Background

Mutations in the gene for apolipoprotein AII (apoAII) have recently been found to cause hereditary renal amyloidosis. In each case amyloid deposition has been associated with a peptide extension at the carboxyl-terminus of apoAII, the result of mutations in the normal stop codon.

Methods

A Caucasian man who has had progressive renal dysfunction since age of 34 was found to have amyloidosis on renal biopsy at age 56. Echocardiogram showed mild intraventricular septal thickness and technetium-99m (^99mTc)-pyrophosphate scintigraphy demonstrated uptake by cardiac muscle consistent with amyloid deposition in the myocardium. His father died of renal failure and his paternal half brother has renal dysfunction.

Results

DNA sequencing of the apoAII gene in the proband showed a T to C transition at the first position of the stop codon indicating replacement of the stop codon by L-arginine (Arg) at residue 78. Western analysis of the proband's plasma under reducing conditions using anti-apoAII revealed an extra band at approximately 10 kD in addition to the normal apoAII band at 8 kD. Western analysis of solubilized amyloid fibrils isolated from rectal biopsy tissue contained only the variant apoAII.

Conclusion

These results indicate that the proband's amyloid fibrils are derived from apoAII and the amyloidogenesis is linked to the peptide extension at the carboxyl-terminus of variant apoAII. Of particular interest is that this novel apoAII variant may cause amyloid cardiomyopathy in addition to renal amyloid.

METHODS

Clinical evaluation

The proband is a 56-year-old Russian male of Armenian origin who emigrated from Russia to the United States at age 49 years. He had been well until he was found to have proteinuria and hypertension at age 34 years. While his serum creatinine (normal <1.5 mg/dL) level was 1.8 mg/dL at the age of 36 years, no extensive medical studies were done at that time. His renal function gradually worsened and at age 50 years he was found to have a serum creatinine level as high as 2.2 mg/dL and 3 g of daily proteinuria. An electrocardiogram (ECG) suggested a previous myocardial infarction but coronary angiography revealed no significant disease. By age 54 years, his serum creatinine was elevated to 3.0 mg/dL and blood urea nitrogen (BUN) (normal <25 mg/dL) level was 40 mg/dL. Physical examination at age 56 years showed that his arterial blood pressure was 150/80. There were no signs of hepatosplenomegaly or polyneuropathy. Laboratory examinations revealed mild normocytic anemia [red blood cell count 4.21 10⁶/mm³ (normal 4.5 to 6.0 10⁶/mm³) and hemoglobin 12.6 g/dL (normal 14 to 18 g/dL)] with weekly erythropoietin injections. Serum levels of creatinine and BUN were elevated to 3.5 mg/dL and 51 mg/dL, respectively. Plasma lipid profile revealed cholesterol 227 mg/dL, HDL cholesterol 55 mg/dL, triglycerides 170 mg/dL, very low-density lipoprotein (VLDL) 34 mg/dL, and low-density lipoprotein (LDL) 138 mg/dL. There was no evidence of monoclonal gammopathy on immunofixation of serum or urine. An ECG disclosed poor R progression in leads V₁ to V₃, and echocardiography showed mild intraventricular septal thickness (1.5 cm, normal range <1.2 cm). Ventricular wall motion was intact. Ejection fraction was estimated at 60%. Stress ECG showed no ischemic changes. Technetium-99m (^99mTc)-pyrophosphate scintigraphy showed slight uptake by cardiac muscle consistent with amyloid deposition. Abdominal ultrasound examination showed no abnormality of liver or spleen. Renal scintigraphy demonstrated moderately decreased flow and function of kidneys, with total glomerular filtration rate (GFR) to be 19.7 mL/min. Renal biopsy performed at age 55 years revealed evidence of focal glomerulosclerosis in addition to amyloid deposition in glomeruli and vascular walls Figure 1. A skin biopsy revealed amyloid deposition in vascular walls in the dermis. Abdominal fat pad aspiration was negative for amyloid deposition. Bone marrow biopsy demonstrated no abnormality and no amyloid deposition. Rectal biopsy revealed amyloid deposition in vascular walls in the submucosa. DNA analysis of a number of genes that are associated with renal amyloidosis, including apoAI, fibrinogen A-chain, and lysozyme, failed to reveal any abnormalities.

Figure 1.

Renal biopsy. (A) Extensive glomerular amyloid deposits and amyloid in walls of blood vessels. (B) Congo red staining.

Full figure and legend (186K)

The proband's father died of renal insufficiency at 53 years of age. A paternal half brother (age 45 years) has suffered from renal failure. The proband has a 21-year-old son who is in good health.

DNA isolation

After full informed consent was obtained, genomic DNA of the proband was isolated from peripheral blood leukocytes using standard phenol/chloroform method. DNAs of other family members were not available for investigation.

Single-strand conformation polymorphism (SSCP) analysis

Exons 3 and 4 of apoAII gene were examined by single-strand conformation polymorphism (SSCP) analysis using oligonucleotide primers described elsewhere⁵. Polymerase chain reaction (PCR) was done in a total volume of 50 L, containing 5 L of 10 PCR buffer (100 mmol/L Tris HCl, pH 8.3, 500 mmol/L KCl, and 12 mmol/L MgCl₂), 8 L of 1.25 mmol/L desoxynucleoside triphosphate (dNTP), 15 pmol of each primer, 2.5 units of Taq polymerase (Sigma Chemical Co., St. Louis, MO, USA), and 100 ng of genomic DNA. Amplification was performed using a Perkin-Elmer ThermalCycler (Norwalk, CT, USA) for 40 cycles consisting of denaturing at 95°C for 30 seconds, annealing at 63°C (exon 3) or 60°C (exon 4) for 1 minute, and extension at 72°C for 30 seconds. SSCP analysis was done as described previously¹⁰.

Direct DNA sequence analysis

DNA was analyzed by direct sequencing of exons 3 and 4 of the apoAII gene. PCR was done as indicated above. The PCR product was purified by QIAquick PCR Purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Sequencing reaction was performed by using 1.0 L of purified PCR product as template and the Thermo Sequence [-³³P]-ddNTP–radiolabeled terminator cycle sequencing kit (USB; Cleveland, OH, USA). Samples were electrophoresed on a 6% polyacrylamide gel at 45 W for 3 hours using a glycerol-tolerant gel buffer, dried, and exposed to Kodak X-Omat film (Rochester, NY, USA).

Western analysis of plasma

Plasma samples were heated in a boiling water bath for 10 minutes with or without 5% 2-mercaptoethanol (2-ME) and electrophoresed on a 16.5% Tris-Tricine Ready gel (Bio-Rad, Hercules, CA, USA). Subsequently, proteins were electrotransferred onto nitrocellulose membrane (Trans-Blot Transfer Medium; Bio-Rad). After blocking the membrane with 5% nonfat dry milk in phosphate-buffered saline (PBS), pH 7.4, the apoAII bands were detected with sheep anti-human apoAII (Roche, Indianapolis, IN, USA). Alkaline phosphatase conjugated donkey anti-sheep IgG (Sigma Chemical Co.) was used as the second antibody. The immunoblots were visualized with a reaction to nitro blue tetrazolium/bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) alkaline phosphatase substrate (Sigma Chemical Co.).

Rectal biopsy tissue was hand homogenized in PBS, centrifuged, and washed twice by resuspension in PBS followed by centrifugation at 14,000 rpm 15 minutes¹¹. The pellet was dialyzed against distilled water and lyophilized. Freeze-dried material was dissolved in 6 mol/L guanidine containing dithiothreitol, alkylated with iodoacetic acid, dialyzed versus water and lyophilized. Western analysis was performed as noted above for plasma samples.

RESULTS

DNA analysis

SSCP analysis revealed an abnormally migrating band for apoAII exon 4 PCR products of the patient in contrast to those of normal controls, which was different from those in patients with apoAII Stop78Gly mutation⁵ and Stop78Ser mutation¹⁰ (data not shown). Direct DNA sequencing of the exon 4 PCR product showed a single-base substitution in the stop codon for the apoAII gene (TGA to CGA) indicating a stop to arginine substitution at codon 78 followed by 60 bases before a new stop codon Figures 2 and 3¹². No other mutations were found in exons 3 and 4 of apoAII gene, which encode the entire pro- and mature apoAII protein¹².

Figure 2.

DNA sequence analysis of exon 4 of apolipoprotein AII (apoAII) gene. Both T and C are present at the first position of apoAII stop codon (arrow) in the DNA sequence of the patient, indicating a replacement of stop codon by arginine (Arg) at codon 78. Abbreviations are: Ser, serine; Gln, glycine; Thr, threonine; Ala, alanine.

Full figure and legend (45K)

Figure 3.

Schema of 3' cDNA sequence and deduced C-terminal amino acid sequence in the patient shows that the Stop78Arg mutation induces an extended peptide composed of 21 amino acids. 

Full figure and legend (27K)

To exclude the possibility that the T to C mutation might be a polymorphism, we analyzed DNA from 50 unrelated individuals (100 alleles) by SSCP analysis. None had the abnormal SSCP pattern as seen in the patient (data not shown).

Characterization of plasma apoAII

Plasma samples, obtained from this patient, patients with variant apoAII proteins described previously (apoAII Stop78Gly and Stop78Ser mutations)^5,10, and a normal individual, were studied by Western analysis using anti-human apoAII Figure 4. In the patient in this study, positive bands migrating with molecular weight corresponding to dimeric normal apoAII, heterodimeric apoAII with normal and variant molecules, and dimeric variant apoAII were identified in nonreducing conditions. In the presence of 2-ME, Western analysis revealed two bands (approximately 8 and 10 kD) corresponding to normal apoAII monomer and the larger variant apoAII monomer, respectively. The molecular weight of the variant apoAII in the patient described here was the same as in patients previously reported^5,10.

Figure 4.

Western blot of patient's plasma with anti-human apolipoprotein AII (apoAII). Nonreduced denatured and reduced denatured plasma samples were run in lanes C (normal control), G78 (a patient with apoAII Stop78Gly variant⁵), S78 (a patient with apoAII Stop78Ser variant¹⁰), and R78 (the patient in this study). Abbreviations are: N, normal apoAII monomeric form; V, variant apoAII monomeric form; V + V, N + V, and N + N, dimeric forms of apoAII.

Full figure and legend (27K)

Characterization of isolated amyloid fibril protein

Western analysis using anti-apoAII of amyloid fibril protein isolated from rectal biopsy tissue identified a protein band migrating at the same level as the variant apoAII in serum. No apoAII band corresponding to wild-type apoAII was identified in the isolated tissue preparation indicating that no significant blood contamination was present Figure 5.

Figure 5.

Western analysis of serum and amyloid fibrils from patient with apolipoprotein AII (apoAII) amyloidosis. Serum of the patient (lane 2) shows both normal (N) and variant (V) apoAII under reducing conditions, whereas a normal control serum (lane 3) contains only normal apoAII. Amyloid fibrils isolated from the patient (lane 1) contain only the variant apoAII, indicating that either reduction of the disulfide linked dimer occurs prior to amyloid fibril formation or only the variant homodimer participates in the amyloid process.

Full figure and legend (31K)

DISCUSSION

Human apoAII amyloidosis is now known as one form of hereditary renal and nonneuropathic amyloidosis, similar to that described by Ostertag¹. Previously two different mutations were identified and, surprisingly, both mutations were located in the stop codon of apoAII (Stop78Gly and Stop78Ser) and induce a C-terminal 21 amino acid extension Figure 6^5,10. Clinically, the most prominent manifestation in all affected patients of both families was renal failure due to amyloid deposition in the kidney Table 1. Symptoms due to amyloidosis of other visceral organs, including the heart, liver, spleen, and peripheral nerves, were absent. The onset of the disease in both families varied from adolescence to the fifth decade^9,10. While autopsy studies in the first reported family with apoAII Stop78Gly variant revealed that amyloid accumulation was present in most visceral organs, except for central nervous system and peripheral nerves, renal amyloid was the most prominent feature⁹. The severity of amyloid deposition in the liver, heart, and spleen was moderate and limited mainly to vascular walls⁹. In the present study, DNA analysis revealed that the patient was heterozygous for a novel stop codon mutation in the apoAII gene (TGA to CGA) that causes replacement of the stop codon by arginine at codon 78. Western analysis of the patient's plasma using anti-human apoAII revealed not only a normal apoAII but also a variant apoAII. On Western analysis, the molecular weight of the variant apoAII in this patient was the same as that of patients with apoAII variant Stop78Gly⁵ and Stop78Ser¹⁰. Therefore, our results suggest that the variant apoAII induced by Stop78Arg mutation in the patient described here consists of a C-terminal 21 amino acid extension as well as a normal apoAII, as has been seen in patients reported previously Figure 6, since the Stop78Arg mutation does not cause a frame-shift.

Figure 6.

Schema of three identified mutant apolipoprotein AII (apoAII) amyloid proteins compared to wild-type apoAII. Each variant is a 98 residue protein differing only at residue 78.

Full figure and legend (28K)

Table 1 - Three families with apoAII mutation.

Full table

The most noticeable clinical manifestation of the patient in this study was nephropathy caused by renal amyloidosis, which resembles that in patients with variant apoAII reported previously, although the onset of the disease varied among the three families Table 1. While the mechanism of how amyloid proteins have organ specificity that leads to variation in clinical features is unknown, the nonneuropathic and nephropathic syndrome is the common feature for apoAII amyloidosis. The clinical features of apoAII amyloidosis are similar to fibrinogen A-chain and lysozyme amyloidosis in terms of nephropathy without neuropathy^3,4,13. However, the latter forms (fibrinogen A-chain and lysozyme) usually show more extensive hepatic and splenic involvement.

On the other hand, in the proband described herein, amyloid deposition in the myocardium was suggested by ^99mTc-pyrophosphate scintigraphy, ECG, and echocardiography. As mentioned above, a previous study⁹ showed that amyloid deposition in the heart was limited to only vascular walls. Also, in the patient with variant apoAII Stop78Ser, there was no clinical evidence of amyloid cardiomyopathy¹⁰. Although the proband has not shown symptoms of cardiomyopathy so far, of particular interest is that this apoAII Stop78Arg variant may cause cardiac amyloid deposition.

Since the discovery of apoAII as an amyloid fibril protein, limited autopsy specimens have become available for biochemical studies of the fibril deposits. The application of microtechniques for amyloid tissue characterization, as used in this study, has expanded the means to correctly analyze the pathology of these diseases¹¹. Both Western analysis and amino acid sequence analysis are now feasible methods for determining the type of amyloid in tissues.

In addition, although not performed in the present study, immunohistochemistry using anti-apoAII, which is used for Western analysis, may be helpful for diagnosis.

While the precise mechanisms of amyloid fibril formation in human apoAII amyloidosis, especially how the apoAII molecule obtains the -sheet configuration during fibril formation, are not clear, all patients among the three families had an extended 21 amino acid peptide at the C-terminus in common; although the first residue of that 21 residue peptide (glycine⁵, serine¹⁰, or arginine at residue 78) is different in each family Figure 6. To date, there have not been reports of human apoAII amyloidosis caused by other mutations except for the stop codon site. Therefore, it is likely that the role of human apoAII as a precursor of amyloid fibrils is linked to the extension of the protein at the C-terminus. This is clearly different from the SAM model, in which amyloidogenesis is linked to difference in the apoAII N-terminal sequence^14,15. The importance of newly induced C-terminal peptide in amyloid formation has also been identified with fibrinogen Aa-chain^16,17, ABri in familial British dementia¹⁸, and ADan in familial Danish dementia¹⁹. The new peptide sequence at the C-terminus in variant apoAII is predicted to contain an -helix that is different from the three class A amphipathic helices, important for lipid binding²⁰. As suggested previously⁵, it is likely that decreased binding capacity to HDL caused by variant apoAII with extended sequence without lipid binding capability could alter its metabolism and accelerate amyloid fibril formation.

References

References

1.	Ostertag B. Familiare Amyloid-erkrankung. Z Menschl Vererbungs Konsit Lehre 1950; 30: 105–115.
2.	Nichols WC, Dwulet FE, Liepnieks J & Benson MD. Variant apoprotein AI as a major constituent of a human hereditary amyloid. Biochem Biophys Res Commun 1988; 156: 762–768. | Article | PubMed | ISI | ChemPort |
3.	Benson MD, Liepnieks J & Uemichi T. et al Hereditary renal amyloidosis associated with a mutant fibrinogen -chain. Nat Genet 1993; 3: 252–255. | Article | PubMed | ISI | ChemPort |
4.	Pepys MB, Hawkins PN & Booth DR. et al Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 1993; 362: 553–557. | Article | PubMed | ISI | ChemPort |
5.	Benson MD, Liepnieks JJ & Yazaki M. et al A stop-codon mutation in the apolipoprotein AII gene associated with human hereditary amyloidosis. Genomics 2001; 72: 272–277. | Article | PubMed | ISI | ChemPort |
6.	Benson MD. Amyloidosis,. inThe Metabolic and Molecular Bases of Inherited Disease 2001; volume III 8th edition), edited by Scriver CR, Beaudet AL, Sly WS, Valle D New York, McGraw Hill Book Co pp: 5345–5378.
7.	Brewer HB, Jr, Lux SE, Ronan R & John KM. Amino acid sequence of human apoLp-Gln-II (apoA-II), an apolipoprotein isolated from the high-density lipoprotein complex. Proc Natl Acad Sci 1972; 69: 1304–1308. | PubMed | ChemPort |
8.	Higuchi K, Yonezu T & Kogishi K. et al Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum: apoSASSAM is an apoA-II apolipoprotein of mouse high density lipoproteins. J Biol Chem 1986; 261: 12834–12840. | PubMed | ChemPort |
9.	Weiss SW & Page DL. Amyloid nephropathy of Ostertag with special reference to renal glomerular giant cells. Am J Pathol 1973; 72: 447–460. | PubMed | ISI | ChemPort |
10.	Yazaki M, Liepnieks JJ & Yamashita T. et al Renal amyloidosis caused by a novel stop-codon mutation in the apolipoprotein A-II gene. Kidney Int 2001; 60: 1658–1665. | Article | PubMed | ISI | ChemPort |
11.	Kaplan B, Murphy CL & Ratner V. et al Micro-method to isolate and purify amyloid proteins for chemical characterization. Amyloid 2001; 8: 22–29. | PubMed | ISI | ChemPort |
12.	Knott TJ, Wallis SC & Robertson ME. et al The human apolipoprotein AII gene: Structural organization and sites of expression. Nucleic Acid Res 1985; 13: 6387–6398. | PubMed | ISI | ChemPort |
13.	Uemichi T, Liepnieks JJ & Benson MD. Hereditary renal amyloidosis with a novel variant fibrinogen. J Clin Invest 1994; 93: 731–736. | PubMed | ISI | ChemPort |
14.	Higuchi K, Yonezu T & Tsumasawa S. et al The single proline-glutamine substitution at position 5 enhances the potency of amyloid fibril formation of murine apoA-II. FEBS Lett 1986; 207: 23–27. | Article | PubMed | ISI | ChemPort |
15.	Higuchi K, Kitagawa K & Naiki H. et al Polymorphism of apolipoprotein A-II (apoA-II) among inbred strains of mice: relationship between the molecular type of apoA-II and mouse senile amyloidosis. Biochem J 1991; 279: 427–433. | PubMed | ISI | ChemPort |
16.	Uemichi T, Liepnieks JJ & Yamada T. et al A frame shift mutation in the fibrinogen A-chain gene in a kindred with renal amyloidosis. Blood 1996; 87: 4197–4203. | PubMed | ISI | ChemPort |
17.	Hamidi Asl L, Liepnieks JJ & Uemichi T. et al Renal amyloidosis with a frame shift mutation in fibrinogen A-chain gene producing a novel amyloid protein. Blood 1997; 90: 4799–4805. | PubMed | ChemPort |
18.	Vidal R, Frangione B & Rostagno A. et al A stop-codon mutation in the BRI gene associated with familial British dementia. Nature 1999; 399: 776–781. | Article | PubMed | ISI | ChemPort |
19.	Vidal R, Revesz T & Rostagno A. et al A decamer duplication in the 3' region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred. Proc Natl Acad Sci 2000; 97: 4920–4925. | Article | PubMed | ChemPort |
20.	Segrest JP, Jones MK & Loof HD. et al The amphipathic helix in the exchangeable apolipoproteins: A review of secondary structure and function. J Lipid Res 1992; 33: 141–166. | PubMed | ISI | ChemPort |

Acknowledgments

This work was supported by NIH grants PHS AG 10133, DK42111, RR-00750, Veteran Affairs Medical Research, the Marion E. Jacobson Fund, and the Machado Family Research Fund.