Amyloidogenicity and Clinical Phenotype Associated with Five Novel Mutations in Apolipoprotein A-I
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.024
ISSN1525-2191
AutoresDorota Rowczenio, Ahmet Doğan, Jason D. Theis, Julie A. Vrana, Helen J. Lachmann, Ashutosh D. Wechalekar, Janet A. Gilbertson, Toby Hunt, Simon Gibbs, Prayman Sattianayagam, Jenny H. Pinney, Philip N. Hawkins, Julian D. Gillmore,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe phenotype of hereditary apolipoprotein A-I amyloidosis is heterogeneous with some patients developing extensive visceral amyloid deposits and end-stage renal failure as young adults and others having only laryngeal and/or skin amyloid, which may be of little clinical consequence. Clinical management and prognosis of patients with systemic amyloidosis depend entirely on correct identification of the fibril protein, such that light chain amyloidosis (AL, previously referred to as “primary”), the most frequently diagnosed type, is treated with chemotherapy, which has absolutely no role in hereditary apolipoprotein A-I amyloidosis. We report five novel apolipoprotein A-I variants, four of which were amyloidogenic and one of which was incidental in a patient with systemic AL amyloidosis. Interestingly, only one of four patients with apolipoprotein A-I amyloidosis had a family history of similar disease. Laser microdissection and tandem mass spectrometry–based proteomics were used to confirm the amyloid fibril protein and, for the first time in apolipoprotein A-I amyloidosis, demonstrated that only mutated protein as opposed to wild-type apolipoprotein A-I was deposited as amyloid. The clinical spectrum and outcome of hereditary apolipoprotein A-I amyloidosis are reviewed in detail and support the need for sequencing of the apolipoprotein A-I gene among patients with apparent localized amyloidosis in whom IHC is nondiagnostic of the fibril protein, even in the absence of a family history of disease. The phenotype of hereditary apolipoprotein A-I amyloidosis is heterogeneous with some patients developing extensive visceral amyloid deposits and end-stage renal failure as young adults and others having only laryngeal and/or skin amyloid, which may be of little clinical consequence. Clinical management and prognosis of patients with systemic amyloidosis depend entirely on correct identification of the fibril protein, such that light chain amyloidosis (AL, previously referred to as “primary”), the most frequently diagnosed type, is treated with chemotherapy, which has absolutely no role in hereditary apolipoprotein A-I amyloidosis. We report five novel apolipoprotein A-I variants, four of which were amyloidogenic and one of which was incidental in a patient with systemic AL amyloidosis. Interestingly, only one of four patients with apolipoprotein A-I amyloidosis had a family history of similar disease. Laser microdissection and tandem mass spectrometry–based proteomics were used to confirm the amyloid fibril protein and, for the first time in apolipoprotein A-I amyloidosis, demonstrated that only mutated protein as opposed to wild-type apolipoprotein A-I was deposited as amyloid. The clinical spectrum and outcome of hereditary apolipoprotein A-I amyloidosis are reviewed in detail and support the need for sequencing of the apolipoprotein A-I gene among patients with apparent localized amyloidosis in whom IHC is nondiagnostic of the fibril protein, even in the absence of a family history of disease. Amyloidosis is a rare disorder characterized by extracellular deposition of fibrillar protein that results in a progressive disruption of structure and function of affected tissues and organs. Amyloidosis is a remarkably heterogeneous disease; it can be acquired or hereditary and systemic or localized. The most common form of systemic amyloidosis is AL (light chain, previously referred to as “primary”), in which the fibrils are derived from monoclonal immunoglobulin light chains and consist of the whole or part of the variable (VL) domain. The prognosis of systemic AL amyloidosis is generally worse than in other amyloid types, although there is marked heterogeneity in the extent of organ involvement and rate of disease progression. Treatment of AL amyloidosis is with chemotherapy for which there is no role in other amyloid types. Hereditary systemic amyloidosis is a rare autosomal dominant condition caused by deposition of variant proteins as amyloid fibrils. The term “hereditary nonneuropathic systemic amyloidosis” (Online Mendelian Inheritance of Man no. 105200) was coined by Ostertag1Ostertag B. Demonstration einer eigenartigen familiaren paraamyloidose.Zentralbl Aug Pathol. 1932; 56: 253-254Google Scholar in 1932 following the discovery of two families with dominantly inherited renal amyloidosis. Mutations in the genes encoding apolipoprotein A-I (apoAI),2Jones L.A. Harding J.A. Cohen A.S. Skinner M. New USA family has apolipoprotein AI (Arg26) variant.in: Natvig J.B. Førre Ø. Husby G. Husebekk A. Skogen B. 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Renal apolipoprotein A-I amyloidosis associated with a novel mutant Leu64Pro.Am J Kidney Dis. 2004; 44: 1103-1109Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Obici L. Palladini G. Giorgetti S. Bellotti V. Gregorini G. Arbustini E. Verga L. Marciano S. Donadei S. Perfetti V. Calabresi L. Bergonzi C. Scolari F. Merlini G. Liver biopsy discloses a new apolipoprotein A-I hereditary amyloidosis in several unrelated Italian families.Gastroenterology. 2004; 126: 1416-1422Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar apolipoprotein A-II (apoAII),13Benson M.D. Liepnieks J.J. Yazaki M. Yamashita T. Hamidi Asl K. Guenther B. Kluve-Beckerman B. A new human hereditary amyloidosis: the result of a stop-codon mutation in the apolipoprotein AII gene.Genomics. 2001; 72: 272-277Crossref PubMed Scopus (129) Google Scholar fibrinogen Aα-chain,14Benson M.D. Liepnieks J. Uemichi T. Wheeler G. Correa R. Hereditary renal amyloidosis associated with a mutant fibrinogen α-chain.Nat Genet. 1993; 3: 252-255Crossref PubMed Scopus (190) Google Scholar, 15Uemichi T. Liepnieks J.J. Benson M.D. Hereditary renal amyloidosis with a novel variant fibrinogen.J Clin Invest. 1994; 93: 731-736Crossref PubMed Scopus (87) Google Scholar, 16Uemichi T. Liepnieks J.J. Yamada T. Gertz M.A. Bang N. Benson M.D. A frame shift mutation in the fibrinogen A α-chain gene in a kindred with renal amyloidosis.Blood. 1996; 87: 4197-4203PubMed Google Scholar, 17Hamidi Asl L. Liepnieks J.J. Uemichi T. Rebibou J.M. Justrabo E. Droz D. Mousson C. Chalopin J.M. Benson M.D. Delpech M. Grateau G. Renal amyloidosis with a frame shift mutation in fibrinogen a α-chain gene producing a novel amyloid protein.Blood. 1997; 90: 4799-4805PubMed Google Scholar and lysozyme18Pepys M.B. Hawkins P.N. Booth D.R. Vigushin D.M. Tennent G.A. Soutar A.K. Totty N. Nguyen O. Blake C.C.F. Terry C.J. Feest T.G. Zalin A.M. Hsuan J.J. Human lysozyme gene mutations cause hereditary systemic amyloidosis.Nature. 1993; 362: 553-557Crossref PubMed Scopus (560) Google Scholar have since been identified as the cause of hereditary renal amyloidosis in different kindreds (Online Mendelian Inheritance of Man no. 105200). The clinical amyloidosis syndromes that accompany the various mutations in these different genes are diverse with respect to age at onset, mode of presentation, pattern of organ distribution, rate of progression, and prognosis. Indeed, certain apoAI variants are associated with neuropathy19Nichols W.C. Gregg R.E. Brewer H.B.J. Benson M.D. A mutation in apolipoprotein A-I in the Iowa type of familial amyloidotic polyneuropathy.Genomics. 1990; 8: 318-323Crossref PubMed Scopus (147) Google Scholar, 20Hazenberg A.J. Dikkers F.G. Hawkins P.N. Bijzet J. Rowczenio D. Gilbertson J. Posthumus M.D. Leijsma M.K. Hazenberg B.P. Laryngeal presentation of systemic apolipoprotein A-I-derived amyloidosis.Laryngoscope. 2009; 119: 608-615Crossref PubMed Scopus (20) Google Scholar such that the nomenclature, which includes the term “nonneuropathic,” is confusing and probably no longer appropriate. Patients with hereditary apoAI amyloidosis typically present with hypertension, proteinuria, and renal impairment6Persey M.R. Booth D.R. Booth S.E. van Zyl-Smit R. Adams B.K. Fattaar A.B. Tennent G.A. Hawkins P.N. Pepys M.B. Hereditary nephropathic systemic amyloidosis caused by a novel variant apolipoprotein A-I.Kidney Int. 1998; 53: 276-281Crossref PubMed Scopus (64) Google Scholar and frequently develop extensive visceral deposits that affect the liver, spleen, and kidneys, with occasional involvement of the heart, nerves, larynx, and gastrointestinal tract. Although the condition may lead to end-stage renal disease and other organ failure, the natural history of the disease is often slow, contrasting systemic AL amyloidosis in which the median patient survival without therapy is approximately 6 to 15 months from diagnosis.21Kyle R.A. Gertz M.A. Primary systemic amyloidosis: clinical and laboratory features in 474 cases.Semin Hematol. 1995; 32: 45-59PubMed Google Scholar, 22Kyle R.A. Gertz M.A. Greipp P.R. Witzig T.E. Lust J.A. Lacy M.Q. A trial of three regimens for primary amyloidosis: colchicine alone, melphalan and prednisone, and melphalan, prednisone, and colchicine.N Engl J Med. 1997; 336: 1202-1207Crossref PubMed Scopus (626) Google Scholar Here we report five novel apoAI variants, four of which were associated with hereditary systemic amyloidosis and one of which was nonamyloidogenic, and an incidental finding discovered in a patient with systemic AL amyloidosis. The importance of correct identification of the amyloid fibril protein is highlighted, and the novel use of mass spectrometry to identify apoAI amyloidosis is reported. We review the UK National Amyloidosis Centre experience and published literature regarding the clinical spectrum and outcome of hereditary apoAI amyloidosis. Five unrelated patients were referred to the UK National Amyloidosis Centre after discovery of amyloid deposits and underwent detailed investigations to elucidate the cause. Informed consent was obtained from all patients, and clinical care was in accordance with the Declaration of Helsinki. A 35-year-old English man presented with hoarseness (patient 1). Vocal cord nodules were discovered at laryngoscopy and excised. A 51-year-old English woman presented with a soft tissue mass growing on her palate (patient 2). A 29-year-old Polish woman presented with edema (patient 3). A 77-year-old English woman presented with hypertension and was noted to have proteinuria and renal impairment (patient 4). A 56-year-old English man presented with proteinuria and progressive renal impairment (patient 5). Biopsy specimens of vocal cord and testicles from patient 1, palatal mass from patient 2, rectum from patient 3, and kidney from patients 4 and 5 were stained at the UK National Amyloidosis Centre. Sections, 6-μm thick, were stained for amyloid deposits with Congo red and viewed under crossed polarized light.23Puchtler H. Sweat F. Levine M. On the binding of Congo red by amyloid.J Histochem Cytochem. 1962; 10: 355-364Crossref Google Scholar Immunohistochemistry (IHC) staining of the amyloid deposits was performed using monospecific antibodies reactive with serum amyloid A protein, lysozyme, apoAI, fibrinogen Aα-chain, transthyretin, and κ and λ immunoglobulin light chains, as previously described.24Tennent G.A. Cafferty K.D. Pepys M.B. Hawkins P.N. Congo red overlay immunohistochemistry aids classification of amyloid deposits.in: Kyle R.A. Gertz M.A. Parthenon Publishing, Pearl River, New York1999: 160-162Google Scholar Specificity of staining was confirmed by prior absorption of the antiserum with pure antigen in each case, and positive and negative controls were included in each run. Genomic DNA from all five patients was extracted from whole blood treated with EDTA.25Talmud P. Tybjaerg-Hansen A. Bhatnagar D. Mbewu A. Miller J.P. Durrington P. Humphries S. Rapid screening for specific mutations in patients with a clinical diagnosis of familial hypercholesterolaemia.Atherosclerosis. 1991; 89: 137-141Abstract Full Text PDF PubMed Scopus (99) Google Scholar Exons 3 (c.5352 to c.5711) and 4 (c.6116 to 6859) of the APOAI gene (National Center for Biotechnology Information accession no. NG_012021) were amplified by PCR performed with Ready-To-Go tubes (Amersham Pharmacia Biotech, Piscataway, NJ). PCR amplification of exons 3 and 4 was performed with the following oligonucleotides: exon 3 forward 5′-GGCAGAGGCAGCAGGTTTCTCAC-3′ (c.5352−5374) and reverse 5′-CCAGACTGGCCGAGTCCTCACCTA-3′ (c.5688−5711) and exon 4 forward 5′-CACTGCACCTCCGCGGACA-3′ (c.6116−6134) and reverse 5′-CTTCCCGGTGCTCAGAATAAACGTT-3′ (c.6835−6859). The total volume of PCR reaction was 25 μL, and it consisted of the following: 2 μL of DNA (at a concentration of 100 ng/μL), 1 μL of each primer (at a concentration of 10 μmol/L), and 21 μL of water. PCR cycling conditions were as followed: initial denaturation at 96°C for 5 minutes; five cycles of 96°C for 30 seconds and 72°C for 30 seconds; 35 cycles at 96°C for 30 seconds, 72°C for 30 seconds, and 72°C for 1 minute; and final elongation step at 72°C for 7 minutes. Negative and positive controls were included. The PCR products were purified with a QIAquick PCR purification kit (Oiagen, Velno, the Netherlands) according to the manufacturer's protocol and sequenced with the ABI BigDye Terminator v 3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The primers 5′-GATCTCAGCCCACAGCTGGCC-3′ (c.5417−5437) and 5′-AGGGCTCACCCCTGATAGGCTG-3′ (c.6144−6166) were used for sequencing of exons 3 and 4, respectively. APOAI gene sequences were analyzed on the ABI 3130xl Genetic Analyzer, using Sequencing Analysis Software, version 5.4 (Applied Biosystems, Carlsbad, CA). The proteome of amyloid deposits was analyzed by combining microdissection and mass spectrometry–based proteomics. Sections (10-μm thick) of formalin-fixed, paraffin-embedded tissues were placed on DIRECTOR slides (Expression Pathology, Rockville, MD). Sections were air dried and then melted, deparaffinized, and stained in hematoxylin followed by Congo red. Congo red–stained sections were examined for the presence of amyloid deposits under fluorescence (B/G/R filter cube; Leica, Wetzler, Germany). Positive areas were microdissected into 0.5-mL microcentrifuge tube caps containing 10 mmol TRIS/1 mmol EDTA/0.002% Zwittergent 3−16 (Calbiochem, San Diego, CA) using a Leica DM6000B Microdissection System (Leica). For each case, two to four different microdissections were collected, and each microdissection contained 50,000 to 60,000 μm2 of the tissue section. Collected tissues were heated at 98°C for 90 minutes with occasional vortexing. After 60 minutes of sonication in a waterbath, samples were digested overnight at 37°C with 1 μg of trypsin (Promega, Madison, WI). The trypsin-generated digests were reduced with dithiothreitol and separated by nanoflow liquid chromatography–electrospray tandem mass spectrometry using a ThermoFinnigan LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron, Bremen, Germany) coupled to an Eksigent nanoLC-2D HPLC system (Eksigent, Dublin, CA). A 0.25-μL trap (Optimize Technologies, Oregon City, OR) packed with Michrom Magic C-8 was plumbed into a 10-port valve. A 75 μm × 15 cm C-18 column was used for the separation using an organic gradient from 6% to 86% in 55 minutes at 400 nL/min. The Thermo-Fisher Scientific (Waltham, MA) MS/MS raw data files were searched using three different algorithms (Mascot, Sequest, and X!Tandem) and the results assigned peptide and protein probability scores. The results were then combined and displayed using Scaffold (Proteome Software, Portland, OR). All searches were conducted with variable modifications and restricted to full trypsin-generated peptides, allowing for two missed cleavages. Peptide mass search tolerances were set to 10 ppm and fragment mass tolerance to 1.00 Da. The search algorithms used to identify peptides interrogate the human SwissProt database, which does not contain the mutated sequences identified in this study. To show that the peptides containing the mutation were part of amyloid deposits, these sequences were added to the human SwissProt database using an in-house script, and a second-round data analysis was performed. Peptide identifications below the 90% confidence level were not considered in our analysis. All five patients underwent whole body anterior and posterior scintigraphic imaging 24 hours after administration of 123I-labeled serum amyloid P component (SAP) using a GE Infinia Hawkeye (GE Healthcare, Chalfont St Giles, UK) gamma camera, as previously described.26Hawkins P.N. Lavender J.P. Pepys M.B. Evaluation of systemic amyloidosis by scintigraphy with 123I-labeled serum amyloid P component.N Engl J Med. 1990; 323: 508-513Crossref PubMed Scopus (426) Google Scholar The labeled SAP study results were interpreted by a panel of physicians with experience of more than 10,000 SAP scans. Patient 1 presented with a hoarse voice and no other significant history. He underwent direct laryngoscopy and excision of multiple nodules on the vocal cords and aryepiglottic folds, which were discovered to be amyloid on histologic analysis. On direct questioning there was no history of hemoptysis, dyspnea, rashes, petechiae, weight loss, anorexia, or edema. There was no history of paraesthesia or postural dizziness. The only medical history was of hypogonadism and azoospermia for which he had undergone a testicular biopsy 8 to 10 years previously. He did not take regular medication, and there was no family history of similar illness, cardiac disease, or renal disease. Clinical examination was unremarkable. Baseline investigations showed normal blood cell count, clotting profile, renal, and liver function. There was no evidence of a plasma cell dyscrasia by sensitive nephelometric serum free light chain assay,27Bradwell A.R. Carr-Smith H.D. Mead G.P. Tang L.X. Showell P.J. Drayson M.T. Drew R. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine.Clin Chem. 2001; 47: 673-680PubMed Google Scholar conventional electrophoresis and immunofixation of serum or urine, or bone marrow examination. There was no proteinuria, and ECG and echocardiography did not reveal evidence of cardiac amyloid infiltration. Patient 2 presented with a 4-year history of a gradually enlarging mass on the palate within an area of scarring from a childhood injury. There had been no bleeding or pain associated with the lump, and the patient remained otherwise well. She experienced an occasional dry mouth and dry eyes and very rare tingling in her fingertips, with no other features to suggest a peripheral neuropathy or carpal tunnel syndrome. There was no history of weight loss or autonomic symptoms. There was no family history of amyloidosis, renal disease, or cardiac disease. Clinical examination was unremarkable. She had a medical history that included celiac disease, dermatitis herpetiformis, and multinodular goiter for which she had undergone a partial thyroidectomy at the age of 29 years. Shortly before her presentation with the lump in the palate, she had developed a recurrent clinically overt goiter. There was no evidence of a plasma cell dyscrasia by serum free light chain assay, conventional electrophoresis and immunofixation of serum or urine, or bone marrow examination. There was no proteinuria, and ECG, echocardiogram, and N-terminal pro-B–type natriuretic peptide were not suggestive of cardiac amyloid infiltration. Patient 3 presented with edema and was discovered to have subnephrotic range proteinuria (1.68 g/24 h) and stage 3 chronic kidney disease (CKD). A kidney biopsy was performed in 2008 and revealed amyloidosis, thought on initial IHC in her local hospital to be AA type. The results of investigations to identify an underlying inflammatory condition were all negative. Treatment with colchicine was commenced empirically, and she was referred to the UK National Amyloidosis Centre. There was no history of chronic inflammatory disease in her or her family and no family history of kidney disease. There was no history to suggest peripheral or autonomic neuropathy and no dyspnea. Baseline investigations revealed normal blood cell count, clotting profile, and liver function. There was no evidence of a plasma cell dyscrasia. An echocardiogram demonstrated a 12-mm interventricular wall thickness but normal diastolic dysfunction and good left ventricular systolic function, but there was mild thickening of her heart valves; overall, there was no definite evidence of cardiac amyloidosis, but it could not be completely excluded. There was insufficient tissue remaining in her renal biopsy specimen to definitively confirm the amyloid type, and there was doubt about the diagnosis of AA amyloidosis; therefore, a rectal biopsy was requested. Patient 4 presented with hypertension and was discovered to be nephrotic (7.9 g/24 h; serum albumin, 24 g/L) with CKD stage 3 on a background of type 2 diabetes mellitus and ischemic heart disease. A renal biopsy specimen revealed amyloid deposits in the glomeruli and interstitium. At presentation, she was experiencing paraesthesia in the feet and minimal dyspnea on exertion. There was no evidence of a plasma cell dyscrasia. Cardiac investigations did not suggest amyloid cardiomyopathy. The patient's sister was subsequently discovered to have amyloid deposits on a renal biopsy specimen, undertaken for proteinuria. Patient 5 presented with edema and was discovered to have proteinuria, advanced CKD, and mildly obstructive derangement of liver function tests. A renal biopsy specimen revealed amyloid deposits. There was mild breathlessness on exertion but no clinical evidence of peripheral or autonomic neuropathy. Nephelometric serum free light chain assay revealed a marked κ excess (κ, 393 mg/L; λ, 30 mg/L). There was no paraprotein by serum or urine immunoelectrophoresis, and bone marrow examination revealed less than 5% clonal plasma cells. Cardiac investigations did not show any evidence of cardiac involvement by amyloid. The patient received 15 days of high-dose dexamethasone for presumed AL amyloidosis, which substantially suppressed the κ serum free light chain concentration. Despite this, however, there was a progressive decline in renal function and the patient commenced hemodialysis. Extensive amyloid deposits were identified in the vocal cord and testes of patient 1, the palate of patient 2, the rectum of patient 3, and the kidneys of patients 4 and 5 by their pathognomonic green birefringence when stained with Congo red and viewed under crossed polarized light (Figure 1A–B). The amyloid from patients 1 through 4 stained specifically with antibodies to apoAI (Figure 1C), and staining was completely abolished by prior absorption of the antiserum with an excess of pure human apoAI. There was no other staining with antibodies to κ light chains and no staining with antibodies against other known amyloid fibril proteins, including lysozyme, transthyretin, serum amyloid A protein, or λ light chains. The amyloid from patient 5 stained with antibodies against both κ light chains and apoAI such that IHC results were inconclusive. Genetic analysis of exons 3 and 4 of the APOAI gene revealed five novel mutations (Figure 2). Four were single nucleotide substitutions, c.595G>C, c.284T>A, c.172G>A, and c.178T>G, resulting in amino acid change: alanine to proline at position 175, A175P in patient 1; phenylalanine to tyrosine at position 71, F71Y in patient 2; glutamic acid to lysine at position 34, E34K in patient 3; and serine to alanine at position 36, S36A in patient 5, respectively. The other novel variant p. His155MetfsX46, discovered in patient 4, resulted from a frameshift mutation caused by a deletion of cytosine at position c.535. The new reading frame created by this single nucleotide deletion results in premature termination, and consequently the mutated protein was 44 nucleotides shorter than wild-type apoAI. The remainder of the APOAI gene in all five patients was identical to the published sequence.28Shoulders C.C. Kornblihtt A.R. Munro B.S. Baralle F.E. Gene structure of human apolipoprotein A1.Nucleic Acids Res. 1983; 11: 2827-2837Crossref PubMed Scopus (82) Google Scholar Laser microdissection and tandem mass spectrometry showed that, in patients 1 through 4, apoAI was one of the most abundant proteins present in the amyloid deposits (Figure 3A). In contrast, there were no apoAI peptides present in the amyloid deposits of patient 5, which were composed of κ light chains. In addition to apoAI, other identified proteins included serum proteins, such as vitronectin and albumin, along with proteins that are known to be associated with all types of amyloidosis, such as apolipoprotein E and serum amyloid P component. To show the presence of mutated proteins in the amyloid deposits, we searched the mass spectrometry raw data files using the human SwissProt database supplemented with the APOAI gene variants identified in this study. The mutated protein (a tryptic peptide carrying the altered amino acid sequence) was present in the amyloid deposits of patients 2 and 3 (Figure 3B). Detailed examination of the apoAI protein coverage showed that certain tryptic peptides, which would have been present had protein encoded by the normal allele been incorporated into amyloid deposits, were absent, indicating that only the protein encoded by the abnormal allele was amyloidogenic (Figure 4). Interestingly, the mutation in patient 1 affected an area of the protein that cannot be detected by the mass spectrometry–based proteomics technology used in the study due to the presence of numerous trypsin cutting sites in this area (Figure 4). In patient 4, the frameshift mutation in APOAI generates a novel C terminus amino acid sequence, which, theoretically, could be detected by mass spectrometry–based proteomics. Although this novel sequence was included in our supplemented database, we did not identify peptides representing the novel sequence, suggesting that this part of the protein was unstable and was most likely cleaved, generating a truncated apoAI protein that was deposited as amyloid. SAP scintigraphy did not show any visceral amyloid deposits in patient 1 and showed only renal amyloid deposits in patients 4 and 5. Subclinical hepatic amyloid deposits were detected in patient 2. A large amyloid load affecting the spleen and liver with an obscured kidney signal was identified in patient 3. It should be noted that amyloid deposits in the skin, vocal cord, heart, nerves, and testes do not show up by this technique. Determining the fibril protein remains a challenge in many patients with amyloidosis. Indeed, AL amyloidosis, the most commonly diagnosed systemic form of amyloid, is not infrequently a diagnosis of exclusion, which is complicated by the fact that there is considerable overlap between the clinical features of the different amyloid types. Furthermore, it is important to recognize that the presence of a plasma cell dyscrasia, which occurs in approximately 3% of the population older than 50 years and approximately 5% of those older than 70 years,29Kyle R.A. Therneau T.M. Rajkumar S.V. Larson D.R. Plevak M.F. Offord J.R. Dispenzieri A. Katzmann J.A. Melton III, L.J. Prevalence of monoclonal gammopathy of undetermined significance.N Engl J Med. 2006; 354: 1362-1369Crossref PubMed Scopus (979) Google Scholar in a patient with amyloidosis does not prove AL type and may be incidental to their amyloidosis.3
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