Orthotopic liver transplantation for familial amyloidotic polyneuropathy: A pathological study

Orthotopic Liver Transplantation for Familial Amyloidotic Polyneuropathy: A Pathological Study BETH HUIZENGA SHAZ, MD, FREDRIC GORDON, MD, W, DAVID LEWIS, MD, ROGER L, JENKINS, MD, MARTHA SKINNER, MD, AND URMILA KHETTRY, MBBS Familial amyloidotic polyneuropathy (FAP), a hereditary form of systemic amyloidosis with clinicallysignificant neuropathy and cardiomyopathy, is caused by a genetic defect of the transthyretin gene, which is mostly synthesized in the liver. Orthotopic liver transplantation (OLT) is thought to eliminate the amyloidogeuic protein and currently is the only definitive treatment for this disorder. The aim of this study was to define the distribution and extent of amyloid deposition in tissues from these patients and evaluate the suitability of the resected FAP rivers for transplantation into non-FAP patients. Surgical specimens from 14 patients removed at the time of OLT and autopsy tissues from 3 of the 14 were examined histologically using hematoxylin and eosin and Congo red-stained sections. The extent of amyloid deposits was evaluated, semiquantitatively graded from negative to marked, and correlated with clinical course and patient outcome. Amyloid deposits were consistently seen in hilar and vagus nerves. Liver lobular involvement was minimal in 1 and absent in the

other 13 cases, with portal arterial amyloid deposits seen in 7 cases. At autopsy, extensive amyloid deposition in the heart was seen in all 3 cases with involvement of the conduction system. The extent of amyloid deposition at OLT did not correlate with the duration of symptoms before OLT or patient outcome after OLT. In conclusion, fiver parenchymal involvement in FAP is minimal, and these explants are suitable for grafting in non-FAP patients. The recipients of such grafts must be carefully observed for the development of any amyloid-related disease, particularly cardiomyopathy. Of the tissues removed at OLT, the histopathologic confirmation of FAP is most consistently made by the examination of hilar and vagus nerves. HUM PATROL31:40-44. Copyright © 2000 by W.B. Saunders Company Key words: liver, transplant, amyloid, neuropathy. Abbreviations: FAP, familial amyloidotic polyneuropathy; TTR, transthyretin; OLT, orthotopic liver transplantation.

Familial amyloidotic polyneuropathy (FAP) is a hereditary form of systemic amyloidosis resulting in clinically significant neuropathy and cardiomyopathy. The symptoms usually begin between the ages of 25 to 35 years but may occur later in life, and death occurs 7 to 15 years later. 1 The genetic defect is in the transthyretin (TTR) gene, and the mutant amyloidogenic protein is formed from one of multiple known single amino acid substitutions. 2 Because approximately 98% of TTR is synthesized in the liver, liver transplantation is thought to eliminate most of the amyloidogenic protein and is currently the only definitive treatment for FAR Because patients with FAP do not develop cirrhosis or chronic liver disease, and livers removed at the time of orthotopic liver transplantation (OLT) from these patients appear grossly unremarkable, attempts have been made to transplant resected FAP livers into other patients in a " d o m i n o " fashion. ~,4 Clinical follow-up on these patients, both FAP and " d o m i n o " recipients, using m o d e r n radiological techniques and nuclear scanning, has been reported from many centers; however, detailed morphological evaluation of their tissues

removed at the time of OLT is not available in the literature. 3,5-7 The aim of this study was to define the distribution and extent of amyloid deposition in tissues from these patients and critically evaluate the suitability of the resected FAP livers for transplantation into non-FAP patients.

PATIENTS AND METHODS

Patients Our study population consisted of 14 patients with FAP, between the ages of 27 to 59 years, who were referred from the Amyloid Treatment and Research Program at Boston University Medical Center to the Beth Israel Deaconess Medical Center for OLT. All patients had clinical symptoms ranging from 1 to 12 years before transplantation. The pre-OLT and post-OLT patient characteristics and clinical evaluation have been published elsewhere. 1 Pretransplantation diagnosis in each patient was based on the following: (1) known family history, (b) symptoms of peripheral neuropathy, (c) genetic studies, and (d) fat-pad biopsy. Sural nerve biopsies were not performed.

Tissue Specimens Examined for This Study

From the Departments of Pathology, Medicine, and Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School; the Department of Medicine, Boston University School of Medicine, Boston, MA; and Lahey Clinic Medical Center, Burlington, MA. Accepted for publication September 21, 1999. Presented in part at the United States and Canadian Academy of Pathology Meeting in San Francisco, CA, March 22, 1999. Address correspondence and reprint requests to Urmila Khettry, MD, Department of Pathology, Lahey Clinic Medical Center, 41 Mall Rd, Burlington, MA 01805. Copyright © 2000 by W.B. SaundersCompany 0046-8177/00/3101-0007510.00/0

Removed at the Time of OLT. Specimens removed at the time of OLT consisted of native livers with attached hilar soft tissue-bearing segments of hepatic artery, portal vein, bile duct, hilar nerves, and recipient's gallbladder (if present). In 11 cases, vagus nerve branches with ramifications close to the liver hilum resected for technical purposes were also available for this study. At the Time of Autopsy. Four of 14 patients died at variable periods after OLT. Two of these 4 had autopsies performed at our medical center. A third patient had a postmortem exami-

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OLT FOR FAMILIALAMYLOIDOTIC POLYNEUROPATHY(Shaz et al)

FIGURE 1. Hilar nerve removed at the time of OLT with sparse (+) ovoid areas of amyloid disrupting the nerve fibers. (Congo red, magnification x850,)

FIGURE 2. Hilar nerve with prominent deposits of amyloid (+ + +) resulting in marked expansion of the nerve. (Congo red, magnification x850.)

FIGURE 3. Liver removed at OLT with portal triad artery showing thickening with amyloid within the wall, Some edema fluid is seen in the portal connective tissue. (Hematoxylin and eosin, magnification x850.)

FIGURE 4. Hilar soft tissue from case LT-292 with amyloid deposition around adipocytes giving them a "'ringed" appearance. (Hematoxylin and eosin, magnification x850,)

outcome. An overall histological grade (semiquantitative) was assigned to each patient based on neural (hilar and vagus) involvement. Furthermore, histological liver parenchymal involvement was critically evaluated to determine its suitability for transplantation into another patient.

nation at another institution. Autopsy slides from these 3 cases were reviewed. Histologic Examination

Formalin-fixed, paraffin-embedded tissue sections stained with hematoxylin and eosin and alkaline modification of Congo red stains were examined by 2 pathologists (B.H.S., U.K.), initially independently followed by a joint review. Green birefringence by polarized light microscopy was used to confirm the presence of amyloid on Congo red-stained sections. The presence or absence of amyloid deposits in all structures examined was recorded. When present, the amyloid deposition was graded on a semiqnantitative scale, as follows: negative ( - ) - - n o amyloid seen on Congo red, focal ( - / + ) - - s c a n t amyloid seen only by Congo red, mild ( + ) rare small ovoid areas seen by hematoxylin and eosin and confirmed by Congo red (Fig 1), moderate ( + + ) - - m u l t i p l e but small areas of deposit easily detected on hematoxylin and eosin, and severe (+ + + )--numerous large deposits (Fig 2).

RESULTS Tissues r e m o v e d

a t OLT

A m y l o i d deposits within the hilar nerves were seen consistently in all 14 patients. T h e s e deposits were usually n o t p r o m i n e n t a n d c o u l d be easily missed o n h e m a t o x y l i n a n d e o s i n - s t a i n e d sections. T h e deposits within the n e r v e h a d a linear o r ovoid to r o u n d c o n f i g u r a t i o n with a "star-burst" a p p e a r a n c e in s o m e areas. T h e areas o f the deposits were h o m o g e n e o u s a n d eosinophilic with i n t e r r u p t i o n o f the n o r m a l n e r v e contours. T h e hilar vessels did n o t show any t h i c k e n e d or h o m o g e n i z e d walls. A m y l o i d in hilar soft tissue was p r e s e n t in only 4 cases. Within the liver p a r e n c h y m a , the lobular architecture was p r e s e r v e d in all cases. N o r m a l hepatic cords a n d sinusoids were p r e s e n t with n o e x p a n s i o n o f subsinusoidal space with amyloid. In the portal tract, involve-

Clinical Correlation

The extent of amyloid deposition in structures removed at the time of OLT was correlated with age at the time of OLI, clinical course until the time of transplantation, and patient

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@

N

FIGURE5. Peripheral nerve at autopsy with congophilic deposits. (Congo red, magnification ×250.)

FIGURE 6. Choroid plexus with globular homogenous subepithelial masses of amyloid. (Hematoxylin and eosin, magnification x850.)

FIGURE 7. Myocardium at autopsy with prominent subendocardial amyloid, Arterial amyloid is seen within the interstitium. (Congo red, magnification ×250.)

FIGURE 8. Atrioventricular node dissected at autopsy with prominent irregular masses of amyloid staining purple in this section. Nodal cells are inconspicuous and atrophic. (Trichrome, magnification x850,)

m e n t of arterial wall was seen in 7 cases (Fig 3). In 6, this was minimal with patchy thickening and scant Congophilia; the percentage of involved portal tracts containing such arteries ranged from 30% to 50%. Bile ducts and veins were not involved with amyloid deposits. Segments o f vagus nerve removed in 11 cases showed involvement by amyloid in 10. The deposits were similar in appearance to those seen in hilar nerves, but somewhat more conspicuous. Of the total 14 cases, LT #292 had the most severe involvement of all structures. H e r vagus nerve was markedly thickened with exuberant endoneurial deposits disrupting the myelinated fibers. Within the liver, rare foci of amyloid confirmed by Congo red stain were f o u n d in the subsinusoidal location in the lobules. These foci were inconspicuous and could be detected on the Congo red stain only. Consistent involvement of arterial walls was seen in all portal triads. Hilar vessel and soft tissue involvement was also extensive. Adipocytes in the soft tissue had massive rimming with eosinophilic h o m o g e n e o u s material, giving them a '°ringed" appearance (Fig 4). This patient also had a 3.0-cm epithelioid angiomyolipoma in the right lobe of

the liver confirmed by positive staining for HMB45 and negative staining for keratin proteins. The amyloid deposition detected during histological review for this study is depicted in Table 1. The overall extent of histological amyloid involvem e n t of tissues removed at OLT, selected clinical parameters, including the outcome and TTR mutations, are shown in Table 2. Tissues E x a m i n e d a t A u t o p s y

Of the 4 patients who died at variable periods after the OLT, 3 u n d e r w e n t postmortem examination. The amyloid deposition was most prominently seen in the vascular system, with small muscular arteries involvem e n t in virtually every organ examined histologically. Eosinophilic, h o m o g e n e o u s material within the arterial muscular walls made these small vessels rather conspicuous; however, in some organs such as lung and gastrointestinal tract, Congo red stain was necessary to highlight the presence of amyloid within the arterial walls. As expected, the other consistently involved tissue was neural (Fig 5); cranial nerves in 2 patients, spinal

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OLT FOR FAMILIALAMYLOIDOTIC POLYNEUROPATHY(Shaz et al)

TABLE 1.

Distribution of Amyloid Deposits in Tissues Removed at OLT

parenchymal involvement. Amyloi d deposits were in the myocardium, most prominent beneath the subendocard i u m (Fig 7 ) , w i t h s h r i n k a g e a n d c y t o p l a s m i c e o s i n o p h i l i a o f m y o c y t e s . V a l v u l a r i n v o l v e m e n t was n o t s e e n i n a n y o f t h e cases. T h e c o n d u c t i o n s y s t e m o f t h e h e a r t w a s dissected and examined in the 2 autopsies that were performed at our medical center. Prominent amyloidosis o f b o t h S A a n d A V n o d e s was s e e n i n b o t h c a s e s ( F i g 8). I n a d d i t i o n , t h e c o n s t i t u e n t c e i l s o f t h e c o n d u c t i o n system showed atrophy with condensation of cytoplasm and nuclear pyknosis. K i d n e y i n v o l v e m e n t was s e e n i n all 3 cases. I n t h e renal cortex, most glomeruli had typical globular deposits o f a m y l o i d i n o n l y 1 case. R e n a l m e d u l l a was d i f f u s e l y i n v o l v e d i n 2 cases. T h y r o i d was n o t i n v o l v e d i n 1 c a s e t h a t was a v a i l a b l e f o r review. S p l e n i c a r t e r i e s w e r e m i l d l y i n v o l v e d i n all 3 c a s e s w i t h o u t a n y r e d p u l p d e p o s i t s .

Liver Portal Hilar Hilar Vagus Soft Patient Lobules Tract* Vessels Nerves Nerve Gallbladder t Tissue LT-249 LT-257 LT-258 LT-266 LT-267 LT-292 LT-304 LT-305 LT-323 LT-368 LT-373 LT-387 LT-402 LT-442

+/-

+/+/+/+/+ +/ +/-

++ -

+ + + + + ++ + + + + + + + +

NA + NA + +++ ++ + + +/NA ++ ++ +

+/+/+ ++ + + + NA

+ NA ++ + + -

NOTE. Amyloid deposition: - = negative, + / - = focal, + = mild, + + = moderate, + + + = severe. Abbreviations: NA, not available; OLT, orthotopic liver transplantation. *Mostly vascular amyloid deposition. tVascular and muscle amyloid deposition.

DISCUSSION OLT has been employed with success in the treatm e n t o f m a n y h e r e d i t a r y m e t a b o l i c d i s o r d e r s , s,9 S o m e of these disorders affect the liver parenchyma, resulting i n h e p a t i c f a i l u r e , w h e r e a s i n o t h e r d i s o r d e r s t h e l i v e r is the predominant source of an abnormal substance, causing debilitating or life-threatening dysfunction of o n e o r m o r e o r g a n s . FAP, o n e o f t h e l a t t e r d i s o r d e r s , is characterized by a variably progressive, inevitably fatal form of hereditary systemic amyloidosis caused by accumulation of a mutant form of transthvretin (TTR), affecting the peripheral and autonomic nervous system, o f t e n w i t h i n v o l v e m e n t o f t h e c a r d i o v a s c u l a r system. L i v e r is t h e p r o d u c t i o n site o f a p p r o x i m a t e l y 9 8 % o f T T R i n t h e body, a n d t h e r e m a i n i n g 2 % is f o r m e d in t h e c h o r o i d p l e x u s , r e t i n a l e p i t h e l i u m , a n d p a n c r e a t i c a l p h a cells. O L T is n o w a w e l l - r e c o g n i z e d t r e a t m e n t f o r F A P a n d is r e p o r t e d t o a c h i e v e a r a p i d a n d l o n g - t e r m diminution in circulating mutant TTR. Although the results of early post-OLT studies are cautiously optimistic w i t h a h a l t i n d i s e a s e p r o g r e s s i o n r e p o r t e d f r o m various centers, clearcut demonstrable improvement in peripheral neuropathy or cardiomyopathy have not b e e n o b s e r v e d yet. 1'1°'11 O L T c e r t a i n l y i n t e r r u p t s o r a l t e r s t h e n a t u r a l c o u r s e o f FAP; t h e r e f o r e , a c l o s e c l i n i c a l f o l l o w - u p o f t h e s e p a t i e n t s is m a n d a t e d , a n d some way to obtain an international standardization has been suggested) 2 With the exception of one comprehensive pathol o g i c a l s t u d y i n v o l v i n g 17 a u t o p s y c a s e s w i t h o u t p r i o r OLT, details of histological examination are sparse in most of the recent post-OLT reports./s Comparing our autopsy data with the results reported in the aforement i o n e d study, w e c o n f i r m t h a t t h e t i s s u e d i s t r i b u t i o n o f amyloid and the severity of involvement were not too dissimilar in the 2 studies, one following the natural course of the disease, and the other with potential d i s r u p t i o n o f t h a t c o u r s e . 13 C e r t a i n m i n o r d i f f e r e n c e s , however, were observed with indeterminant statistical significance because of the low number of autopsy cases i n o u r s e r i e s . I n 2 o f o u r a u t o p s y cases, w h e r e b r a i n a n d spinal cord were examined, involvement of dorsal

n e r v e r o o t s a n d s y m p a t h e t i c c h a i n i n all 3, p e r i p h e r a l n e r v e ( p h r e n i c ) i n all 3, a n d s m a l l e r n e r v e s w i t h i n t h e v a r i o u s v i s c e r a l o r g a n s m all 3 as well. C r a n i a l n e r v e s a n d b r a i n w e r e n o t e x a m i n e d a t a u t o p s y i n o n e c a s e (LT #2661. performed at the outside institution. The amyloid deposits were endoneurial and could be seen on h e m a t o x y l i n a n d e o s i n s t a i n s as a n i r r e g u l a r a r e a o f homogeneous pink material disrupting the normal wavy p a t t e r n o f a n e r v e t r u n k o r b r a n c h . T h e p a t t e r n a n d e x t e n t o f t h i s i n v o l v e m e n t was i d e n t i c a l t o t h a t s e e n in the hilar and vagus nerves removed at the time of OLT. N o a m y l o i d d e p o s i t was s e e n i n b r a i n p a r e n c h y m a or spinal cord. Sections of choroid plexus were available in 1 of 2 cases performed at our institution and showed g l o b u l a r d e p o s i t s o f a m y l o i d ( F i g 6). I n all 3 p a t i e n t s , t h e h e a r t h a d t h e m o s t d i s c e r n i b l e

TABLE 2.

ClinicopathoIogic-Genetic Correlation in 14 FAP Patients

Age at Overall OLT Duration of Histological TTR Severity Mutation Patient /yr) Disease (yr) LT-249 LT-257 LT-258 LT-266 LT-267 LT-292 LT-3O4 LT-305 LT-323 LT-368 LT~373 LT-387 LT-402 LT-442

35 35 40 38 42 42 34 34 59 44 47 29 27 39

4 2 5 I2 I0 3 3 4 17 1 9 2 6 3

--±~ --

Met30 Gly42 Met30 Met30 Gly42 Gly42 Gly42 Met30 His58 Met30 Met30 Met30 Met30 Met30

Outcome (days) Alive (2495) Alive (2412) Alive (2397) Dead* (239) Alive (2331) Dead* (1443) Dead* (103) Alive (1936) Dead* , 15) Alive (1345) Alive (1262) Alive ~1108) Alive (988) Alive (580

NOTE. ~- = mild. - - - moderate, ~-~ - severe amyloid deposition dnclusive of all nerves examined). *All 4 deaths resulted from fatal arrythmias causing sudden heart failure and not from complications of transplantation.

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ventral root ganglia was conspicuously absent. Peripheral nerve involvement was just as p r o m i n e n t as in that earlier autopsy study on 17 patients and also similar to the a m o u n t of amyloid seen in most of our hilar and vagus nerve specimens removed at the time of OLT. This lack of reduction in neural amyloid perhaps correlates with n o n i m p r o v e m e n t in peripheral neuropathy after OLT. Amyloid deposition within the heart was concentrated in the subendocardial region (extensive in one patient) and was consistent in all 3 autopsy cases. Demonstration of amyloid in the conduction system in 2 cases was strongly suggestive of arrhythmia being the cause of sudden death. The organ involvement was somewhat similar in the 3 autopsy cases; variability in the extent of involvement seemed to be the rule. Because the m i n o r sites of a mutant TTR production are unaffected after OLT, some concern has been raised regarding changing the pattern of amyloid deposits in long-term post-OLT FAP survivors. 12 In some studies, the mutant TTR p r o d u c e d by choroid plexus has been implicated as t h e sole source of neural amyloid deposits in FAP patients, ls,14 Because the overall number of FAP patients undergoing OLT is low, standardized autopsies must be conducted to evaluate any post-OLT altered amyloid distribution in these patients. The concept of " d o m i n o d o n o r " is being evaluated at certain liver transplantation centers to circumvent, partially, the dire shortage of d o n o r organs. Explanted livers from FAP patients have been used for such purposes based on their normal gross appearance and absence of liver dysfunction in these patients before OLT. The histological evaluation of explanted FAP livers, however, in these reports is limited. Minimal or noninvolvement of liver with amyloid is vital if these livers are to be used as " d o m i n o donors. ''B In one study, 14 FAP explanted livers were essentially normal, with 6 showing only traces of amyloid in the large hilar nerves. 3 The previously m e n t i o n e d autopsy study on 17 patients reported amyloid only within the vascular walls in the Glisson's capsule in the liver. 1B We carefully evaluated, with employment of special stains, all tissues removed at OLT. As shown in Table 1, minimal hepatic lobular involvement was seen in only one patient, who had extensive amyloid deposits in neural, vascular, and soft tissues. The adipose tissue at the hilum in this case had a characteristic " r i n g e d " appearance easily discernible on routine hematoxylin and eosin sections, a finding that could be used for intraoperative evaluation of severe disease if livers from such cases were to be excluded as " d o m i n o " donors. However, it must be noted that this patient with the most severe disease still had minimal liver parenchymal involvement. Involvem e n t of vagus and hepatic hilar nerves was the most consistent finding. These neural deposits were somewhat subtle and were missed in some cases at the time of initial pathological examination. In this study, we confirm that hepatic lobular involvement in FAP is absent in most cases, and minimal to mild involvement of vessels in the portal triad may be seen. Given the long latent period before e m e r g e n c e of clinically significant disease in FAP patients, utilization of their explanted livers seems medically sound. The

evolution of mutant TTR-related changes in " d o m i n o recipients" may shed some light on the pathogenesis of FAP neuropathyP If TTR synthesized in choroid plexus is the only source of amyloid neuropathy, long-term " d o m i n o recipients" may show no polyneuropathy but different disease manifestations from the mutant T F R than the d o n o r patients. As reported in a clinical study from our medical center, clinical outcome does not correlate with age at OLT. ~ The survival after OLT is variable and in our pathologic study did not relate to the severity of amyloidosis. The patient with the most severe involvement lived for 1,443 days posttransplantation. Furthermore, the histopatho!ogic severity of amyloid deposition does not correlate with duration of disease before transplantation. In conclusion, liver parenchymal involvement in FAP is minimal, usually restricted to portal vessels and hilar nerves, and, therefore, the exp!ants are suitable for grafting into non-FAP patients.

Acknowledgment. The authors thank Dr Harvey Goldman for reviewing the manuscript, Edith Hurney for her excellent secretarial help, and Dr William Quist for assisting with the illustrations. REFERENCES 1. Pomfret EA, Lewis WD,Jenkins RL, et al: Effect of orthotopic liver transplantation on the progression of familial amyloidotic polyneuropathy. Transplantation 65:918-925, 1998 2. Benson MD: Amyloidosis, in Scriver CR, Beaudet AK, Sly WS (eds): The Metabolic and Molecular Basis of Inherited Disease (ed 7). NewYork, NY, McGraw-Hill, 1995, pp 4159-4190 3. Stangou AJ, Heaton ND, Rela M, et al: Domino hepatic transplantation using the liver from a patient with familial amyloid polyneuropathy. Transplantation 65i1496-1498, 1998 4. Hemming AW, Cattral MS, Chari RS, et al: Domino liver transplantation for familial amyloid polyneuropathy. Liver Transplant Surg 4:236-238, 1998 5. Dubrey SW, Davidoff R, Skinner M, et al: Progression of ventricular wall thickening after liver transplantation for familial amyloidosis. Transplantation 64:74-80, 1997 6. Suhr OB, Holmgren G, Steen L, et al: Liver transplantation in familial amyloidotic polyneuropathy. Transplantation 60:933-938, 1995 7. Rydh A, Suhr O, Hietaia S-O, et al: Serum amyloid P component scintigraphy in familial amy!oid polyneuropathy: Regression of visceral amyloid following liver transplantation. Eur J Nucl Med 25:709-713, 1998 8. Pratschke J, Steinnmller T, Bechstein WO, et al: Orthotopic liver transplantation for hepatic associated metabolic disorders. Clin Transplant 12:228-232, 1998 9. Mowat AP: Orthotopic liver transplantation in liver-based metabolic disorders. EnrJ Pediatr 151 :$32-38, 1992 10. Parrilla P, Ramirez E Bueno FS, et al: Clinical improvement after liver transplantadon for type I familial amyloid polyneuropathy. BrJ Surg 82:825-828, 1995 11. Suhr OB, Wiklund U, Ando Y, et al: Impact of liver transplantation on autonomic neuropathy in familial amyloidotic polyneuropathy: An evaluation by spectral analysis of heart rate variability. J Intern Med 242:225-229, 1997 12. Lewis WD, Skinner M: Liver transplantation for familial amyloidotic polyneuropathy: A potentially curative treatment. Amyloid: IntJ Exp Clin Invest 1:143-144, 1994 13. Takahashi K, Yi S, Kimura Y, et al: Familial amyloidotic polyneuropathy type 1 in Kumamoto, Japan: A clinicopathologic, histochemical, immunohistochemical, and ultrastructural study. HUM PATHOL22:519-527, 1991 14. Shimada K, Maeda S, Murakami T, et al: Transgenic mouse model of familial amyloidotic polyneuropathy. Mol Biol Med 6:333343, 1989

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