Juris J. Liepnieks

Variant apolipoprotein AI as a major constituent of a human hereditary amyloid

Amyloid fibrils were isolated from spleen and liver of a patient who died with Familial Amyloidotic Polyneuropathy Type III (Iowa). The major protein constituent of the fibrils was found to be the amino terminal portion (residues 1-83) of apolipoprotein AI with an arginine for glycine substitution at position 26. This is the first report of an apolipoprotein as a major amyloid constituent in a form of autosomal dominant hereditary amyloidosis in humans.

Direct sequencing of the gene for Maryland/German familial amyloidotic polyneuropathy type II and genotyping by allele-specific enzymatic amplification.

Direct genomic DNA sequencing has been used to characterize the mutation associated with familial amyloidotic polyneuropathy in the Maryland/German kindred. A mutation of thymine to adenine in the prealbumin (transthyretin) gene at the position corresponding to the second base of codon 58 in the prealbumin mRNA gives a histidine for leucine substitution in the plasma protein. Since the mutation does not result in a change in the restriction pattern of the prealbumin gene, a new method for the direct detection of single base changes in genomic DNA was developed using the polymerase chain reaction and an allele-specific oligonucleotide primer.

Progression of cardiac amyloid deposition in hereditary transthyretin amyloidosis patients after liver transplantation

It has been hypothesized that transthyretin (TTR) amyloidosis may progress after orthotopic liver transplantation (OLT) as a result of continued amyloid fibril synthesis and deposition from normal TTR. To test this hypothesis amyloid fibrils were isolated from cardiac tissues of three patients who died 1½ to 5½ years after OLT: two with Val30Met and one with Thr60Ala TTR. The ratio of variant to normal TTR in each case was determined and compared with the ratio of variant to normal in cardiac tissues from seven patients who died with TTR amyloidosis but who had not had liver transplantation. Tissues from patients with TTR amyloidosis without OLT included three with Val30Met, two with Thr60Ala, one with ΔVal122, and one with Val122Ile. All tissues from patients without OLT had greater amounts of variant TTR than normal TTR except for the Val122Ile in which the ratio was 50:50. The overall median variant to normal ratio was 60:40 with a range of 50–70% variant. In contrast, the mean percentage of variant TTR in the three tissues from patients after OLT was 25% (range 20–35). These data are consistent with the continued deposition of normal TTR in cardiac tissue after liver transplantation.

Hereditary renal amyloidosis with a novel variant fibrinogen.

Two families with hereditary renal amyloidosis were found to have a novel mutation in the fibrinogen A alpha chain gene. This form of amyloidosis is an autosomal dominant condition characterized by proteinuria, hypertension, and subsequent azotemia. DNAs of patients with amyloidosis were screened for a polymorphism in fibrinogen A alpha chain gene by single-strand conformation polymorphism analysis, and affected individuals from two kindreds were found to have a mutation. Both of these kindreds are American of Irish descent presenting with non-neuropathic, nephropathic amyloidosis in the fifth to the seventh decade of life. DNA sequencing showed a point mutation in the fibrinogen A alpha chain gene that is responsible for substitution of valine for glutamic acid at position 526. By restriction fragment length polymorphism analysis, 7 affected individuals and 14 asymptomatic individuals in these two kindreds were positive for the fibrinogen A alpha chain Val 526 gene. Fibrinogen was isolated from plasma of a heterozygous gene carrier and shown to contain approximately 50% variant fibrinogen. Discovery of this new mutation confirms the association between fibrinogen A alpha chain variant and hereditary renal amyloidosis and establishes a new biochemical subtype of amyloidosis.

Hereditary amyloid cardiomyopathy caused by a variant apolipoprotein A1.

Autosomal dominant hereditary amyloidosis with a unique cutaneous and cardiac presentation and death from heart failure by the sixth or seventh decade was found to be associated with a previously unreported point mutation (thymine to cytosine, nt 1389) in exon 4 of the apolipoprotein A1 (apoA1) gene. The predicted substitution of proline for leucine at amino acid position 90 was confirmed by structural analysis of amyloid protein isolated from cardiac deposits of amyloid. The subunit protein is composed exclusively of NH2-terminal fragments of the variant apoA1 with the longest ending at residue 94 in the wild-type sequence. Amyloid fibrils derived from four previously described apoA1 variants are composed of similar fragments with carboxyl-terminal heterogeneity, but contrary to those variants, which all carry one extra positive charge, the substitution Leu90Pro does not result in any charge modification. It is unlikely, therefore, that amyloid fibril formation is related to change of charge for a specific residue of the precursor protein. This is in agreement with studies on transthyretin amyloidosis in which no unifying factor such as change of charge for amino acid residues has been noted.

Characterization of amyloid A protein in human secondary amyloidosis: the predominant deposition of serum amyloid A1

Serum amyloid A protein (SAA) is the plasma precursor for amyloid A protein (AA), the subunit protein in amyloid deposits of secondary or reactive amyloidosis. Several forms of acute phase SAA have been identified in human plasma. To elucidate whether one of these forms of SAA predominates in the formation of AA amyloid deposits, the amino acid sequence of the subunit protein in six cases of reactive amyloidosis was investigated. Minimal heterogeneity was present at the N-terminus as all samples started with residue 1, 2, or 3 of SAA. The C-terminus, however, was more heterogeneous with the AA protein in each case terminating at multiple sites from residue 58 to 84 of SAA. Since less than 20% of the AA protein in each case contained sequence past residue 67 of SAA, the sequence and recovery of tryptic peptides containing residues 52, 57, and 60 where human SAA1 and 2 differ was used to determine the relative amounts of SAA1 and 2 present. One sample contained only SAA1 sequence, four contained approx. 11% or less of SAA2 sequence, and the sixth contained 24-33% of SAA2 sequence. Thus, while five of the six AA samples contained both SAA1 and 2, the predominant form in all cases was SAA1. In three of the six cases, the protein defensin was isolated along with the AA protein from the fibrils. This may suggest neutrophil involvement in SAA processing to AA fibrils.

Progression of transthyretin amyloid neuropathy after liver transplantation

Objective: To biochemically characterize peripheral nerve amyloid in subjects with transthyretin (TTR) amyloidosis and assess effect of orthotopic liver transplantation (OLT) on progression of neuropathy. Methods: Amyloid fibrils were isolated from peripheral nerve tissues of 6 patients with TTR amyloidosis who were heterozygous for an amyloid-associated TTR mutation. Ratio of variant to wild-type TTR in the fibrils was determined by amino acid sequencing of tryptic peptides containing either the variant amino acid residue or the corresponding normal amino acid. Results: Amyloid fibrils from 3 subjects who died without having received a liver transplant were composed of 60%–65% variant TTR and 35%–40% wild-type. Amyloid fibrils from a subject who died 5 years after liver transplantation contained 25% variant and 75% wild-type TTR. Conclusion: Ratios of variant to wild-type TTR in amyloid patients heterozygous for an amyloid-associated TTR mutation are similar to published ratios for amyloid fibrils in cardiac tissue. Survival after liver transplantation for TTR amyloidosis may be associated with progression of neuropathy due to continued deposition of amyloid derived from wild-type TTR.

Hereditary fibrinogen A α-chain amyloidosis: phenotypic characterization of a systemic disease and the role of liver transplantation

Variants of fibrinogen A alpha-chain (AFib) cause the most common type of hereditary renal amyloidosis in Europe and, possibly, the United States as well. Variant fibrinogen is produced in the liver, and solitary renal allografts fail within 1 to 7 years with recurrent amyloidosis. We assessed 22 AFib patients for combined liver and kidney transplantation (LKT) and report the clinical features and outcome. Twenty-one had E526V and 1, the R554L variant. Coronary atherosclerosis was identified in 68% and systemic atheromatosis in 55%. Vascular atheroma excised at endarterectomy and endomyocardial biopsies contained purely variant fibrinogen amyloid. Half had autonomic neuropathy. Six of 9 patients who underwent LKT are alive (67%), with good allograft function and no amyloidosis at median 67 months (range, 33-155 months) of follow-up. Serial technetium-99m-labeled dimercaptosuccinic acid ((99m)Tc-DMSA) renal scintigraphy in 2 cases of preemptive LKT demonstrated preserved native kidney residual function at 5 years. Four 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.

A Cell Culture System for the Study of Amyloid Pathogenesis: Amyloid Formation by Peritoneal Macrophages Cultured with Recombinant Serum Amyloid A

A murine macrophage culture system that is both easy to employ and amenable to manipulation has been developed to study the cellular processes involved in AA amyloid formation. Amyloid deposition, as identified by Congo red-positive, green birefringent material, is achieved by providing cultures with recombinant serum amyloid A2 (rSAA2), a defined, readily produced, and highly amyloidogenic protein. In contrast to fibril formation, which can occur in vitro with very high concentrations of SAA and low pH, amyloid deposition in culture is dependent on metabolically active macrophages maintained in neutral pH medium containing rSAA2 at a concentration typical of that seen in acute phase serum. Although amyloid-enhancing factor is not required, its addition to culture medium results in larger and more numerous amyloid deposits. Amyloid formation in culture is accompanied by C-terminal processing of SAA and the generation of an 8.5-kd fragment analogous to amyloid A protein produced in vivo. Consistent with the possibility that impaired catabolism of SAA plays a role in AA amyloid pathogenesis, treatment of macrophages with pepstatin, an aspartic protease inhibitor, results in increased amyloid deposition. Finally, the amyloidogenicity exhibited by SAA proteins in macrophage cultures parallels that seen in vivo, eg, SAA2 is highly amyloidogenic, whereas CE/J SAA is nonamyloidogenic. The macrophage culture model presented here offers a new approach to the study of AA amyloid pathogenesis.

Prion proteins with different conformations accumulate in Gerstmann-Sträussler-Scheinker disease caused by A117V and F198S mutations

Gerstmann-Sträussler-Scheinker disease (GSS) is characterized by the accumulation of proteinase K (PK)-resistant prion protein fragments (PrP(sc)) of approximately 7 to 15 kd in the brain. Purified GSS amyloid is composed primarily of approximately 7-kd PrP peptides, whose N terminus corresponds to residues W(81) and G(88) to G(90) in patients with the A117V mutation and to residue W(81) in patients with the F198S mutation. The aim of this study was to characterize PrP in brain extracts, microsomal preparations, and purified fractions from A117V patients and to determine the N terminus of PrP(sc) species in both GSS A117V and F198S. In all GSS A117V patients, the approximately 7-kd PrP(sc) fragment isolated from nondigested and PK-digested samples had the major N terminus at residue G(88) and G(90), respectively. Conversely, in all patients with GSS F198S, an approximately 8-kd PrP(sc) fragment was isolated having the major N terminus start at residue G(74). It is possible that a further degradation of this fragment generates the amyloid subunit starting at W(81). The finding that patients with GSS A117V and F198S accumulate PrP(sc) fragments of different size and N-terminal sequence, suggests that these mutations generate two distinct PrP conformers.