explanted livers were used successfully for domino transplantation. Fibrinogen amyloidosis is a systemic amyloid disease with visceral, vascular, cardiac, and neurologic involvement. LKT is curative; however, cardiovascular amyloidosis may preclude this option. Our data encourage evaluation of preemptive solitary liver transplantation early in the course of amyloid nephropathy to prevent hemodialysis and kidney transplantation. (Blood. 2010;115(15):2998-3007)

Introduction

Amyloidosis is a protein misfolding disorder, in which normally soluble proteins undergo conformational changes and are deposited in the extracellular space as abnormal insoluble brils that progressively disrupt tissue structure and function.1 The clinical syndromes of autosomal dominant hereditary renal amyloidoses were rst described by Ostertag in 1932.2 To date, more than 25 different mutations in lysozyme,3 apolipoprotein AI (ApoAI),4,5 ApoAII,6,7 and brinogen A -chain genes8-11 have been identied that share in common the manifestation of amyloid nephropathy in middle age. Given the absence of overt peripheral neurologic disease, these forms are collectively referred to as nonneuropathic hereditary renal amyloidoses.12 Fibrinogen amyloidosis due to mutations in the brinogen -chain gene (AFib), rst described by Benson et al in 1993,8 is emerging as the most common type of all hereditary renal amyloid diseases in the United Kingdom and Europe,11,13,14 whereas our data (M.D.B.) from a tertiary US amyloid reference center suggest AFib is the leading cause of hereditary renal amyloidosis associated with nephrotic syndrome in the United States.8,15,16 There are currently no treatments that can lead to resolution of amyloid deposits. Management is restricted to attempting to interrupt further supply of precursor amyloidogenic proteins, in combination with supportive care of failing organs, including transplantation. The role of transplantation may be either potentially curative, as in liver replacement to eliminate the source of the variant transthyretin in familial amyloid polyneuropathy, or merely supportive to restore failing organ function.1,17,18 Fibrinogen production is exclusively hepatic.19 Isolated renal transplantation as a treatment for renal failure in AFib amyloidosis is of limited value. Kidney amyloid recurrence and subsequent allograft loss is almost universal, and occurs within 1 to 7 years. Ten-year graft survival among the 18 reported kidney transplants for AFib to date is 5.5%.11,20-23 This outcome compares poorly with the current half-life of a cadaveric renal transplant for all causes of chronic kidney disease (CKD) of at least 10 to 12 years, or 10-year graft and patient survival of 64% and 68%, respectively.24,25 The lack of success of isolated kidney transplantation in brinogen amyloidosis in the current climate of organ shortage prompted us to evaluate hepatorenal transplantation at the Amyloidosis Treatment Center at Kings College Hospital to manage the underlying disorder and prevent disease recurrence. Twenty-one of the cases presented here were initially evaluated at the UK National Amyloidosis Center, Royal Free Hospital, and appeared to have exclusively renal disease.22

Submitted May 31, 2009; accepted June 26, 2009. Prepublished online as Blood First Edition paper, November 30, 2009; DOI 10.1182/blood-2009-06-223792. An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked advertisement in accordance with 18 USC section 1734. 2010 by The American Society of Hematology
BLOOD, 15 APRIL 2010 VOLUME 115, NUMBER 15
BLOOD, 15 APRIL 2010 VOLUME 115, NUMBER 15 HEREDITARY SYSTEMIC A -CHAIN AMYLOIDOSIS 2999
We report major additions to the current phenotypic description of brinogen amyloidosis disease, previously unrecognized disease manifestations and risks, and the rst systematic evaluation for liver transplantation in this series of 22 patients with brinogen amyloidosis managed in our center; and we present the long-term outcome of 9 combined hepatorenal transplantations and 4 sequential (domino) liver transplantations using the explanted livers from patients with AFib who underwent transplantation.
Immunohistochemical characterization of cardiac amyloid deposits Immunohistochemistry was performed by standard technique. Sections were prepared and incubated sequentially in 1.5% goat serum for 30 minutes, rabbit antihuman brinogen 1:200 (Dako Cytomation Inc) for 30 minutes, biotinylated goat antirabbit immunoglobulin G (1:200; Vector Laboratories) for 30 minutes, ABC reagent (Vector Laboratories) for 45 minutes, and substrate for 3 to 7 minutes. Horseradish peroxidase substrate was prepared using FAST diaminobenzidine and urea H2O2 tablets (Sigma-Aldrich). Tissues were counterstained with hematoxylin. Representative sections were photographed on a Nikon Microphot-SA microscope with a SPOT RT-KE digital camera (Diagnostic Instruments). Biochemical analysis of amyloid atheromatous plaque Fibrils were isolated from an atheroma excised at endarterectomy from the carotid artery of a brinogen A -chain E526V patient by hand homogenization in 2 mL of 0.1M sodium citrate, 0.15M sodium chloride, and centrifugation. The pellet was homogenized and centrifuged again 2 more times. The nal pellet was solubilized in 1 mL of 8M guanidine hydrochloride, 0.5M tris(hydroxymethyl)aminomethane, pH 8.2, containing 10 mg of dithiothreitol/mL with magnetic stirring at room temperature overnight. The sample was alkylated with iodoacetic acid (25 mg/mL) and centrifuged. The supernatant was chromatographed on a Sepharose CL6B column (0.cm) equilibrated and eluted with 4M guanidine hydrochloride, 50mM tris(hydroxymethyl)aminomethane, pH 8.2. Fractions from approximately 25 kDa to the column volume elution area were pooled for analysis, exhaustively dialyzed in Spectra Por 6 dialysis tubing (Spectrum Laboratories) against water, and lyophilized. The pool was digested with trypsin in 0.1M ammonium bicarbonate overnight at room temperature, and the resulting peptides were fractionated by reverse-phase high-performance liquid chromatography on a Synchropak RP8 column (0.cm; Eprogen) equilibrated with 0.1% triuoracetic acid in water and eluted with a 0% to 60% acetonitrile gradient over 90 minutes. High-performance liquid chromatography fractions were dried in a Speed Vac concentrator (Savant Instruments) and analyzed by Edman degradation27 on an Applied Biosystems Model 491 cLC protein sequencer using the manufacturers standard cycles and methods. Statistical analysis Statistical analysis was performed using SPSS 17.0 for Windows software (SPSS Inc) for estimation of median and mean values and bivariate analysis for correlation of variables. Survival was estimated by Kaplan-Meier analysis.

Methods

Twenty-two patients with brinogen amyloidosis and stage III-5 CKD with median creatinine clearance of 0.26 mL/s/m2 (range, 0-0.86 mL/s/m2, SI system) and median glomerular ltration rate (GFR) of 16 mL/min (range, 0-52 mL/min) were assessed for combined liver and kidney transplantation between 1996 and 2007. Three patients had been misdiagnosed as primary systemic AL (light-chain) amyloidosis in association with B-cell dyscrasias13: 1 had received inappropriate chemotherapy and 2 had each received 2 renal allografts that all failed within 58 months. Twenty patients were British and 2, New Zealanders; 21 had the E526V and 1, the R554L AFib variant.22 None were diabetic. Median age at presentation was 55 years (range, 33-63 years), and at assessment 57 years (range, 49-68 years). Patient demographics are summarized in Table 1. Four patients aged 58 to 63 years with hepatitis C, cryptogenic cirrhosis, or alcoholic liver cirrhosis complicated by hepatocellular carcinoma underwent sequential (domino) liver transplantation in which we used as liver grafts the functionally and morphologically normal explanted livers from AFib patients who underwent combined liver and kidney transplantation. The domino recipients received full information and comprehensive pretransplantation counseling regarding the potential risks of transmission of AFib through domino transplantation and consented to the procedure as well as the requirements for regular long-term follow-up. Patients were managed in accordance with the Declaration of Helsinki. The Kings College Hospital Research Ethics Committee had sight of the study and indicated approval. Unrelated liver transplant regulatory authority (ULTRA, UK transplant) approval was obtained for all domino donors and recipients prior to placement on the transplant list. Written informed consent was obtained for diagnostic investigations, for tissue transfer, and for use of material in this publication. Clinical assessment Cardiovascular investigations comprised 12 lead electrocardiogram and transthoracic or transesophageal echocardiography at baseline. Patients with one or more risk factors of ischemic heart disease subsequently underwent coronary angiography. Endomyocardial biopsies were performed in the R554L case, and in 3 E526V patients with abnormal echocardiography. Iliac and carotid vessels were assessed with Doppler ultrasound. Cardiac autonomic function was assessed by serial measurements of heart rate variability and blood pressure monitoring at rest and after stress challenges, and 24-hour blood pressure monitoring. Peripheral neurologic assessment comprised sensory topographic mapping and the modied polyneuropathy disability score for evaluation of motor function. Patients who were accepted for transplantation and placed on the liver transplant list underwent re-evaluation at 6- to 12-month intervals during waiting time. Routine histology was carried out in all explanted livers from AFib patients undergoing combined liver and kidney transplantation before being sequentially used for domino transplant, to exclude parenchymal amyloid deposition and to assess the degree of possible steatosis and graft suitability. Posttransplantation follow-up comprised 3 monthly dual allograft function, creatinine clearance, and 24-hour urine protein levels. Neurologic evaluation, allograft Doppler ultrasound scans, and echocardiography were performed at least annually. Two AFib patients who had residual native kidney function with GFR of 15 and 12 mL/min, respectively, at the time of dual organ transplantation underwent dynamic and static 99mTc-DMSA renograms annually postoperatively.

Morphologic ndings

Kidneys. Extensive amyloid deposits were present in all renal biopsies, showing enlarged glomeruli replaced by amyloid with minimal interstitial involvement. Spleen. Spontaneous splenic rupture occurred in 1 patient during hemodialysis and in 3 further cases during transplantation. The excised spleens demonstrated widespread amyloid with predominantly trabecular and subcapsular distribution. Atheromatous plaque. The atheromatous carotid lesion exhibited Congo redpositive deposits that on transmission electron
Nine patients received combined liver and kidney allografts between January 1996 and September 2009 in our center. Transplantation procedures were complicated by spontaneous perioperative splenic rupture in 3 and postoperative deep vein thrombosis in 2 cases. At median follow-up of 67 months (range, 33-155 months), 6 of 9 patients are alive and well (cumulative survival, 67%) with normal liver function. Five patients have good renal function, creatinine 120M (range, 87-159M), and median GFR of 54 mL/ min (range, 45-69 mL/min). The sixth patient developed renal failure after 68 months and renal biopsy demonstrated chronic allograft nephropathy. All patients who underwent LKT before or within 6 months of initiating renal replacement therapy are alive and well (survival 100%), but outcome of LKT was successful in only 50% of patients who were on long-term hemodialysis at the time transplantation. Two fatal transplantation outcomes occurred in long-term hemodialysis patients and were due to biliary dyskinesia and acute necrotizing pancreatitis complicated by fatal bradyarrhythmia in a 62-year-old man, and biliary leak, sepsis, subendocardial infarct, and multiorgan failure in a 55-year-old woman. A further patient on peritoneal dialysis, previously misdiagnosed and inappropriately treated as AL amyloidosis in another facility, developed hepatic artery thrombosis requiring retransplantation but died 4 months after the initial transplantation. Median intensive treatment unit and overall hospital stay was 35 days (range, 2-130 days) and 40 days (range, 18-130 days), respectively. Patients received standard immunosuppression with tacrolimus and steroids and additionally mycophenolate mofetil. There were no episodes of rejection. Twenty-three echocardiography examinations in 8 patients after LKT have documented stable cardiac indices and no evidence of progressive or de novo cardiac amyloidosis, at up to 12 years of follow-up. Symptoms of gut dysmotility had continued in the 2 patients who had received previous isolated kidney transplants,
Table 1. Patient demographics, clinical characteristics, presenting features, diagnosis, and course of renal disease
Symptoms history to diagnosis, mo Mode of diagnosis Renal bx Renal bx Graft bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx Renal bx E526V E526V E526V E526V R554L E526V E526V E526V E526V E526V E526V E526V E526V Negative Negative Negative Negative Positive Positive Negative Negative Negative Negative Positive Positive Negative E526V Negative E526V Negative E526V Negative E526V Negative E526V Negative Negative Positive Positive Negative Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Negative Positive Positive Positive E526V Unknown Unknown E526V Negative Positive E526V Positive Positive E526V Negative Positive At CKD 3 At CKD 4 AFib variant 12 1.50 2.9 1.53 4.8 1.72 2.2 0.41 8.0 1.40 3.5 0.90 11.6 0.78 9.8 1.50 2.8 0.46 4.6 0.61 3.8 0.41 9.8 0.33 6.7 0.16 7.25 0.41 7.9 0.36 8.0 1.16 9.4 0.30 7.8 0.75 4.8 0.46 11.8 0.45 1.0 0.82 7.5 Cr Cl, mL/s/m2 Urinary protein, g/24 h Renal family history Cardiovascular family history Diagnosis to ESRF, mo

3002 STANGOU et al

Table 2. Findings during assessment for LKT, patient selection, and transplantation outcome
Echo ndings (IVS and LVPW in mm) ITU stay, d Listed Rejected Rejected Listed 8 2 130 Normal Normal Dilated LA EF 45%, IVS 13 Dilated RA, LA Normal Normal Normal Normal Normal Normal Normal Normal EF 45%, LVH IVS, LVW 13 Restrictive IVS 13, AV 18 Dilated CMP IVS, LVW 14 IVS, LVW 12.5 Wall akinesis* Impaired relaxation Mild None MI-stent None MI-CABG None None None IHD Carotid IHD Aorta Rejected Rejected IHD Carotid Listed IHD None Listed IHD, MI Iliac Listed IHD Carotid Listed None None Listed IHD, MI Carotid Listed 42 None Aorta Listed 34 IHD None Listed 8 None Femoral Listed 26 None None Listed 0 IHD Carotid Rejected IHD Carotid Rejected Previous MI Iliac Rejected IHD Aorta Rejected None None Listed 36 None None Listed 19 130 Coronary disease Systemic vascular disease LKT listing decision Duration of dialysis at LKT, mo
Duration of dialysis at assessment for LKT, mo

CrCl, mL/s/m2

Autonomic neuropathy

Hospital stay, d

LKT outcome survival, mo Alive 74 Alive 155 Alive 68 Alive 33 Dead 2.5 Dead 1.3 Alive 66 Alive 51 Dead 4.3 Awaiting LKT

Failed 2nd Tx

Cardiac, GI

Cardiac

LKT indicates liver and kidney transplantation; Cr Cr, creatinine clearance; Echo, echocardiography; IVS, intraventricular septum; LVPW, left ventricular posterior wall (thickness in millimeters); GI, gastrointestinal; EF, ejection fraction; CAGB, coronary artery bypass graft; MI, myocardial infarct; LA, left atrium; RA, right atrium; restrictive, restrictive physiology with impaired relaxation; IHD, ischemic heart disease; CMP, cardiomyopathy; and , nonapplicable because the patients did not receive LKT. *Patient no. 21 underwent echocardiography in another facility and echo ndings are excluded from analysis. Both patients received hemodialysis at end-stage renal disease before kidney transplantation and for varying periods between subsequent failed kidney transplants. Patient no. 22 was initially listed for preemptive isolated liver transplantation, but transplantation status was changed to listing for LKT when GFR fell below 50 mL/min during follow-up on the waiting list. Patient nos. 13, 14, 15, and 16 were initially assessed as suitable candidates and were listed for LKT, but were removed from the waitlist due to deteriorating cardiac function or progressive IHD 11 to 34 months later.
BLOOD, 15 APRIL 2010 VOLUME 115, NUMBER 15 HEREDITARY SYSTEMIC A -CHAIN AMYLOIDOSIS 3003

LAD Calcium

Figure 1. Morphologic ndings in the common brinogen A -chain amyloidosis E526 variant. (A) Congo red stain in renal biopsy (100 magnication) in brinogen A -chain amyloidosis. Extensive amyloid inltrate with glomerular enlargement and replacement of the normal glomerular architecture by amyloid deposition, with almost no amyloid in the interstitium. (B) Same section as panel A viewed between crossed polars shows apple green birefringence typical of amyloid deposits. (C) Congo red stain in histologic sample from a ruptured spleen (100 magnication) in AFib E526V exhibits extensive splenic amyloid deposits with predominantly trabecular and subcapsular distribution. (D) Endomyocardial biopsy specimen (100 magnication) in a patient with AFib E526V and renal failure, with abnormal echocardiography (patient 19, Tables 1 and 2). Cardiac histology stained with Congo red demonstrates extensive, diffuse amyloid deposition in the myocardium. An endomyocardial vessel is demonstrated at the center of the section, showing complete replacement of its entire wall thickness by amyloid deposition. (E) Bright apple green birefringence of the amyloid deposits in myocardium and endocardial vessels of section in panel D under cross-polarized light. (F) Endomyocardial biopsy (40 magnication) hematoxylin and eosin stain, in a patient with AFib E526V variant (patient 16, tables), on hemodialysis for 18 months, who had severe carotid atherosclerosis, and whose echocardiography showed normal wall thickness with impaired left ventricular relaxation. (G) Congo red stain demonstrates amyloid deposition at the periphery of an atrophic muscle ber from the same sample (100 magnication). (H) Apple green birefringence under polarized light in previous section. (I) Arterial intima atheroma (100 magnication) excised during endarterectomy for the indication of 80% carotid stenosis in the same patient as in panels D and E. Congo red stain demonstrates extensive amyloid deposition within the intima and atheromatous plaque (arrows). The Congo red material exhibited strong apple green birefringence. Immunostaining with antibrinogen antibodies was not performed because normal (wild-type) brinogen is expected to be part of the thrombus, and therefore a positive immunohistochemistry could not be reliably positive for a diagnosis of variant brinogen amyloidosis. (J) Transmission electron microscopy (magnication) images of the atheroma in panel F demonstrates brillar material with bril diameter of approximately 10 nm, compatible with amyloid. Fibril extraction and characterization revealed the amyloid atheromatous plaque to consist wholly of variant E526V brinogen. (K) Images from subsequent coronary angiography in the same patient, carried out to exclude signicant asymptomatic coronary atherosclerosis as part of generalized amyloid angiopathy, in context of ndings in panels F through I. Left anterior oblique cranial angiogram of left coronary artery shows diffuse atheroma in the left anterior descending coronary artery (LAD) with heavy calcication (arrows). (L) Left anterior oblique angiographic projection of the right coronary artery (same patient as in panel K) shows diffuse atheroma throughout its course. Leads from dual chamber pacemaker inserted for the indication of bradyarrhythmias are also shown (arrow).

but resolved in all patients after hepatorenal transplantation. All patients have resumed normal everyday activities and those of nonretiring age have been able to return to full employment. After elimination of the source of amyloid production through hepatorenal transplantation, whole-body 123I-labeled serum amyloid P component (SAP) scintigraphy demonstrated regression of systemic visceral amyloid deposits as early as at rst annual follow-up scan after transplantation. None of the
AFib patients who underwent transplantation presented evidence of de novo or progressive amyloid deposition at up to 13 years of follow-up as previously reported.23 In contrast, SAP scintigraphy documented progressive systemic amyloidosis and amyloid deposition in the kidney grafts in 2 AFib patients in this series who had previously undergone isolated kidney transplantation.22,23 One domino recipient had SAP scans and echocardiography examinations for up to 5 years with no evidence of
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Figure 2. Dilated amyloid cardiomyopathy in brinogen A -chain amyloidosis R554L variant. (A) Endomyocardial biopsy (100 magnication) in a 55-year-old patient with the AFib R554L variant and stage II CKD, whose echocardiography during evaluation for combined LKT showed dilated cardiomyopathy with ejection fraction of only 25% (patient 18, tables). Endomyocardial histology shows bright apple green birefringence of diffuse Congo redpositive endocardial and interstitial amyloid deposits. No additional pathology or potential causes for dilated cardiomyopathy other than amyloidosis were identied. (B) Strongly positive brinogen immunohistochemistry using rabbit antihuman brinogen 1:200 (Dako Cytomation Inc), in the same section of panel A. (C) Apical 4-chamber 2-dimensional echocardiographic view at end diastole, demonstrating a dilated and globular left ventricular cavity. (D) Imaging from M-mode echocardiogram in the same patient, demonstrating increased left ventricular dimensions and reduced function. (E) Parasternal long axis echocardiographic images of increased left ventricular end diastolic dimensions. (F) Invasive angiogram of the left coronary artery (right anterior oblique projection), showing a 40% stenosis in the proximal anterior descending artery (black arrow), lumen irregularity, and more minor narrowing in the intermediate vessel (dotted white arrow) and narrowing in the rst septal perforator (white arrow). The patients right coronary had minor irregularity (not shown).

de novo amyloid deposition. The remaining 3 patients did not have SAP scans. One has normal liver and renal function at 2.5 years, and 2 were lost to follow-up after returning to their country of origin after successful liver transplantation (LT). Serial dynamic and static 99mTc-DMSA renal scintigraphy after preemptive LKT in 2 cases demonstrated stable native renal function, with consistent contribution to the overall kidney function of 14% and 20% at up to 5 years of follow-up (Figure 3).

Discussion

Fibrinogen is a plasma protein with a crucial role in the coagulation cascade through its conversion to brin, and is composed of 2 identical sets of 3 polypeptide chains termed A , B , and , joined by disulde bridging. Each polypeptide is encoded by a distinct gene, FGA, FGB, and FGG. The gene for the brinogen A -chain with 610 amino acid residues is localized on chromosome 4 and has 6 exons.30 Mutations in any of the 3 genes encoding for brinogen polypeptides can cause dysbrinogenemias, and recently identied mutations in the A -chain gene can lead to hereditary systemic amyloidosis.12 Six amyloidogenic mutations in the brinogen A -chain gene have been described to date. The rst variant, identied in a Peruvian-Mexican family and 2 unrelated African-American
and French kindreds, is caused by a point mutation in the -chain gene encoding for substitution of arginine to leucine (R554L) in the brinogen molecule.8,16 The E526V and E540V variants, 2 frameshift mutations (4904delG and 4897delT), and more recently an insertion/deletion (1636-1650del, 16491650insCA) variant in the brinogen -chain gene were identied in kindreds of Irish, British, Portuguese, French, German, and Far Eastern origin with amyloid nephropathy due to variant brinogen.9-12,31 Renal amyloidosis in AFib universally progresses to complete ESRF and the focus of clinical intervention has been to provide adequate renal replacement therapy including kidney transplantation.13,22 Age of onset varies with different mutations, from childhood in the insertion deletion mutation to middle life in the most common E526V variant.8-10,31 The onset of the disease is usually heralded by the development of renal amyloidosis, and penetrance is reportedly low.13,22 Our data provide valuable insights into the disease phenotype and suggest that hereditary brinogen amyloidosis is neither solely nephropathic nor solely nonneuropathic, but is a disease with a diverse and complex phenotype. All reported cases to date have been diagnosed through renal pathology, during investigation for hypertension, kidney impairment, and proteinuria, frequently identied during routine medical screening. However, in light of the ndings in this series, corroborated by another recent report,32 a

BLOOD, 15 APRIL 2010 VOLUME 115, NUMBER 15 HEREDITARY SYSTEMIC A -CHAIN AMYLOIDOSIS 3005
Figure 3. Serial 99mTc-DMSA scintigraphy shows stable renal graft: native kidneys divided function at 1 and 4 years after preemptive combined liver and kidney transplantation, in AFib E526. (A-B) Serial 99mTc-DMSA scintigraphy in an AFib E526V patient who received preemptive combined liver and kidney transplantation at stage 5 CKD, 1 month before scheduled commencement of hemodialysis. (A) Posterior view of dynamic renal scintigraphy at 1 year after combined liver and kidney function, 3 hours after injection of 80 MBq 99mTc-DMSA. Regions of interest (ROI; circles) shown are used in the calculation of divided function. The percentage of divided function is as follows: transplant kidney 86% and total native 14%. (B) DMSA scintigraphy in the same patient at 4 years after LKT. The panel shows posterior view scintigraphy 3 hours after injection of 80 MBq 99mTc-DMSA, and same regions of interest are used in the calculation of divided function. The percentage of divided function is as follows: transplant kidney 87% and total native kidney function 13%. We have conrmed by mass spectrometry19 in this patient that variant brinogen A -chain has been completely eliminated from the plasma and is replaced by normal (wild-type) brinogen A -chain after LKT.
bias toward underdiagnosing cases with predominantly cardiovascular amyloidosis, in the absence of readily accessible tissue for histologic sampling, cannot be excluded and may account for the apparent reduced penetrance. We have identied a high incidence of cardiovascular atheromatous disease among our patients, often predating evolution of proteinuria or renal impairment by many years. There was also a strong family history for coronary/vascular disease among carriers even in the absence of overt renal disease (Table 1). Our ndings are unlikely to be due solely to the vascular effects of renal failure,33 because the specic form of brinogen amyloid was present in the vascular walls and atheromatous plaques. Biochemical analysis indeed revealed that amyloid atheromatous plaques, and cardiac and vascular amyloid deposits in AFib were composed wholly of variant brinogen, and excluded the presence of other amyloid precursor proteins with inherent atherogenetic properties such as senile transthyretin or apolipoprotein ApoAI.34 This is the rst report of a link between variant brinogen amyloidosis and atheromatosis. A plausible explanation for the syndrome of systemic amyloid angiopathy in AFib lies principally with direct amyloid deposition in vascular walls and myocardial vessels. The associated, but likely not necessarily prerequisite, manifestations of nephrotic syndrome, hyperlipidemia, hypertension, and declining renal function facilitate atheroma formation on a background of vascular amyloid deposition and impaired endothelial function, further accelerated by commencement of renal replacement therapy.33,35,36 The well-described predilection for renal amyloid localization to the glomerulus rather than interstitium largely represents a form of amyloid vascular disease within the kidney, and is in accord with our observation of vascular deposition of variant brinogen.13,22 We are further investigating the mechanisms through which vascular amyloidosis may be proatherogenic in AFib and other types of systemic amyloidosis associated with nephrotic syndrome

such as systemic AL and AA amyloidosis, as well as the possibility that coronary amyloid atheromatous lesions may per se mimic atherosclerotic appearances on conventional angiography. The presence of amyloid deposits in the vascular walls of endomyocardial vessels, as well as systemically, suggests that a specic type of global coronary amyloid cardiomyopathy may exist in brinogen amyloidosis. This syndrome can result in clinical presentation with dilated amyloid cardiomyopathy as in the R554L case presented here, rather than the typical features of restrictive hypertrophic cardiomyopathy universally seen in the systemic amyloidoses.28 Cautious interpretation of echocardiographic ndings as well as coronary angiography imaging ndings is thus required to avoid misdiagnosis or underdiagnosis of disease features. Our observation emphasizes the importance of awareness regarding cardiovascular symptoms for those at potential risk such as AFib carriers and nonscreened family members, and of counseling regarding risk factors such as smoking, obesity, hyperlipidemia, and hypertension. Conversely, screening for proteinuria is inexpensive and readily available, and we recommend its routine use in the primary care monitoring of families with history of cardiovascular events. Neuropathic features have been observed in association with uremia, and may be reversible after successful renal transplantation. The clinical pattern of cardiac parasympathetic and systemic autonomic neuropathy in this series, corroborated by previous demonstration of cardiac denervation on iodine-131-metaiodobenzylguanidine scintigraphy and resolution of symptoms after hepatorenal but not after kidney transplantation, suggest this is a true amyloid-related autonomic manifestation.20,37 Splenic involvement in AFib may be clinically signicant as manifested by resistant anemia,20 in the absence of demonstrable splenomegaly. Spontaneous or intraoperative splenic rupture in 4 cases due to extensive splenic amyloid is a specic risk, and has led us to alter the transplantation surgical technique to using superior mesenteric venous bypass, to avoid even transient rises in

3006 STANGOU et al BLOOD, 15 APRIL 2010 VOLUME 115, NUMBER 15
portal pressure. AFib patients receive prophylactic triple vaccination for meningitis, pneumococcal pneumonia, and inuenza upon placement on the waiting list. Liver transplantation is the standard treatment for a number of genetic hepatic metabolic disorders including Wilson disease, hemochromatosis, homozygous hypercholesterolemia, ornithine transcarboxylase deciency, and primary hyperoxaluria type I, and more than 1200 liver transplantations have been performed worldwide for transthyretin-related familial amyloid polyneuropathy.38,39 Fibrils isolated from amyloid deposits in AFib have consistently been shown to contain exclusively variant brinogen. Circulating total brinogen in AFib consists of a mixture of wild-type variant brinogen in a ratio of 1:1 to 3:2; however, only the variant brinogen molecule is incorporated in the amyloid brils, suggesting that unlike transthyretin, wild-type brinogen does not perpetuate amyloid disease.9,20 We have previously shown by mass spectrometry (Table 2, patients 2, 7, and 11) that after liver transplantation the variant brinogen is eliminated and promptly replaced by wild-type brinogen.19 The long-term AFib E526V hepatorenal transplant recipients have no amyloid progression at up to 12 years of follow-up, suggesting that liver transplantation for brinogen amyloidosis may be truly curative.19-21,23,26 We have used 4 of the explanted AFib livers for sequential (domino) transplantation in patients with end-stage liver disease and hepatocellular carcinoma, without evidence of AFib disease transmission at up to 5 years of follow-up. The unexpected salvage of residual native kidney function in the 2 patients who received LKT preemptively at stage IV CKD is of particular interest. Stable contribution of 15% to 20% of total (native and graft) renal function is maintained at 4 and 5 years after LKT. We suggest that the apparent long-term stabilization of residual native amyloidotic kidney function in both cases is attributed to arresting the disease through liver replacement. In conclusion, we have shown that AFib is a systemic amyloid disease with multivisceral and neurologic involvement, and is associated with cardiac amyloid deposition and amyloid angiopathy and atheromatosis. Hepatic amyloidosis is rare but can lead to liver failure. The addition of liver transplantation to kidney transplantation in AFib with ESRF is curative. We support AFib as a new indication for liver transplantation, and suggest that lowcardiovascular-risk patients are a favorable group for combined liver and kidney transplantation. Long-term hemodialysis patients, or those cardiovascularly unsuitable for the combined approach, could undergo kidney transplantation as the best form of renal replacement therapy.40 The characterization of the disease phenotype as systemic amyloid disease justies earlier intervention to arrest amyloid disease progression, and renal outcomes of preemptive hepatorenal transplantation encourage evaluation of isolated liver transplantation early in the course of amyloid nephropathy to prevent systemic and renal progression to ESRF and requirements for dialysis and kidney transplantation. Amyloidotic kidneys may be exceptionally vulnerable to perioperative hemodynamic changes and possible nephrotoxicity of immunosuppression. It is thus recommended that AFib patients who are listed for preemptive isolated LT should be monitored monthly while on the waiting list, to ensure that GFR is maintained at levels greater than 50 mL/min at the time of a suitable liver graft being available for LT. The transplantation status of patients whose GFR falls below the safety cutoff of 50 mL/min on the LT waiting list should be altered to LKT, as in

case 22 in this series. The explanted liver grafts can be used in domino transplantation, thus neutralizing the impact on the supply of liver allografts. We encourage all centers involved in the management of amyloid disease to report transplantation and domino procedures for brinogen A -chain amyloidosis to the Familial Amyloid Polyneuropathy World Transplant Registry (FAPWTR, www. FAPWTR.org), to enable international centralized data collection and meaningful analysis of long-term outcomes in this novel indication.

Acknowledgments

We acknowledge and thank the scientists and staff in Merrill Bensons Amyloid Research Group, Pathology and Laboratory Medicine, Indiana University School of Medicine; Mr Hisham Rashid, Vascular Surgery Department; Mr Benjamin Corcoran, Nuclear Medicine Department at Kings College Hospital; Mr Bart Wagner in the Electron Microscopy Department, Northern General Hospital; Mrs Katharine Bleasdale-Barr and technicians in the Neurovascular Medicine Department, NHNN, Queen Square; Dr Margaret Burke and Dr Alex Bell, Histopathology Department, Hareeld Hospital; Profs Ian Simpson and Edward Gane, Auckland City Hospital; Profs Mark Pepys and Philip Hawkins and Staff in the National Amyloidosis Center, Royal Free Hospital; the Nephrology Departments of the Queen Elizabeth, Heartlands, and Wolverhampton Hospitals; the Glasgow Western Inrmary Hospital, the Moriston Hospital; Wirral Hospital; the Preston and Blackpool Victoria Hospitals; the Bournemouth Hospital; the John Radcliffe Hospital; and Nuffield Department of Surgery.

Authorship

Contribution: A.J.S., J.O., N.D.H., and M.D.B. are responsible for the conception, design, organization, and execution of the study; A.J.S. wrote the paper; J.O. and N.D.H. contributed to editing of the paper; N.R.B. contributed to the design and organization of the study and drafting and editing of the paper and carried out some of the cardiovascular investigations; B.M.H. contributed to the renal management of the patients and the editing of the paper; M.R. devised the surgical technique for liver transplantation in brinogen amyloidosis and performed the combined liver and kidney transplantations; B.P. examined and typed the histologic samples, and contributed to editing of the paper; J.W. has been responsible for expert intensive care management and has seen and approved the paper; M.M. and P.M. contributed to the cardiovascular investigations; M.B.-T. was responsible for the nuclear medicine investigations; C.J.M. has been responsible for all neurologic investigations and has seen and approved the paper; J.J.L. and M.D.B. carried out the amyloid bril characterization and typing of amyloid deposits; and M.D.B. contributed to drafting and editing the paper. Conict-of-interest disclosure: The authors declare no competing nancial interests. Correspondence: Arie J. Stangou, Institute of Liver Studies and Amyloidosis Treatment Center, Kings College Hospital, Denmark Hill St, London, SE5 9RS, United Kingdom; e-mail: arie.stangou@kcl.ac.uk.

30. 31.

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