AA- (Secondary) amyloidosis SAA 104 aa AA 1

Plasma cell dyscrasia and renal amyloidosis: What we can learn from basic science

Per Westermark Rudbeck Laboratory Uppsala University, Sweden

Definition of Amyloid • Characteristic ultrastructure • Cross b-Sheet Fibrils (X-ray diffraction) • Binding of Congo red with resulting green birefringence • Usually extracellular deposits • In vivo

Folding intermediate

Toxic oligomer

Newly synthesized Folded, functional protein protein Normal function and metabolism

Amyloid fibrils

Merlini & Westermark, JIM 2004

Importance of seeding Steady state

SEED Elongation

Lag phase

TIME

The word ’amyloid’ is used widely:

• • • •

Amyloid in deposits (”real” amyloid) Synthetic ”amyloid” Amyloid-like material in nature (silk etc) ”Functional amyloid”

Amyloid fibril proteins and their precursors in human

_______________________________________________________________________________________ AL Immunoglobulin light chain Systemic AH Immunoglobulin heavy chain Systemic Aβ2M β2-microglobulin Systemic ATTR Transthyretin Systemic AA (Apo)serum AA Systemic AApoAI Apolipoprotein AI Systemic AApoAII Apolipoprotein AII Systemic AApoAIV Apolipoprotein AIV Systemic AGel Gelsolin Systemic ALys Lysozyme Systemic AFib Fibrinogen α-chain Systemic ACys Cystatin C Systemic ABri ABriPP Systemic ADan* ADanPP Systemic ALect2 Leucocyte chemotactic factor 2 Systemic Aβ Aβ protein precursor (AβPP) Brain APrP Prion protein Brain ACal (Pro)calcitonin C-cell thyroid tumors AIAPP Islet amyloid polypeptide (amylin) Islets of Langerhans, insulinomas AANF Atrial natriuretic factor Cardiac atria APro Prolactin Aging pituitary, prolactinomas AIns Insulin Skin (Iatrogenic) AMed Lactadherin Aortic media AKer Kerato-epithelin Cornea, familial ALac Lactoferrin Cornea AOaap Odontogenic ameloblast-ass. protein Odontogenic tumors ASemI Semenogelin I Vesicula seminalis

Validation of the 3D profile method for prediction of fibrillizing segments.

Goldschmidt L et al. PNAS 2010;107:3487-3492

©2010 by National Academy of Sciences

Some protein factors important for amyloid fibril formation • • • • • •

b-sheet propensity Hydrophobicity Charge Destabilisation, e.g. by mutation Concentration Degradation

Systemic amyloidosis • 15 different proteins known • Precursor circulating in plasma • The precursor may be modified before deposition, e.g. by truncation • Depositions in many organs

Human systemic amyloid fibril proteins and their precursors AL AH Ab2M ATTR AA AApoAI AApoAII AApoAIV AGel ALys AFib ACys ABri ADan ALECT2

Immunoglobulin light chain Immunoglobulin heavy chain b2-microglobulin Transthyretin Serum AA Apolipoprotein AI Apolipoprotein AII Apolipoprotein AIV Gelsolin Lysozyme Fibrinogen a-chain Cystatin C ABri PP ADan PP Lecocyte chemotactic factor 2

Primary; myeloma associated Primary; myeloma associated Hemodialysis Familial; senile systemic Secondary, reactive Familial Familial Sporadic? Familial Familial Familial Familial Familial Familial Sporadic

Other components in amyloid: • Amyloid P-component: AP = SAP is a glycoprotein, produced by the liver. Up to 10% of the dry substance can consist of SAP

• Proteoglycans:

Heparan sulfate proteoglycan (HSPG)

• Apolipoprotein E • Other components

Systemic AL amyloidosis • Extreme variation in deposition pattern • Sometimes enormous amounts of amyloid (kilograms) • Life threatening • Early diagnosis important

Immunoglobulin molecule Antibody molecule

Light chain structure

Fab: antigen binding Variable (VL) heavy chain , H light chain , L VH variable region, V

VL

CH

constant region, C

CL

CH

Fc: mediates the effect

FR

CDR

Constant (CL)

AL amyloidosis = primary and myeloma associated amyloidosis • Primarely a plasma cell disorder: monoclonal expansion • Monoclonal immunoglobulin light chain • Manifests with extreme variations: heart, liver, kidneys, peripheral nerves etc. • Treatment with melphalane + prednisolone and/or peripheral stem cell transplantation

AL amyloid Each protein is unique; no two sequences are identical: About 50 different genes for light chains Somatic mutations Therefore it is not surprising that each patient has a unique disease Properties of the amyloid vary from patient to patient Affinity for Congo red varies from very weak to strong. The same is true for the green birefringence in polarized light

Germ line

Germ line

Enqvist et al., PLoS ONE 2007

Immunoglobulin light chain and AL protein

Variable segment

Constant segment

N

C

Major part of amyloid fibril AL protein is encircled

Fragmentation pattern of monoclonal light chains in six different AL proteins of kappa type

Enqvist et al., J. Path. 219:473; 2009

Distribution of amyloid in patients with AL-amyloidosis coming from the two genes O18-O8 and L2-L16

Enqvist et al., PLoS ONE 2007

Examples of questions to be answered • What determines fibril formation from an immunoglobulin light chain? • Is cleavage of the light chain of importance? • What initiates fibril formation? Nidus? • What determines the deposition pattern? • Are there toxic oligomers in AL amyloidosis • Which is the role of HSPG in AL amyloidosis?

Amyloid Diagnostics

Biopsy Rectum

Subcutis (abdomen) surgical needle (not too thin) Organ with symptom: kidney, liver, skin

Source of errors: Under or over diagnostics Insufficient material Incorrect staining Errors in microscope (light, polarizers) Unexperienced examiner

Determination of amyloid type

• Direct method necessary

N Engl J Med 2002, 346:1786-1791. Misdiagnosis of hereditary amyloidosis as AL (primary) amyloidosis Lachmann HJ, Booth DR, Booth SE, Bybee A, Gilbertson JA, Gillmore JD, Pepys MB, Hawkins

Out of 350 patienter with preliminary diagnosis of AL amyloidosis 34 (9.7%) had familial forms! 18 AFib 2 AApoAI 13 ATTR 1 ALys

Typing of amyloid • Clinical findings: Not enough! • Immunohistochemistry: Sometimes good but there are pitfalls • Immune electron microscopy • Immunochemistry: Western blot, ELISA • Mass spectrometry and/or N-terminal sequence analysis after protein extraction

Surgical Fat Tissue Biopsy For Typing and for Protein Studies

Immunohistochemistry Limitations with commercial antibodies Light chain antibodies: directed against the Cterminal part Own antibodies: Mab Pwlam AA ATTR

AA-amyloid, MAB Sne5

AL-amyloid lambda, MAB PWlam

AL-amyloid lambda, MAB PWlam

AL-amyloid lambda, MAB PWlam

ATTR-amyloid, polyklonal 1898

Mass spectrometry Identification of specific peptides, e.g. after tryptic digestion Laser Dissection Microscopy

Enqvist, Hellman et al., unpublished

Amyloid fibril proteins and their precursors in human

_______________________________________________________________________________________ AL Immunoglobulin light chain Systemic AH Immunoglobulin heavy chain Systemic Aβ2M β2-microglobulin Systemic ATTR Transthyretin Systemic AA (Apo)serum AA Systemic AApoAI Apolipoprotein AI Systemic AApoAII Apolipoprotein AII Systemic AApoAIV Apolipoprotein AIV Systemic AGel Gelsolin Systemic ALys Lysozyme Systemic AFib Fibrinogen α-chain Systemic ACys Cystatin C Systemic ABri ABriPP Systemic ADan* ADanPP Systemic ALect2 Leucocyte chemotactic factor 2 Systemic Aβ Aβ protein precursor (AβPP) Brain APrP Prion protein Brain ACal (Pro)calcitonin C-cell thyroid tumors AIAPP Islet amyloid polypeptide (amylin) Islets of Langerhans, insulinomas AANF Atrial natriuretic factor Cardiac atria APro Prolactin Aging pituitary, prolactinomas AIns Insulin Skin (Iatrogenic) AMed Lactadherin Aortic media AKer Kerato-epithelin Cornea, familial ALac Lactoferrin Cornea AOaap Odontogenic ameloblast-ass. protein Odontogenic tumors ASemI Semenogelin I Vesicula seminalis

Future Directions Other amyloid markers (e.g. polyelectrolytes) Proteomics Molecule-specific diagnosis? Individe-specific treatment?

Thanks to Knut Sletten, Oslo, Norway Ulf Hellman, Uppsala Charles Murphy, Knoxville, TN Gunilla T. Westermark, Uppsala Stina Enqvist, Uppsala All former and present graduate students All other coworkers

Informa Healthcare Informahealthcare.com