Vox Sang. 24: 481-488 (1973)

A New Genetic Marker of Human Immunoglobulins Determined by an Allele at the a 2 Locus

ERNA VAN LOGHEM, A. C. WANG and J. SHUSTER

Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, Department of Medicine, Sectionof Hematologyand Immunology, University of California, San Francisco, Calif., and Division of Clinical Immunology and Allergy, and McGill

University Medical Clinic, Montreal General Hospital, Montreal, Que.

Abstract. A new antigenic determinant occurring on human IgA2 protein is des- cribed. In family studies this marker showed to be dominant, non-sex-linked and anti- thetical to the first known marker of a2 chains. It is proposed to call this marker A,m(2), indicating that it is the second marker of IgA2 proteins.

Introduction

Human IgA proteins are divisible into two subclasses within this immuno- globulin class as a consequence of marked antigenic and chemical differences [4, 9, 171. Allotypes have only been found in the minor subclass. The first genetic marker on heavy chains of human IgA2 proteins was discovered independently by VYAS and FUDENBERG [19] and by KUNKEL et al. [lo] and called Am( 1) and Am(2), respectively. Subsequently, it was shown that Am( 1) and Am(2) are related to an identical antigenic determinant [ 141.

The allele Am(l) which is inherited as a dominant factor and is closely linked to Gm alleles [12] is found in high frequency in Caucasians. Lower frequencies are found in several Negroid and Mongoloid populations so that these populations offer better opportunities for genetic studies. In this paper we report the discovery of another allotype at the Am locus which behaves as the allele to the Am(1) antigenic specificity. Taking into account that Am is associated with a2 chains, and thus the Am locus is associated with the a2 locus, we propose to call the first allele A,m(l) and the newly found one A,m(2).

Accepted: November 23, 1972.

482 VAN LOGHEM/WANG/SHUSTER

Material and Methods

The present studies were performed on isolated myeloma proteins and random serum samples from Caucasians, Chinese, Eskimos, Amerindians, Pygmies, Bantus and some non-human primates (several samples from gorillas, chimpanzees, orangutans, gibbons, baboons, macacas, C. fulguris, Loris and Tupaia). 47 Japanese families [I21 and two additional ones, provided by Dr. H. MATSUMOTO, Osaka, consisting of a total of 98 parents and 122 offspring of known Gm, Am and Inv types and Bush Negro families (62 parents and 108 offspring) which were typed for Gm(z), (a), (x), (f) , (n), (g), (b”), (bl), (b]), (b4), (b”, (Pa) [13], (s), ( t ) , (c?), (13, Inv(l) and A,m(l) were studied for the presence of this new genetic marker. The Bush Negrofamilies werecollected inSurinam by Dr. C. ZUKCHER, Amsterdam.

The antiserum (Ta), detecting the new marker, was obtained from an IgA deficient patient suffering from pernicious anemia, and co-existent nodular hyperplasia of the small bowel. This patient has been treated with regular injections of vitamin B,, and y-glo bu I i n.

The protein (Fo), carrying the new marker, was isolated from the serum of a patient hith multiple myeloniatosis by a three-step procedure [20] consisting of precipitation with 18% sodium sulphate, ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex (3-200.

The usual agglutination-inhibition method for genetic typing \\as performed using the CrCI, method of GOLD and FUDENBERG [6] for sensitization of red cells. Typing was performed in serum samples either diluted 1 : 10 and 1 :30, or, if necessary, in a series of twofold dilutions starting at I : 2 for serum and at 1.5 nig/ml for isolated proteins.

Results

Isolated Mj*elonia Proteins None of the proteins of the IgM, IgD or IgE class, nor any of the four

IgG subclasses inhibited the A,m(2) system at 1.5 mg/ml. Ten lgAl proteins and ten IgA2 proteins, positive for A,m(l), all failed to inhibit the test system. However, one IgA2 protein which was negative for A,m(l), gave significant inhibition at concentrations as low as 0.001 nig/ml. The antigen showed to be present in F(ab’), fragment obtained by pepsin digestion at pH 4.5 from protein Fo.

Random Serum Samples The data of A,m( 1 ) and A,m(2) typing in normal human sera are presented

in table I . Random serum samples gave inhibition of dilutions ranging from 1 :20 to 130. Failure to detect the phenotype A,m(l-2-) in the various normal human populations suggests that A,m( 1) and A,m(2) are comple- mentary alternatives in a genetic system with two alleles, that is, the sum

A New Genetic Marker of Human Immunoglobulins Determined.. . 48 3

Tubk I. Random sera tested for A,m(l) and A,m(2)

Number A,m(l)+ A,m(l)+ A,m(l)- A,m(l)- A d 2 ) - A,m(2)+ A,m(2)+ A,m(2)--

Caucasians Amerindians Eskimos Austr. aborigines Papuans Chinese Pygmies Bantus

100 40 21 19 48 24 77 24

97 39 16 17 14 0 8 1

3 1 4 2

25 6

27 4

0 0 1 0 9

18 42 19

Table Ira. Data from 49 Japanese families tested for A,m( I ) and A,m(2)

Mating Number of A,m phenotype of offspring families

(1 t2-) ( 1 t 2 t ) (1-2t)

(It2-) x (112-) 6 (1+2-)X(1+21) 14 (It2-) x (I-2+) 4 (1 t2+) x ( I t2-1) 14 (1+2+) x (I-2+) 7 (1-2t) x (1-21) 4

15 0 0 18 19 0 0 9 0 5 21 10 0 11 4 0 0 10

Table IIb. Data from 31 Bush Negro families tested for A,m(l) and A,m(2)

Mating Number of A,m phenotypes of offspring families

(1 +2-) ( I t2+) (1-2t)

(1 +2-) x ( 1 +2-) ( 1 +2-) x (1 +2+) ( 1 +2-) x (1-2+) (1 +2+) x (1 +2+) (1+2+) x (1-2+) 10 (1-2+) x (1-2t) 12

0 1 2 6

0 0 0 3 1 0 0 6 0 8 12 7 0 14 18 0 0 39

484 VAN LOGHEM/WANG/SHUSTER

of their gene frequencies approaches unity [ 151. The only samples negative for both markers were found among IgA deficient patients. None of the non- human primates inhibited the A,m(2) system.

Family Studies a ) Evidence ,for A,m(I) and A,m(2) being alternative alleles. The data

from Japanese families are presented in table I la and those from the Bush Negro families in table 11 b.

If Am( 1) and Am(2) are alternative alleles, the matings of the respective homozygotes are expected to have only heterozygous offspring. There were a total of 15 offspring as expected from such matings in the Japanese and Bush Negro families. The distribution of the offspring of the matings of heterozygotes with homozygotes and with heterozygotes (shown in table 111) gives a fair agreement between the observed and expected frequencies.

b) Linkage between the heavy chain cistrons. The linkage relation between yl-y2-y3- and a2-cistrons, as was demonstrated in the Japanese families with the A,m(l) allotype, was confirmed by typing for the A,m(2) marker. The number of informative families when typing for A,m( I) only, was 10, when typing for both A,m(l) and A,m(2) there were 16 informative families. As well, there were 8 informative Negro families. No recombination was observed (table IV). This is further evidence for the extremely close linkage of the three y-cistrons and the a2-cistron.

c) Relationship of A,m to Gm. I n Japanese, there are four main Gm haplotypes za;n-;gPa, zax;n-;gPa, za;n-;bostb3b5 and ,fa;n;bob1b3b4b5Pa. In the Japanese families, A,m(l) was shown to be most closely associated with the Gm haplotypes za;n-;gPa and zax;n-;gPa. Conversely, A,m(2) in- vestigated in this study was most closely associated with the Gm haplotypes za;n- ;bostb 3b and ,fa;n;bob ' b 3b4b 5Pa.

The Gm haplotypes in Negroid populations, which are different from those present in the Japanese, consist of four main Gm haplotypes : za;n-; bob 'b 3b4b jPa, za;n-;bob 'c3c5Pa, za;n-;bob lc3b 4b 5Pa and za;n-;bosb3b and a fifth minor one zax;n-;bob1b3b4b5Pa. In contrast to the Japanese, the association of A,m(l) and Am,(2) with particular C m haplotypes is less evident in the Negroid population. The gene frequencies for unrelated individuals could be calculated by the gene count method [3] from the pheno- types of the parents except for some cases where no distinction could be made between Gm(zax) and Gm(za). The data are presented in table V.

A New Genetic Marker of Human Immunoglobulins Determined.. . 485

Table III. A,m phenotype of offspring of matings with at least one heterozygous parent. Comparison with expected numbers

(1 +2-) (1t2t) (1-2+)

Observed Expected

34 36.25

78 75.5

39 39.25

Table IV. Gm yl-yZ-y3 and A,m linkage informative families

Number Number Lod scores at recom- of families of recombinants bination fraction 0.0

Japanese Negroes

16 0141 8.127 8 0128 6.020

~ ~~ ~~

Table V. Distribution of A,m(l) and Azm(2) in Gm haplotypes

Gene frequencies

Japanese (n = 98) 1) Gm (za; n-; gPa) 2) Gm (zax; n-; gPa) Undecided 1) or 2) 3) Gm (za; n-; b0stb3b5) 4) Gm (fa; n; bob1b3b4b5 Pa)

Total

Negroes (n = 62) 1) Gm (za; n-; bob1b3b4b6 Pa) 2) Gm (za; n-; b0b1c3c5 Pa) 3) Gm (za; n-; b0b1c3b4b6Pa) 4)Gm(za; n-; bosb3b6) 5 ) Gm (zax; n-; bob1b3b4b5 Pa)

0.349 0.057 0.406 0.125 0.026 0.151 0.026 0.005 0.03 1 0.047 0.214 0.261 0.010 0.141 0.151

0.557 0.443 1 .Ooo

0.137 0.476 0.613 0.040 0.234 0.274 0.024 0.Ooo 0.024 0.024 0.057 0.08 1 0.008 0.Ooo 0.008

Total 0.233 0.767 1 .Ooo

486 VAN LOGHEM/WANG/SHUSTER

Discussion

Antibodies to IgA in humans can be found in patients after immunization with incompatible blood or blood products [5, 181. In patients with IgA deficiency, antibodies have also been found without any obvious stimulation such as transfusion or pregnancy [ 161. We have also observed this pheno- menon. In the first mentioned group, the antibodies produced are often monospecific, while in the latter group antibodies of more than one specificity are common. Exceptionally, sera containing anti-IgA have precipitating properties, but the usual method for demonstrating the presence of anti- bodies to IgA and for investigating its specificity is the agglutination- inhibition method. When a number of cells coated with different IgAl and IgA2 proteins all are agglutinated by the serum to be tested and when each of these agglutinations is inhibited by any of the panel of IgA proteins, but not by any other class protein, the antibodies have anti-a specificity since they detect a determinant common to al and a2 chains. When not all cells are agglutinated, the specificity is more restricted. Then there may be present subclass specific (anti-a1 or anti-a2) or anti-allotype antibodies.

When all or several coated cell preparations are agglutinated, but one or more show a different pattern of inhibition, several antibody specificities are present. The antiserum Ta, obtained from an IgA deficient patient, contained both anti-A2m(2) antibodies and anti-A,m( 1) antibodies which were demonstrable with A,m(2) and A,m( I ) coated cells respectively, as well as anti-a demonstrable with several IgAl coated cells. When using antiserum Ta for A,m(2) typing the anti-A,m(l) antibodies cannot interfere in the reaction if the coat used does not carry A,m( I ) antigen. The anti-a antibodies do not disturb the typing results since these are present in low titre and any IgA protein, whether A,m(2) poFitive or negative, has a determinants that will neutralize the anti-a antibodies. These antibodies must be absorbed (e.g. with IgAl proteins) when a A,m(l) or A,m(2) reagent is required for typing protein fragments.

A,m( I ) positive IgA2 proteins are lacking in disulphide bridges between heavy and light chains [7] in contrast to A,m(l) negative IgA2 proteins [8]. It is conceivable that this disulphide bridge is concerned in the A,m(2) specificity. This question may be answered by A,m(2) typing of enzymatic fragments and isolated heavy and light chains of the appropriate IgA molecules. The antigen was still present in F(ab’), fragment prepared by pepsin digestion from protein Fo. However, further investigation is required. The frequency distribution of A,m(2) is complementary to that of A,m(l),

A New Genetic Marker of Human Immunoglobulins Determined.. . 487

A,m(2) and A,m(l) being alternatives in a bi-allelic system. It can be con- cluded from A,m( 1) typing results done in our laboratory that the frequency of A,m(2) is highest in Africa, decreases in Australia and Asia and is lowest in Europe. Studies on evolution of Gm markers [l 11 agree with the hypothesis of African origin of the human species, followed by migration over the world.

We postulate that A,m(2) arose first and that A,m(l) was generated by mutation. High frequencies of A,m( 1) as found in other continents may have resulted from selective advantage or genetic drift. Recent developments in population genetics methodology may soon allow to discriminate between the relative weight of these two evolutionary forces [I] . Europeans have become nearly homogenous at the a2 locus. This may have happened if this population passed through a bottle neck of numbers or if the a2 allele (or a closely linked gene) had a higher fitness in the European environment.

Obviously, the close linkage of y- and a-chain cistrons prevents G m haplotypes and A,m markers to reach equilibrium of distribution in a relatively short evolutionary time. The disequilibrium is less evident in Negroes as in Japanese. This may be explained by the assumption that the polymorphisms at the A,m and Gm loci were older in Negroid populations and had more time to reach equilibrium, while in Japanese the coexistence of the different haplotypes happened more recently and thus their frequencies are still more far away from equilibrium. Alternatively, different haplotypic combinations have had different fitness in the two ethnic groups [2].

Investigation of more populations is necessary before conclusions can be drawn.

A cknowledgernents

The authors wish to express their thanks to Dr. M. SELIGMAN, Paris and to Dr. F. SKVA- RIL, Berne, for some of the myeloma proteins, to Dr. H. H. FUDENBERG, San Francisco, for supplying the serum of Fo, to the Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP), New York, for providing the non-human primate sera, to Dr. H. MATSUMOTO, Osaka, for providing the Japanese sera and to Dr. C. ZURCHER, Amster- dam, who collected the Bush Negro sera. The excellent technical assistance of Miss GERDA DE LANCE is gratefully acknowledged.

This study was financially made possible by NIH Research grant AI-09813, National Science Foundation Research grant GB-27666, American Cancer Society grant IC 76F and the Jane Coffin Child Fund.

References

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488 VAN LOGHEM/WANG/SHUSTER

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Request reprints from: Dr. ERNA VAN LOGHEM, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, POB 9190, Amsterdam (The Netherlands)