GENOMICS
8,318-323
(1990)
A Mutation in Apolipoprotein of Familial Amyloidotic
A-l in the Iowa Type Polyneuropathy
WILLIAM C. NICHOLS,* RICHARD E. GREGG,t H. BRYAN BREWER,JR.,* AND MERRILL D. BENSON*$II Departments
of *Medical Genetics and SMedicine and “The Richard L. Roudebush Veterans Administration Medical Indiana University School of Medicine, Indianapolis, Indiana 46202; tDepartment of Cellular Biology, Squibb institute for Medical Research, Princeton, New Jersey 08543-4000; and $Molecular Disease Branch, NHL Bl, National Institutes of Health, Bethesda, Maryland 20892 ReceivedJune
Center,
14, 1990
al., 1989; Nichols
et al., 1989a). Most of these forms of autosomal dominant amyloidosis present as polyneuropathy in adult life with survival usually dictated by the incidence and progression of nephropathy or cardiomyopathy. Sometimes neurologic involvement is minimal or absent, as in the Danish amyloid cardiomyopathy (Frederickson, et al., 1962). One type of systemic amyloidosis which presents as a polyneuropathy similar to Portuguese FAP type I was described in an Iowa kindred (Van Allen et al., 1969). A high incidence of renal amyloidosis and severe gastric ulcer disease in affected individuals led to a separate designation for this type of amyloidosis (FAP type III). Recently we have studied the amyloid from a patient in the original Iowa kindred (Fig. 1) and discovered that the fibrils do not contain transthyretin but, instead, an amino-terminal fragment of apolipoprotein A-I (Nichols et al., 1988). We have now been able by protein and DNA analysis of affected and at-risk individuals in the family to define a mutation associated with this disease and show its inheritance.
Immunoblotting of isoelectric focusing gelsof plasmaand direct genomicDNA sequencinghave been usedto characterize a mutation in apolipoprotein A-I associatedwith the familial amyloidotic polyneuropathy originally described by Van Allen in an Iowa kindred. An arginine for glycine substitution in apolipoprotein A-I identified in the proband’s amyloid flbrils was determined to be the result of a mutation of guanine to cytosine in the apolipoprotein A-I geneat the position corresponding to the first baseof codon 26. Direct sequencing of genomic DNA of three affected individuals who died in the 1960s confirmed the inheritance of the disorder. Immunoblot analysis detected the variant apolipoprotein A-I in the proband’s plasma and in several at-risk members of the kindred. In addition, allele-specific amplification by the polymerase chain reaction was usedto detect carriers of the variant gene. q 1990 Aoademic
Press, Inc.
INTRODUCTION
Numerous types of hereditary amyloidosis have been recognized, including both systemic (familial amyloidotic polyneuropathies) and localized forms (hereditary cerebral hemorrhage with amyloidosis, hereditary Alzheimer disease) (Benson and Wallace, 1989). In some types, single amino acid changes in the amyloid fibril precursor proteins have been shown to predict expression of the disease; however, in others a genetic basis has yet to be defined. The most common type of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy (FAP) in which several single base changes in the transthyretin (prealbumin) gene have been discovered, each resulting in amyloid fibril formation. To date, eight amyloid-associated mutations have been reported, and there is evidence for more (Dwulet and Benson, 1983,1986; Nakazato et al., 1984; Wallace et al., 1986; Nordlie et al., 1988; Wallace et al., 198813; Gorevic et 0888-7543/90 $3.00 Copyright 0 1990 hy Academic Press, All rights of reproduction in any form
MATERIALS
METHODS
Isoelectric Focusing, Electrophoresis, and Immunoblotting
The detection of the isoforms of apoA-I in whole plasma was performed as previously described (Menzel and Utermann, 1986). Briefly, 20 ~1 of plasma was delipidated overnight with ethanol/ether and resuspended in sample buffer. A 5~1 aliquot was electrophoresed on an isoelectric focusing gel (pH 4-6.5, Pharmolyte), and the proteins were then electrotransferred to Immobilon membranes (Towbin et al., 1979). The apoA-I bands were detected with a mouse anti-apoA-I monoclonal antibody (a gift from Dr. Yves Marcel, Montreal, Canada) using an Auroprobe second antibody (Janssen). 318
Inc. reserved.
AND
APOLIPOPROTEIN
n l q 0 q @ FIG. (111-11)
1. Pedigree is indicated
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AMYLOIDOSIS
pathologic verification of amyloidosis clinical findings of disease presumed affected
of Iowa kindred. by the arrow.
Generations
are indicated
DNA Isolation Total genomic DNA was isolated from whole blood (Madisen et al, 1987). DNA was extracted from histological sections or paraffin-embedded specimens using modifications of published procedures (Goelz et al., 1985). Histological slides of either stomach or liver were first soaked briefly in xylenes to remove the coverslip. The slide was rinsed in 100% ethanol, and the tissue section was scraped off using a razor blade. The section was placed in 500 ~1 TE9 (500 mM Tris, pH 9.0,20 mM EDTA, 10 n&f NaCl), 0.5 mg/ml proteinase K, and 1% SDS. Sections of a paraffin block containing nasal polyps material were sliced off using a razor blade, placed on a slide, and heated briefly to melt the paraffin. The freed tissue sections were placed in 1 ml TE9,0.5 mg/ml proteinase K, and 1% SDS. After vortexing, the preparations were incubated for 24 h at 48°C. The proteinase K concentration was increased to 1.0 mg/ml, and the SDS concentration was increased to 2%. After further incubation at 48°C for 4 h, the preparations were extracted three times with 4:3:2 chloroform:phenol:TE9 and two times with Sevag (241 chloroform:isoamyl alcohol), and the nucleic acids were precipitated with 10 M ammonium acetate and 100% ethanol overnight at -20°C. The DNA was collected by centrifugation for 30 min in a microcentrifuge at 4”C, rinsed once with 80% ethanol, and air-dried at 37°C for 30 min. The resulting pellets were dissolved in 50 ~1 1X TE (10 mM Tris, pH 8.0, 1 nnJ4 EDTA). Enzymatic Amplification A-I Gene Region
IN
of Apolipoprotein
One microgram of leukocyte DNA or 5 ~1 of a fixed tissue DNA prep was amplified using the GeneAmp
in Roman
numerals
at left;
individuals
are as numbered.
The
proband
DNA Amplification Reagent Kit (Perkin Elmer Cetus) and primers PCRl (5’-CCACCTGCAGGGAGCCAGGCTCGGC-3’) and PCR2 (5’-TAGGCTGCAGCTCGGCCAGTCTGGC-3’), which define an exon a-containing 256-bp region of the apolipoprotein A-I gene. Thirty cycles of amplification consisted of denaturing at 94°C for 1 min, annealing at 65°C for 1.5 min, and extending at 72°C for 1.5 min using a DNA thermal cycler (Perkin-Elmer/Cetus). After amplification, the samples were extracted with phenol and chloroform and precipitated as described above. The amplification products were collected by centrifugation, dissolved in 10 ~1 1X TE, and electrophoresed through 4% Nusieve agarose (FMC Bioproducts) to separate the major amplification product from any nonspecific products as well as remaining primers. The gel was stained in 1 pg/ml ethidium bromide and viewed with a uv light source. Using a Pasteur pipet, a plug of agarose containing a portion of the desired amplification product was removed from the gel. Fifty microliters of 1X TE was added to each agar plug, and the samples were incubated for 15-20 min at 75’C to melt the agarose. After melting, an additional 950 ~1 1X TE was added to each plug. Asymmetric
Amplification
Single-stranded templates suitable for sequencing were generated as previously described (Gyllensten et al., 1988). Forty picomoles of PCR2 and 0.8 pmol of PCRl (5O:l ratio) were used to amplify 5 ~1 of a 1:50 dilution of the l-ml agar-extracted samples (above). Forty cycles of amplification were performed, with each cycle being 94°C for 1 min, 65°C for 1.5 min, and 72°C for 2 min. The samples were extracted with phenol and chloroform and subjected to spin dialysis
320
NICHOLS
ET
AL.
using the Centriconmicroconcentrator (Amicon). Two milliliters of 1X TE was added to each sample, which was then centrifuged at 5000 rpm for 35 min in a Beckman JA-20 rotor. This was repeated a total of four times. Final volume after spin dialysis was approximately 40 ~1. Sequencing
of ssDNA
Templates
Seven microliters of each retentate was mixed with 1 ~1 of a 5 PM solution of PCRl and 2 ~1 of 5X sequencing buffer (200 mM Tris-HCl, pH 7.5,lOO mM MgCl,, 250 mM NaCl). After annealing at 65°C for 3 min, 50°C for 5 min, and 37°C for 10 min, the mixture was brought to 6.25 mM dithiothreitol, 37.5 nM dGTP, 37.5 nM dCTP, and 37.5 nM dTTP. Deoxyadenosine 5’-[a-35S]thiotriphosphate (6.25 &i, 1350 Ci/mmol, NEN Research Products) and 3 units of modified T7 DNA polymerase (Sequenase Version 2.0, U.S. Biochemicals) were added, and the labeling reaction was continued for 2 min at 20°C. Each reaction (16 ~1) was divided among four tubes containing one of four (G, A, T, or C) termination mixes (80 PM of each deoxynucleoside triphosphate and 8 PM of the appropriate dideoxynucleoside triphosphate) and incubated at 45°C for 3 min. The reactions were stopped with the addition of 4 ~1 stop solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyan01 FF), heated to 75°C for 3 min, and loaded on an 8% polyacrylamide/8.3 M urea sequencing gel. The gel was electrophoresed for 2.5 h at 60 W, dried overnight, and exposed to Kodak X-Omat film for 48 h at -70°C. Direct
DNA
Test for the Iowa FAP Gene
One microgram genomic DNA was amplified for 30 cycles using primers A26 (5’-TGTGCTCAAAGACAGCC-3’) and PCR2 as described above. One cycle consisted of denaturing at 94°C for 1 min, annealing at 64°C for 1.5 min, and extending at 72°C for 1 min. After amplification, the samples were extracted once with Sevag to remove the mineral oil. One-tenth (10 ~1) of each sample was electrophoresed through 4% composite (3% FMC Nusieve, 1% Bethesda Research Labs) agarose until the bromphenol blue in the loading dye had migrated to the bottom of the gel. The gel was stained in 1 pg/ml ethidium bromide and viewed with a uv light source. A permanent record was made using Polaroid photography. RESULTS
Immunoblotting of isoelectric focusing gels of serum was used to show that the amyloid fibril variant apolipoprotein A-I was also present in the proband’s serum. As shown in Fig. 2, two major forms of apoli-
Cl2
3
4
5
FIG. 2. Immunoblot for apolipoprotein A-I of an isoelectric focusing gel of whole plasma from members of the apoA-I Iowa kindred. The major mature isoform of normal apoA-I is designated 0 while proapoA-I migrates in the +2 position. The deaminated forms of mature apoA-I migrate in the -1 and -2 positions. ProapoA-I Iowa and mature apoA-I Iowa migrate in the +3 and +l positions, respectively. Lane C is a normal control plasma while lanes l-5 are from members of the apoA-I Iowa kindred with lane 5 being from the proband of this family. Lanes C and l-3 do not have the mutant apoA-I in the plasma, while lanes 4 and 5 are heterozygous for apoA-I Iowa.
poprotein A-I were detected: normal apolipoprotein A-I (compared to controls) and an apolipoprotein A-I having a +1 charge compared to normal apoA-I. A substitution of arginine for glycine, as was detected in the amyloid fibrils, would be expected to produce the observed +1 shift. Also shown in Fig. 2 is immunoblotting for apolipoprotein A-I of the sera of members of the kindred at risk for development of Iowa type hereditary amyloidosis by virtue of having had an affected parent. Of 11 at-risk individuals tested, 6 were positive for being heterozygous for the variant apolipoprotein A-I having a +l shift. On basis of the reported apolipoprotein A-I gene sequence (Law and Brewer, 1984), the only base change that could account for a glycine to arginine-26 substitution is a guanine (G) to cytosine (C) transversion in the nucleotide corresponding to the first base of the codon for residue 26. This substitution was confirmed by sequencing a 256-bp PCR product containing all of exon 2 of the apolipoprotein A-I locus of our proband. Both the normal guanine and a cytosine were detected in the position corresponding to the first base of codon 26 (Fig. 3). Shown also in Fig. 3 is the sequencing of asymmetrically amplified genomic DNA extracted from fixed tissues of affected individuals of the Iowa kindred who died in the mid 1960s. All three of these individuals were also heterozygous at the first base of codon 26. Amplification of genomic DNA using the arginine26 allele-specific oligonucleotide showed a 150-bp amplification product for the proband and for 6 of 11 at-risk subjects (Fig. 4). These results show 100% concordance with the immunoblotting data and are
APOLIPOPROTEIN
III-11 TGCA
At-risk TGCA
II-6 TGC
A
AI-IOWA
Ill-5
II-5
TGCA
TGCA
FIG. 3. Autoradiogram of direct sequencing gel of singlestranded DNA generated by asymmetric amplification of exon 2 of the apolipoprotein A-I gene. Individuals sequenced are indicated as in the pedigree. Both guanine and cytosine are present in the first position of codon 26 (arrow).
proof that those individuals demonstrating the +l shift have the Arg-26 apolipoprotein A-I gene. DISCUSSION
When amyloidotic polyneuropathy in the Iowa kindred (Fig. 1) was described in 1969, the similarity of the peripheral neuropathy to the Portuguese syndrome (FAP I) suggested a common etiology (Van Allen et al., 1969). The high incidence of gastric ulcer and renal insufficiency, however, led to a separate classification of the Iowa syndrome as FAP III (some publications have referred to it as type IV). The discovery of transthyretin (prealbumin) in the amyloid fibrils of FAP I (Costa et al., 1978) and subsequent identification of eight different single amino acidvariants of this plasma protein associated with hereditary amyloidosis produced speculation that all FAP amyloids might be composed of mutant transthyretin. However, biochemical characterization of the amyloid fibrils isolated from an individual with Iowa type hereditary amyloidosis revealed a variant fragment of apolipoprotein A-I, the major protein constituent of high-density lipoprotein. The amyloid protein contained the amino-terminal 83 residues of the mature 243 amino acid long apolipoprotein A-I, but had an arginine for glycine substitution at position 26. Sequencing revealed only arginine at position 26 of the amyloid subunit protein; no normal apolipoprotein A-I (glycine at position 26) was found in the amyloid. Unfortunately, sufficient plasma was not available to isolate and characterize the apolipoprotein A-I of the proband and determine whether the proband was homozygous or heterozygous for the variant apoA-I or had normal circulating apoA-I. To answer this question we used isoelectric focusing of plasma from the proband and several at-risk members of the kindred. Immunoblotting after isoelectric focusing gel electrophoresis of the proband’s serum showed both normal apolipoprotein A-I and an apolipoprotein A-I
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having a +l charge shift from normal, which would be expected from an arginine for glycine substitution. Demonstration of both the normal and variant proteins in the affected proband’s serum predicts heterozygosity at the apolipoprotein A-I locus and is consistent with the autosomal dominant nature of Iowa type amyloidosis. Serum electrophoresis also enabled the detection of a number of asymptomatic gene carriers among those individuals at risk for development of the disease. The arginine for glycine-26 substitution predicted a guanine to cystosine transversion in codon 26 (GGC + CGC). This was confirmed by direct genomz DNA sequencing of asymmetrically amplified 256-bp PCR products that contained the second exon of the fourexon apolipoprotein A-I gene. The apoA-I gene was sequenced from our proband as well as an asymptomatic gene carrier identified by the serum protein studies. Both the normal guanine and the variant cytosine were detected at the nucleotide that corresponds to the first position of codon 26, proving that each affected individual is heterozygous for the arginine-26 variant apolipoprotein A-I gene. To demonstrate a correlation between the variant apoA-I and development of Iowa type hereditary amyloidosis, DNA was obtained from biopsy specimens (slides and paraffin blocks) of three affected individuals who died in the 1960s (Fig. 1: 11-3, stomach; 11-5, nasal polyps; 111-5, liver). While the nucleic acid material obtained from the histological slides and paraffin blocks was highly degraded, it was possible to detect the desired PCR product after amplification and agarose gel electrophoresis, enabling the subsequent asymmetric PCR step. Direct genomic sequencing of all three individuals revealed sequences for both the normal apolipoprotein A-I gene and the arginine-26
FIG.
4.
DNA was subjected to amplification with primers A26 and PCRB, electrophoresed through 4% composite agarose, stained with ethidium bromide, and photographed under uv light. Lane 2, proband; Lane 3, normal control; Lanes 4-14, individuals at risk for carrying the arginine-26 gene. Only those individuals positive for the a&nine-26 gene demonstrate the 150-bp amplification product. Lane 1 is BRL 1-kb ladder.
322
NICHOLS
apolipoprotein A-I gene. This demonstrates inheritance of the variant gene through three generations of affected individuals and helps to establish its association with development of the amyloidotic polyneuropathy. Although direct DNA tests have been reported for detection of variant transthyretin genes associated with hereditary amyloidosis, these are based on changes in the restriction pattern of the gene (Wallace et al., 1986, 1988a,b; Jacobson et al., 1988a,b; Nichols and Benson, 1990; Nichols et al, 1989a). The guanine to cytosine transversion reported here results in neither the creation nor loss of a known restriction enzyme site in the apolipoprotein A-I gene; therefore, direct detection of the arginine-26 variant gene by restriction analysis is not possible. We have developed a direct DNA test by PCR using an allele-specific oligonucleotide primer (Nichols et al., 1989c; Wu et al, 1989; Okayama et al., 1989). The primer, a 16-mer, was designed so that it is complementary to the negative strand of the arginine-26 apolipoprotein A-I gene with the l-base difference from normal (cytosine for guanine) being the 3’ nucleotide. When the primer anneals to the negative strand of the normal gene, the 3’ nucleotide remains unpaired preventing primer elongation by Taq polymerase. Thus, only those DNA samples containing the arginine-26 variant gene show the expected 150-bp product after amplification and agarose gel electrophoresis. DNA samples that do not contain the arginine-26 variant gene do not show a specific amplification product (Fig. 4). Although the samples may show some faint bands of nonspecific amplification, these can actually serve as an internal control for the amplification reaction and help to prevent the occurrence of false negatives. Complete concordance was observed between the serum protein, genomic DNA sequencing, and allele-specific PCR studies for detection of arginine-26 gene carriers. Thus, the allele-specific PCR test serves as a quick and reliable means for detection of the arginine-26 apolipoprotein A-I gene. The identification of a new variant of apolipoprotein A-I and its association with autosomal dominant amyloidosis indicate that familial amyloidotic polyneuropathy is heterogeneous in terms of the proteins involved in amyloid fibril formation. While the majority of FAP syndromes are caused by variant transthyretins, these findings suggest that other proteins may be identified in the amyloid deposits of other kindreds with FAP. The allele frequency of the arginine-26 apoA-I must be very low. While we have not performed random population testing for this gene, it has not been identified in large series of A-I screenings. In studies by von Eckardstein et al. (1989, 1990), the results of electrophoretic mutation screening of 32,000 new-
ET
AL.
borns failed to detect this mutation of apoA-I. Since the GlyZ6 to Arg,, mutation results in a charge difference, this mutation is readily detected by electrophoretie analysis of plasma (Klaus Altland, personal communication) . The factors that result in only the amino-terminal 83 amino acids of the 243 amino acid apolipoprotein A-I polypeptide being deposited in the amyloid remain to be determined. The arginine for glycine substitution may make the protein more susceptible to proteolytic cleavage, thus producing the 83-residue fragment that is then deposited as amyloid fibrils, or the entire variant protein may be deposited with subsequent proteolysis, leaving only the fragment in the fibrils. It is of interest that the secondary structure of apolipoprotein A-I does not predict it to be an amyloidogenie protein. The majority of amyloid proteins have extensive P-sheet configuration which contributes to their deposition as amyloid fibrils (Glenner, 1980). In contrast, apolipoprotein A-I is composed mostly of 22 amino acid repeats of amphipathic helix (Segrest et al., 1974; Fitch, 1977), and these helical repeats are in the carboxyl-terminal75% of the molecule (Law and Brewer, 1984; Boguski et al,, 1985). Therefore, the amino-terminal 83 amino acid apolipoprotein A-I peptide that is found in the amyloid fibrils contains one 22 amino acid amphipathic helix at the carboxylterminus of the peptide while the structure is not well defined for the amino-terminal three-fourths of the peptide. Similar structural factors must be considered for reactive amyloidosis in which the fibrils contain amyloid A protein. Amyloid A is the amino-terminal portion (most frequently 76 residues) of a serum apolipoprotein that is not predicted to have extensive @ configuration. Investigations are now under way to determine what alterations in the structure of apolipoprotein A-I may result from the glycine to arginine substitution at amino acid 26, and how this substitution results in the development of dominant familial amyloidosis. In addition, studies are being conducted to determine whether there are any alterations in the metabolism of lipids and lipoproteins in subjects with this mutation, and if so, how these abnormalities may also play a role in the expression of this syndrome.
ACKNOWLEDGMENTS We thank Ms. Marie Rindt for technical assistance. Also we thank Dr. Thomas H. Kent and Dr. Robert T. Cook for help with retrieving pathology materials. This work was supported by VA Medical Research, the United States Public Health Service (RR00750, NIDDK-34331, NIAMS-AR20582, AR7448), the Arthritis Foundation, the Grace M. Showalter Trust, and the Marion E. Jacobson Fund.
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