

LPL inhibition. Bovine LPL catalytic activity, in the presence of apoC-III was measured using an emulsion with the same composition as Intralipid 10% (Fresenius-Kabi, Sweden) containing [3H]triolein substrate56. The incubation medium contained 2% (v/v) of the emulsion, 0.1 M NaCl, 60 mg ml 1 BSA, 0.15 M

Tris-HCl, pH 8.5, and 16.7 U ml 1 heparin. The medium was pre-incubated with or without each apoC-III sample for at least 10 min before addition of bovine LPL(0.25 mg ml 1) in a nal 200 ml mixture. After 30 min incubation under agitation at 25 C, the reaction was stopped by adding 2 ml of isopropanol, heptane and 1 M H2SO4 (40:48:3:1) and 0.5 ml of water. Free fatty acids were extracted using sequential centrifugations in glass tubes for 3 min at 2,580g to separate each mixture in two phases. After the rst centrifugation, 800 ml of the upper phase was transferred to other vials containing alkaline ethanol and then added with 3 ml of heptane before mixing and centrifuging again. After removal of the upper phase, 3 ml of heptane were mixed to the lower phase, which was then centrifuged as above. Supernatant was removed and 800 ml of the lower phase transferred to a vial containing 2 ml of scintillation liquid to count radioactivity and quantify the [3H] free fatty acids (nmol ml min 1). The LPL activity was then expressed as percentage of the enzyme activity in the absence of apoC-III.

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NMR spectroscopy. NMR spectra were acquired for 0.5 mM samples of wild-type and D25V apoC-III (10 mM sodium acetate (pH 5.0), 180 mM SDS, 10% D2O) at 315 K, using a 700-MHz BrukerAvance III spectrometer equipped with a TXI cryoprobe, and processed with nmrPipe and CCPN Analysis.

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Acknowledgements

Supported by grants from lAssociation Franaise contre lAmylose, the Institut National de la Sant et de la Recherche Mdicale (INSERM) and the French National Reference Center for AL amyloidosis, the UK NHS Research and Development funds, the University College London Amyloidosis Research Fund and grants from the UK Medical Research Council (MR/K000187/1), the Rosetrees Trust/Royal Free Charity PhD programme (M427), the British Heart Foundation (PG08/008), the Wellcome Trust Investigator Award (097806/Z/11/Z), the Cariplo Foundation Projects (20140700 and 2013-0964), the Telethon Grant GG14127, the INBB (National Institute of Biostructures and Biosystems), the Italian Ministry of Health and the Italian Ministry of University and Research (Projects FIRB RBFR109EOS). Core support for the Centre for Amyloidosis and Acute Phase Proteins is provided by the UK National Institute for Health Research Biomedical Research Centre and unit Funding Scheme. We thank Professors Habib and Laurent Daniel for cardiac and salivary gland biopsies, Drs Gayet and Lebrun for their help in the clinical follow-up of proband III.3 with Dr Noemie Jourde, Ms Lacombe for help with immunohistology analyses, Professor Touchard for helpful discussions, Dr JulieA. Vrana and Jason D. Theis for assistance in proteomics analysis with Ahmet Dogan at the Mayo Clinics, Dr Magali Colombat and Franois Guillonneau and the Plateforme protomique de lUniversit Paris Descartes (3P5), Sorbonne Paris Cit, 75015 Paris, France, for their proteomic analysis of salivary gland from subject IV.3. We thank all the family members for their participation in this study. We thank Alejandra Carbajal and the Division of Medicine Electron Microscopy Unit, Royal Free Campus, University College London, London for imaging of amyloid brils in vitro. We acknowledge the

UCL Biological NMR Facility and the Medical Research Council for access to the Biomedical NMR Centre at the Crick Institute, London, UK, and the staff for their support.

Author contributions

S.V. initiated, conceived and supervised all the clinical, genetic and lipidic studies. B.N. performed all the genetic analysis, A.D.K., M.C., A.K., M.L. and C.D. performed and analysed the lipoprotein studies. N.J.-C. provided blood samples of the patients and coordinated the follow-up of patients; J.-M.G. performed histological analysis on amyloid sample tissues, and F.B. contributed to the experimental design and data analysis of amyloid deposit analysis. A.D. performed the proteomics analysis and interpreted the data. A.M. performed the MS analysis of apoC-III. M.D. and G.G. contributed to the discussion. All the in vitro experimental study was conceived, designed and supervised by V.B. G.V., P.P.M., R.P., C.A.W., A.R. and O.K. performed the research. P.J.T., G.O., M.S., J.C., J.D.G. and P.N.H. contributed to experimental design and discussion. All the authors analysed and interpreted the data. The paper was written by S.V., A.D.K., J.D.G., V.B. and reviewed and approved by all co-authors.

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How to cite this article: Valleix, S. et al. D25V apolipoprotein C-III variant causes dominant hereditary systemic amyloidosis and confers cardiovascular protective lipoprotein prole. Nat. Commun. 7:10353 doi: 10.1038/ncomms10353 (2016).

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