Novel clinical manifestations and treatment of hereditary apoA-I amyloidosis: When a good protein turns bad

Abstract

Amyloidoses are life-threatening diseases caused by the deposition of various proteins including apoA-I, the major protein of plasma HDL. Timely diagnostics of amyloidoses is crucial for their treatment. Colombat et al. report novel aspects of the hereditary apoA-I amyloidosis, including its unexpected clinical presentation, genetic origins, as well as a life- and vision-saving hepatorenal transplant as treatment. This study improves the diagnostics of AApoAI, optimizes its treatment, and expands our understanding of the molecular basis of this multipronged disease.

Amyloid diseases, or amyloidoses, represent a major public health challenge. These hitherto incurable protein deposition disorders are notoriously difficult to diagnose and treat despite decades of research. Amyloidoses can be caused by nearly 40 unrelated human proteins and peptides¹ including exchangeable (water-soluble) apolipoproteins (apos).² Soluble functional forms of these proteins can convert into insoluble cross-beta-sheet-rich amyloid fibrils that deposit in various organs and cause damage. While neurodegenerative amyloidoses such as Alzheimer’s, Parkinson’s and prion diseases have received much attention in recent years, systemic amyloidoses, which can affect various organs except the brain and afflict tens of thousands of patients worldwide, remain relatively underexplored and under-diagnosed.³ Pioneering studies by several teams, including Valleix and colleagues,^4,5 help to fill in this gap.

The diagnostics of systemic amyloidoses remains challenging since amyloid deposits can impair a wide range of organs (kidney, liver, heart, spleen, testes, nerves, etc.), the impairment may range from mild to severe, and the deposition of unrelated proteins often leads to overlapping clinical presentations.^3-5 However, fibril typing to identify the protein precursor of amyloid is paramount to devising the appropriate treatment strategy. For example, bone marrow transplantation can be performed to halt the generation of the aberrant immunoglobulin light chains and thereby block the progression of light chain amyloidosis. In contrast, liver transplantation can be warranted to block the generation of the aberrant protein variants in hereditary apoA-I amyloidosis (AApoAI) described in the current study.⁵

Exchangeable apolipoproteins are prominent in amyloidoses. These proteins are synthesized by the liver and gut and enter the circulation in the form of lipoprotein nanoparticles. Several members of the apolipoprotein family have been identified as direct precursors of pathogenic fibrillar deposit in humans; these include apoA-I, apoA-II, apoC-II, and apoC-III, along with serum amyloid A and alpha-synuclein.^2,4 The propensity of these sticky, lipid surface-binding proteins to misfold and aggregate in amyloid stems from their high hydrophobicity compounded by their transient release from the lipoprotein surface in a labile free form.² Compared to their lipid-bound counterparts, free apolipoproteins such as apoA-I have much less stable and more dynamic structures⁶ and hence are more susceptible to misfolding and proteolysis.^2,7 The latter can cause rapid protein degradation; however, proteolysis may also generate protein fragments that misfold, aggregate and form fibrils faster than they are cleared in vivo. For example, such a misfolding and cleavage can generate N-terminal fragments of apoA-I found in AApoAI amyloid deposits.

There are two forms of human apoA-I amyloidosis, hereditary and acquired, with distinctly different etiology and clinical presentation. The acquired form, which involves the deposition of the wild-type protein in the arterial wall, contributes to atherosclerosis and is linked to aging, high plasma triglycerides, and oxidative stress. In contrast, the hereditary form, termed AApoAI, is caused by the apoA-I gene mutations that can lead to organ failure at a young age.⁸ The latter form is in the focus of the current study.⁵

AApoAI is an autosomal dominant disorder wherein 9-11 kDa N-terminal fragments of the variant protein deposit as fibers in kidneys, liver, nerves, heart, testes, spleen, skin and other organs, causing organ damage^5,7 (Fig. 1). The list of affected organs is probably incomplete; importantly, the current study adds retina to this list.⁵ The clinical presentation of AApoAI can vary from mild to severe depending, in part, on the location of the mutation,⁷ which complicates the diagnostics. Over 20 amyloidogenic apoA-I gene mutations have been identified to-date. Most of those are point substitutions, such as the Glu34Lys variant described in the current study.⁵ Notably, this rare variant features the only known amyloidogenic charge inversion in apoA-I.⁹ Other AApoAI variants involve deletions and frameshift mutations, such as the p.His179Profs*47 and a novel p.Thr185Alafs*41variant described in the current study.⁵

Figure 1. Aberrant deposition of apolipoprotein A-I in systemic hereditary amyloidosis, AApoAI.

ApoA-I is secreted by the liver into plasma and circulates on HDL as its major structural and functional protein. ApoA-I on HDL directs reverse cholesterol transport and plays other cardioprotective roles. A fraction of apoA-I can be transiently released from HDL in a structurally labile free form that is thought to form the protein precursor of amyloid. Specific mutations (orange) or post-translational modifications in apoA-I promote its misfolding and aggregation, which culminates in the deposition of the variant protein as amyloid in various organs (kidney, liver, heart, spleen, skin, testes, etc.). Colombat et al.⁵ reveal the neonatal character of the AApoAI mutations in most families explored, report one novel AApoAI mutation, and add retina to the list of organs impaired by the apoA-I amyloid deposition. The authors also report a combined hepatorenal transplantation as a vision- and life-saving treatment for a 32 year old AApoAI patient.

To stop the progression of AApoAI, Colombat et al. performed liver transplantation in a 32-year-old patient to block the generation of the aberrant Glu34Lys apoA-I variant.⁵ This helped arrest retinal degeneration and save the patient’s vision. The authors show that retinal degeneration in this and other patients stemmed from the apoA-I amyloid depositions, representing a previously unknown important hallmark of AApoAI. Moreover, liver transplantation in this patient was combined with a life-saving kidney transplantation to alleviate renal dysfunction that resulted from the deposition of variant apoA-I. Interestingly, in this and several other patients from the current study, renal amyloidosis was restricted solely to the glomerular compartment, expanding the spectrum of the clinical presentation of AApoAI. Favorable clinical outcome of the double transplantation extends the treatment options for AApoAI,⁵ while the expanded clinical presentation, including novel renal and ophthalmologic features reported here, will help diagnose this multipronged disease.

Another novel aspect of AApoAI is the spontaneous character of the neomutations, which was revealed in five out of six families of the current study.⁵ The implications of this unexpected finding are three-fold. First, this finding encourages the clinicians to consider sporadic AApoAI in the absence of the family history of amyloidosis, which may help improve the early diagnosis. Second, it suggests that the frequency of AApoAI in the general population is relatively high despite the strong negative selective pressure resulting from the early-onset male infertility caused by testicular amyloidosis. Third, it is reasonable to speculate that, similar to the amyloidogenic mutations, the frequency of the spontaneous non-amyloidogenic neomutations in apoA-I is also relatively high. Since many apoA-I mutations cause abnormal metabolism of plasma lipids and cardiovascular disease,^7,8 the current study may have important implications for understanding the genetic basis of these metabolic and cardiovascular disorders and aid their diagnostics. Therefore, the impact of this study is expected to extend beyond the amyloidosis field.

Normally, apoA-I (a single chain of 243 amino acids) plays an important beneficial role as the major structural and functional protein of plasma HDL and a carrier of “good cholesterol” (Fig. 1). In this capacity, apoA-I accepts excess cellular cholesterol and phospholipids and transports them to the liver for excretion via bile in a process termed reverse cholesterol transport. Over 95% of all circulating apoA-I is bound to HDL, yet a small protein fraction can be released from the lipoprotein particle in a transient free form. This metabolically active, structurally labile free protein can bind to the existing HDL particles or generate them de novo; alternatively, free apoA-I can be rapidly proteolyzed and cleared from circulation; finally, it can be converted from the soluble, largely alpha-helical native conformation into the insoluble fibrillary form. Such a fibrillation does not readily occur in native non-modified apoA-I; however, protein modifications such as the oxidation of specific Met residues or certain mutations can shift the delicate balance among the lipid binding, clearance, and protein misfolding, leading to amyloid deposition.^2,6,7 Three of such naturally occurring amyloidogenic mutations are described in the current study.

Why are some amino acid mutations amyloidogenic while others are not? The answer is beginning to emerge from comparative structural and dynamic studies in vitro and in silico of the naturally occurring apoA-I variants.^6,9 The results suggest that the effects of the protein mutations can propagate across the molecule and influence local conformation and dynamics in sensitive regions. In apoA-I, such regions include the flexible residue segment 121-142 (central repeat 5), which is highly susceptible to proteolytic cleavage, or the well-ordered residue segment 11-22, which has a high propensity to initiate amyloid formation (amyloid hot spot).⁶ AApoAI mutations explored to-date increase the solvent accessibility of this hot spot. This apparently characterizes the Glu34Lys substitution described in the current study, which is predicted to enhance solvent exposure and dynamics of Tyr18 in the middle of the amyloid hot spot⁹ Although the frameshift mutations such as those described in the current study have not been explored by biophysical methods, one can speculate that the disordering and premature truncation of the polypeptide chain near residue 185 at the bottom of the apoA-I helix bundle causes subtle local perturbations that increase solvent exposure of the nearby amyloid hot spots. Future biophysical studies of additional apoA-I variants, both amyloidogenic and non-amyloidogenic, will test these ideas.

Another unresolved issue in this and other types of systemic amyloidoses is organ and tissue targeting, which is thought to stem from the differences in the local microenvironment such as the pH, proteoglycan composition, and other factors, including interactions with specific proteins and lipids. Resolving these fundamental issues, together with the cutting-edge clinical studies such as the work by Colombat et al.,⁵ will ultimately help improve our understanding of the molecular basis of the amyloid diseases and improve their therapeutic targeting.