Apolipoprotein AI and transthyretin as components of amyloid fibrils in a kindred with apoAI Leu178His amyloidosis

Abstract

We found a new C-terminal amyloidogenic variant of apolipoprotein AI (apoAI), Leu178His in a French kindred, associated with cardiac and larynx amyloidosis and skin lesions with onset during the fourth decade. This single-point mutation in exon 4 of the apoAI gene was detected by DNA sequencing of polymerase chain reaction amplified material and restriction fragment length polymorphism analysis in two siblings. Blood, larynx, and skin biopsies were available from one sibling. Anti-apoAI immunoblotting of isoelectric focusing of plasma showed a +1 alteration in the charge of the protein. Extraction of fibrils from the skin biopsy revealed both full-length and N-terminal fragments of apoAI and transthyretin (TTR). ApoAI and TTR co-localized in amyloid deposits as demonstrated by immunohistochemistry. The present report, together with the first recently described C-terminal amyloidogenic variant of apoAI, Arg173Pro, shows that amyloidogenicity of apoAI is not a feature exclusive to N-terminal variants. The most striking characteristic of amyloid fibrils in Leu178His is that wild-type TTR is co-localized with apoAI in the fibrils. We have previously determined that a fraction of plasma TTR circulates in plasma bound to high-density lipoprotein and that this interaction occurs through binding to apoAI. Therefore we hypothesize that nonmutated TTR might influence deposition of apoAI as amyloid.

Figure 1.

Larynx histology and immunohistochemistry of the proband. A: Anti-apoAI immunohistochemistry revealed deposition of apoAI; amplified images of a specific region (in the square). B: Congo red staining under polarized light. C: Staining of amyloid deposits with polyclonal anti-TTR; co-localization with apoAI staining and amyloid deposition. D: Lack of staining of amyloid deposits with polyclonal anti-TTR preabsorbed with excess TTR, demonstrating specificity of the antibody.

Figure 2.

Analysis of amyloid fibrils extracted from a skin biopsy. Coomassie staining of the extracted fibrils separated on 15% SDS-PAGE gels (lane 1); anti-apoAI immunoblot of the apoAI standard (lane 2); and of the extracted fibrils (lane 3). Both high molecular weight aggregated forms of apoAI, full-length protein and fragments can be observed. Anti-TTR immunoblot of the extracted fibrils (lane 4) and isolated TTR (lane 5). In the fibrils, high molecular weight aggregates of TTR and a fragment of the protein are immunoreactive. The figure derives from a single gel but the lanes in the immunoblots are organized in a different order for convenient presentation.

Figure 3.

Analysis of the apoAI gene of the proband. A: Nucleic acid sequencing of part of exon 4. The proband is a heterozygote for a single-base change of T to A in codon 178 (arrow). B: Confirmation of the mutation by RFLP. Part of exon 4 (codons 155 to 239) was amplified by PCR and subsequently cut with NlaIII. Lane 1, 1-kb DNA ladder; lane 2, normal individual; lane 3, uncut PCR from the proband; lane 4, RFLP from the proband.

Figure 4.

Isoelectric focusing and immunoblotting of plasma apoAI. Lane 1, plasma from the proband showing the variant protein with +1 alteration in charge (arrow); lane 2, IEF from the normal individual. + and − indicate the positive and negative poles, respectively.