Am J Hum Genet 30:359- 365, 1978

Alpha1-Antitrypsin: Further Genetic Heterogeneity Revealed by Isoelectric Focusing F. KUEPPERS1 AND M. J. CHRISTOPHERSON2

The human serum protein alpha1-antitrypsin displays a remarkable degree of genetic heterogeneity. More than 20 electrophoretic variants are currently known. Starch gel electrophoresis is often used to demonstrate the genetic variability of a1-antitrypsin [1]. This procedure may be followed by crossed immunoelectrophoresis for the identification of variants that are present in concentrations too low to be detected by conventional protein staining [2]. Under these conditions and in isoelectric focusing, a1-antitrypsin forms a characteristic pattern of five to eight protein bands of different intensities [1]. The application of isoelectric focusing to the analysis of a1-antitrypsin phenotypes has revealed an even greater genetic heterogeneity than previously detected by starch gel electrophoresis. Using a narrow pH gradient that included pH 3.5-5.0, the starch gel-electrophoretic phenotype Pi M could be further classified into common phenotypes M1, M1M2, and M2 [3-6].* Pi M2 as used here and by Frants and Eriksson [3] is identical to Pi M1 [4], Pi Mc [5], Pi MN [6], and Pi N used by Constans [7] but not that of Cox et al. [8]. The isoelectric point of Pi M2 is only slightly higher than that of the most frequent type, Pi M1. In this paper, we report another common variant Pi M3 with an isoelectric point between those of Pi M1 and Pi M2. This variant was recognized by a modified procedure of a previously reported technique. MATERIALS AND METHODS

The ampholine-containing acrylamide gel was prepared as described previously [9] except that only 0.02 ml of N,N,N1,Nl tetramethyl ethylenediamine (Eastman, no. 8178, Rochester, N.Y.) was added, and the ampholine (LKB 1809-11 1, batch 13 and 14) concentration was increased to 3 ml per gel. A Buchler power supply and a LKB Multiphor apparatus were used. The gel (124 x 260 x 1 mm) was prefocused for 1 hr using electrode wicks made of two strips of filter paper (Whatman no. 17, 10 mm x 245 mm). The anodal wick was soaked with 1 N H3PO4; 1 N NaOH was used for the cathodal wick. Initially with a constant current of 50 MA applied to the gel, the voltage was 300 V, that increased to 800 V in 20 min. Then constant voltage of 800 was applied for the duration of the prefocusing run. Samples were applied to filter papers 6 mm x 8 mm (Schleicher and Schuell, no. 470, Keene, Received November 10, 1977; revised February 27, 1978. This work was supported by grants HL 19262 and HL 21567 from the National Institutes of Health. ' Temple University School of Medicine, 3401 North Broad Street, Philadelphia, Pennsylvania 19140. 2Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901. * This nomenclature is recommended by the Pi Committee (A. M. Johnson, personal communication, August 4, 1977). © 1978 by the American Society of Human Genetics. All rights reserved.

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KUEPPERS AND CHRISTOPHERSON

N.H.) by soaking in serum and then blotting briefly to remove excess. The sample papers were placed on the surface of the gel 20 mm from the cathodal edge. With sample papers in position, a constant voltage of 800 was applied. The initial current of 30 MA increased to 50 MA within 2 At this point a constant current was applied and the voltage dropped from 800 to a minimum min. of 520-550 and increased again to 800 V within 5 min. For the remainder of the electrofocusing the voltage was kept constant at 800. Sample papers were removed after they had been on the gel

for 20 min. The focusing continued for 2.5-3 hr. A LKB 2113 power supply was used for some runs. This instrument can be set so that 800 V are not exceeded. The gels were stained with Coomassie Brilliant Blue (LKB application note 75, supplied by the manufacturer with the multiphor apparatus). Crossed immunoelectrophoretic runs were performed by a modified procedure of Laurell [2]. After completion of the isoelectric focusing run, portions of the acrylamide gel slabs containing the a1-antitrypsin region were placed on top of antiserum-containing agarose plates (83x 102 mm). The transfer of the gel slab to the agarose plate must be done quickly to minimize diffusion of the protein bands. The antiserum was specific for a1-antitrypsin; 1.2 ml was added to 15 ml of agarose for one plate. An electrophoresis was performed at 400 V for 30 min with the electric field at a right angle to the direction of the isoelectric focusing run. After electrophoresis the acrylamide slab was removed, and the plates were placed in saline solution for 12 to 20 hr. The plates were then dried and stained with amido black. The serum samples were from the following groups: (1) 128 (80 males and 48 females) healthy white blood donors and hospital employees from Rochester, Minnesota (mean age: 33.5 years; range 18-65); (2) 112 (86 males and 26 females) healthy white blood donors from southeastern Pennsylvania (mean age: 27 years; range: 18-62); (3) 231 (161 males and 70 females) healthy blacks from Evans County, Georgia who participated in a blood lipid study (mean age: 39 years; range: 28-82); (4) 73 (72 males and 1 female) healthy black blood donors from Philadelphia (mean age: 31 years; range: 18-54). Additional serum samples from first-degree relatives of selected blood donors were also collected. Alpha1-antitrypsin concentrations were determined by radial immunodiffusion [10, 11]. For comparison two normal serum pools from Dr. J. A. Pierce, Washington University, St. Louis, Mo., and Dr. M. K. Fagerhol, Ullevaal Hospital, Oslo, Norway were used. Absorption experiments for the identification of the newly observed protein bands as a,-antitrypsin were performed as described previously [4]. RESULTS AND DISCUSSION

The modified isoelectric

focusing technique revealed an additional set of bands in a

position cathodally of the M1 bands but anodally of M2. The distinction of the M1 bands and the new components is usually best for the major cathodal bands and for the most

cathodal minor bands (fig. 1). To determine if these additional bands consisted of a1-antitrypsin, absorption experiments with antibody specific for a1-antitrypsin were performed [4]. When antibody was added in slight excess, the new bands disappeared along with the typical

M-bands, indicating that the additional bands consisted of

a1-antitrypsin.

Crossed

immunoelectrophoresis showed consistently wider bands when the new component was present together with M1. In phenotype M1MK, the distance between the major

cathodal bands was sufficient so that two bands could be clearly distinguished on modified crossed immunoelectrophoresis (fig. 2). Studies of several families were consistent with the hypothesis that the new a1-antitrypsin bands were genetically determined by a codominant allele at the Pi locus (table 1). In agreement with Klasen et al. [12], we use the designation Pi M3 for this variant. Other investigators recently reported similar findings.

HETEROGENEITY OF ALPHA 1-ANTITRYPSIN

2M3

M1

M1M3

Ml

-

1A1M2

M2M3 M1M2 M2

-

M2

Ml M2

Ml M3 M2M3

-

361

M.

M3

-

C FIG. 1.-Alpha1-antitrypsin types on isoelectric focusing pH 3.5-5.0 of whole serum: A, M2M3, M1, M1M3, M2M3, M1M2, and M2; B, M1M2, M1, and M3. Anode is at top. Area with alpha1-antitrypsin bands is indicated by the line at the right. C, schematic drawing of alpha,-antitrypsin phenotypes as seen on the gels in figs. IA, IB, and 2. Only the five major bands are drawn. TABLE 1 PHENOTYPES IN FAMILIES WITH SEGREGATING PiMI, PiM2, PiM3 OFFSPRING

MOTHER

FATHER

M,

ml

.................

M2M3 ................. *

M3

.............

M2 M1M2 *

..................

.................. .................

Not available for study.

M1M3

M2

M2S M2M3 M2M3

M2M3

I

M1

M1M3

M1M3 I I

I .. .

4 5

I

1 6 1

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KUEPPERS AND CHRISTOPHERSON

FIG. 2. -Modified crossed immunoelectrophoresis of sera with Pi phenotypes MI, M1M2, and M1M3. M1M3 shows a wider major cathodal band. Anode is at top.

Genz et al. [5] identified variant Mb that is almost certainly identical to M3, judging from the position of the al-antitrypsin bands in the pH gradient. M3 is identical to "M intermediate" by direct comparison on isoelectric focusing (R. R. Frants, personal communication, October 10, 1977). Another recent report of M3 by Klasen et al. [12] described the same variant, although a clear distinction of phenotypes M1, M1M3, and M3 from each other and of M2M3 from M2 was not possible by their technique. The allele frequency of PiM3 in the Dutch population sample is the same (.11) as that of U.S. whites reported here. Genz et al. [5], however, reported an allele frequency of only 0.6 forPiM3 (=piMb) in a German population. This low figure may represent a true difference in allele frequency between the two populations, but it is more likely due to insufficient resolution of the Ma and Mb bands, which could lead to a low estimate of the piM3 frequency. The M3 variant is apparently constant under usual laboratory conditions. Repeated freezing and thawing does not change the typical isoelectric focusing pattern. Repeat samples that had been in the mail for three to four days also did not change their original Pi phenotype. Studies of several groups of blood donors were consistent with the genetic interpretation. Sera from 240 whites and from 304 blacks were examined. In these serum samples, we have seen most of the combinations of the different variants in the proportions expected under Hardy-Weinberg equilibrium. The agreement with ex-

HETEROGENEITY OF ALPHA 1-ANTITRYPSIN

363

pected values was very good: P-.4 for whites and P > .95 for blacks. There was also good agreement of allele frequencies between the samples within each group collected at different locations. It was therefore justified to combine the data of groups 1 and 2 for whites and groups 3 and 4 for blacks to calculate the prevalence of phenotypes and the allele frequencies (tables 2 and 3). The allele frequency of PiM2 is now higher for whites than that previously reported (. 19 as compared to .09) [4]. This difference can best be explained by the improved resolution of the a1-antitrypsin bands with our current isoelectric focusing method. We found no exception from allelic behavior for any of the variants including PiM3. There were never more than two variants present in any sample. Heterozygotes had less concentrated isoelectric a1-antitrypsin bands than homozygotes. Isoelectric focusing of mixtures of equal volumes of serum from homozygotes resulted in patterns that were indistinguishable from those of the respective heterozygotes. The M3 variant is present in serum in the same concentration as a1-antitrypsin of the M1 and M2 type. We found very similar levels given in mg/ml (+ SD) of a1-antitrypsin in sera with the following phenotypes: M1 (40 samples), 2.24 ± 0.22; M2 (13 samples), 2.33 + 0.41; M2M3 (6 samples), 2.37 + 0.15; M1M3 (11 samples), 2.26 + 0.26; M1M2 (15 samples), 2.45 + 0.41; and M3, 2.05 and 1.98 in two samples. Two serum pools from St. Louis and Oslo that are used in some laboratories as standards for the normal concentration (100%) of a1-antitrypsin had levels of 2.15 (+ 0.07) and 2.23 (+ 0.1) mg/ml, respectively. Other investigators have found that the gene frequencies in the Pi system differ markedly between blacks and whites [13-16]. Our data show that this difference extends to the alleles PiM2 and Pim3 (tables 2 and 3). When all known variants were included, 18% of the blacks were heterozygous at the Pi locus, and 15% were heterozygous for PiMl or Pim3. Among whites the proportion of all heterozygotes was 53%, and 44% were heterozygous for PiM2 orPim3. These results show that the genetic heterogeneity of a1-antitrypsin as demonstrated by isoelectric focusing is approximately five times greater for whites and 10 times greater for blacks than was apparent with earlier electrophoretic protein separation techniques [17]. SUMMARY

Alpha,-antitrypsin is a major human serum protein that shows an extensive polymorphism. Genetic heterogeneity has previously been demonstrated by starch gel electrophoresis. By applying analytical isoelectric focusing (pH 3.5-5.0) to this system, we found a common variant, Pi M3, with an isoelectric point between those of Pi M1 and Pi M2. The gene frequency of this variant was . 11 in U. S. whites and .054 in blacks. When Pi3 and PiMi are included in the Pi system, the heterozygosity at the Pi locus is five times greater in whites and 10 times greater in blacks than that detected by earlier electrophoretic techniques. ACKNOWLEDGMENTS We thank Barbara Harpel for technical assistance and Dr. R. Frants for comparing M3 to his M intermediate. We also thank Drs. J. A. Pierce and M. K. Fagerhol for providing samples of their normal serum pools and Dr. C. G. Hames for serum samples of group III.

KUEPPERS AND CHRISTOPHERSON

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