Understanding the Complex World of Amyloid Disorders: Where Does AA Amyloidosis Fit?
Laura Obici, MD
Amyloid Center – Biotechnology Research Laboratories University Hospital “San Matteo” Pavia, Italy
Amyloid
H&E
Congo red staining under polarized light
Courtesy of Professor CL Pirani.
Electron microscopy showing 7.5–10 nm rigid, nonbranching amyloid fibrils
Merlini G, Bellotti V. N Engl J Med. 2003;349:583-596.
Amyloid Structural Features
Congo red
Courtesy of Professor Merlini.
Atomic Force Microscopy of Amyloid Fibrils
600
400
200
0
200
400
600
0 nm
Merlini G, Bellotti V. N Engl J Med. 2003;349:583-596.
Relini A. et al, BBA 2004;1680:33-41.
Merlini G, Bellotti V. N Engl J Med. 2003;349:583-596.
Merlini G, Bellotti V. N Engl J Med. 2003;349:583-596.
Amyloidoses Are Protein Misfolding Diseases
Modified from Dobson C. Nature. 2003;426:884-890
Point Mutations Destabilize Lysozyme and Influence Amyloidogenicity
T70N D67H
0.8
1.2
1
F57I
W64R I56T
Fraction Unfolded
0.6 0.4 0.2 0 -0.2 0
I56T
T70N WT
D67H
1
2
3 GdnHCl (M)
4
5
6
5 natural variants have been isolated: I56T, F57I, W64R, D67H, and T70N Esposito G, et al. J Biol Chem. 2003;278:25910-25918.
b2 Microglobulin in Dialysis-related Amyloidosis
Dialysis-related amyloidosis requires an increased concentration of free b2-microglobulin
Proteolytic Remodeling Makes Proteins Amyloidogenic
100
(% b-aggregation per residue)
80 60 40 20 0 -20 0 5 10 15 20 25 30 35 40 45 Amino Acid Sequence
Ab42 has a significantly higher aggregation propensity compared to Ab40
Nussbaum, RL, Ellis CE. N Engl J Med. 2003;348:1356-13564. Fernandez-Escammilla. A Nature Biotechnology. 2004;22:1302-1306.
TANGO Score
Fibrillogenesis Is a Nucleation-Dependent Process
Merlini G, Bellotti V. N Engl J Med. 2003;349:583-596.
Merlini G, Bellotti V. N Engl J Med. 2003;349:583-596.
SAP Protects Amyloid Fibrils From Proteolysis
B
AAf + pronase
AAf + 50 mg/l SAP + pronase
AAf + 50 mg/l SAP + 7 nM MObDG + pronase
14
16
18
20
22
24
26
%125I released
Emsley J, et al. Nature. 1994;367:338-345.
Tennent GA, et al. PNAS. 1995;92:4299-4303
GAGs May Promote Amyloid Deposition
• Heparan sulfate (HS) is a universal component of amyloid • HS can increase the b-sheet content of murine SAA1.1 and Ab • HS co-deposits both temporally and spatially with AA fibrils in spleen
• Binding sites for HS have been identified for SAA, Ab, b2 microglobulin, immunoglobulin light chains
• GAGs mimetics inhibit amyloid fibril formation
Elimova E, et al. FASEB Journal. 2004;18:1749-1751.
Conditions Associated With AA Amyloidosis
Chronic Inflammatory Diseases
Rheumatoid arthritis Psoriatic arthritis Chronic juvenile arthritis Ankylosing spondylitis Behcet’s syndrome Reiter’s syndrome Adult Still's disease Inflammatory bowel disease Hereditary periodic fevers
Chronic Infections
Tuberculosis Osteomyelitis Bronchiectasis Leprosy Pyelonephritis Decubitus ulcers Whipple’s disease Acne conglobata
Neoplasia
Hepatoma Renal carcinoma Castleman's disease Hodgkin's disease Adult hairy cell leukemia Waldenström's disease
Common variable immunodeficiency Hypo/agammaglobulinemia
Cystic fibrosis
Hereditary Periodic Fevers
FMF mode of inheritance age at onset length of the acess abdominal pain musculoskeletal chest pain rash recessive <20 years 1-4 days very common (serositis) monoarthritis pleuritis, often unilateral rare (<5%) crysipelaslike on lower limbs pericarditis, scrotal attacks, splenomegaly recessive childhood 3-7 days very common athralgias unusual very common (>90%) or several types: maculopapular, papular headache cervical lymph nodes hepatosplenomegaly HIDS variable often more than 1 up to several weeks common myalgias yes common crysipelas-like including upper limbs orbital edema TRAPS dominant M-W_FCAS/CINCA dominant childhood neonatal variable rare arthritis/destruction no urticaria/crythema
other signs
deadness-cold sensitivity/dysmorphy papillitis
amyloids
treatment chromosomal locus gene Protein
yes
colchicine 16p13.3 MEFV Ma renostrin/pyrin
yes
none effective TNF inhibitors? 12q44 MVK Mevalonate
yes
Steroids TNF inhibitors? 12p13 TNFRSFIA Type 1 TNF receptor (55p)
yes
Steroids interleukin-1 inhibitor? 1q44 CIASI cryopyrin
Grateau G. Rheumatology (Oxford). 2004;43:410-415.
Serum Amyloid A Is a Polymorphic Protein
SAA1 Type I acute phase reactants
SAA2
SAAP3 not expressed in humans SAA 4 constitutive
SAA1
SAA1.1 SAA1.2 SAA1.3 SAA1.4 SAA1.5
In mice only SAA1.1 is deposited as AA-amyloid
In human SAA genotype may influence susceptibility to AA
Röcken C, Shakespeare. Virchows Arch. 2002;440:111-22. Husby G, et al. Amyloid. 1994;1:119-147.
Yamada T. Clin Chem Lab Med. 1999;37:381-388. Sipe J. Amyloid. 2000;7:10-12.
AA Amyloidogenesis
Tissue injury, infections IL-1, IL-6, TNF-
SAA1 (>70%) and SAA2
Plasma concentration changes from
HDL-SAA
3–10 mg/L 100–1000 mg/L
Specific receptors on macrophages (spleen, liver, kidney)
SAA released from HDL
AA fibrils
Modified from Röcken C, Shakespeare A. Virchows Arch. 2002;440:111-122.
Presenting Clinical Features in AA Amyloidosis
Feature
Proteinuria/renal insufficiency
Diarrhea/obstipation/malabsorption Goiter Neuropathy/carpal tunnel syndrome Hepatomegaly Lymphadenopathy Cardiac
%
91
22 9 3 5 2 1-2
Modified from Gertz, Kyle. Medicine. 1991;70:246.
AL Amyloidosis
IF anti l Kidney (74%)
Heart (58%)
Bone marrow plasma cell clone synthesizing amyloidogenic light chain No of organs involved: 1 (31%) >1 (69%)
Liver (28%)
GI (8%)
Updated and adapted from N Engl J Med. 2003;349:583-596.
Diagnosis Should Not Rely Only on Clinical Grounds
• 60-year old woman • Nephrotic syndrome and slowly progressing renal failure over the past 4 yrs • Renal biopsy: amyloidosis
• Proteinuria (11.8 g/24 h) • Serum creatinine 2.87 mg/dL • Serum high resolution IF: IgGk • SAA 71.1 mg/L (r.r.: <6.4) • IL-6 80 pg/mL (r.r. <3.12) • No known inflammatory diseases • DNA analysis for familial and hered. periodic fever s.: negative • June 2003, progressive renal failure (serum creatinine 2.3 mg/dL)
U S
IF anti-k
IF anti-g
U
S
U
S
Plasma-cell-Type Castleman’s disease with IL-6 release and increased SAA synthesis
Plasma-cell-type Castleman’s disease (H & E)
Amyloid in the lymph node, green birefringence in polarized light
University of Pavia and University Hospital San Matteo Biotechnology Research Laboratories
Giampaolo Merlini Vittorio Bellotti Vittorio Perfetti Laura Obici Giovanni Palladini Monica Stoppini Irene Zorzoli Palma Mangione Sabrina Marciano Sofia Giorgetti Alessia Andreola Simona Casarini Simona Donadei Milena Quaglia Francesca Lavatelli Valentina Navazza Sara Marini Alberto Bovera