Am J Hum Genet 28:77-86, 1976
Immunoglobulin Allotypes in Sardinia A. PIAZZA,1 E. VAN LOGHEM,2 G. DE LANGE,2 E. S. CURTONI, L. ULIZZI,3 AND L. TERRENATO3 INTRODUCTION
The presence of inherited, serologically detectable antigens on immunoglobulin molecules provides a useful tool for studying the genetic control, structural characteristics, and biological activity of human immunoglobulins. It is also a powerful index of discrimination between different ethnic groups. The genes specifying Y1, y3, and y2 chains appear to be closely linked to one another and to the gene specifying a2 chains [1]. Linkage data are minimal for markers controlled by the y4 gene [2]. No evidence of linkage has been found between the heavy chain or K light chain genetic regions and the genetic regions controlling red and white cell antigens [3]. Only recently has linkage been found between the genes for heavy chains and genes controlling al-antitrypsin [4]. Allotypic and isoallotypic antigens are expressed on the heavy chain of the four IgG subclasses as sets of markers specified by several regions of the homologous chromosomes and inherited as supergenes or "epistatic blocks" (Fisher, [5]), now referred to as haplotypes. Haplotypes vary among racial groups. Their distribution is roughly described in table 1. (See Steinberg [6], Grubb [3], van Loghem [7], and Natvig and Kunkel [8] for review.) A problem of major interest for the student of human biology is the distinction between adaptive and nonadaptive differentiation of the various ethnic groups. Study of microgeographic variation of Gm and Km (formerly Inv) allotypes can be of great relevance to this approach. The island of Sardinia seemed well suited to this kind of analysis because of its geography and sociology. The following is a report on the immunoglobulin allotype results which were part of a more extensive project related to the genetic and demographic structure of the Sardinian population. Received February 11, 1975; revised June 27, 1975. This investigation was supported in part by CNR, International Biological Program (Istituto di Genetica, Facolth di Scienze, Roma) and by CNR, Centro di Studio per l'Immunogenetica e l'Istocompatibilita, Torino, Italy. 1 Istituto di Genetica Medica, Torino, Italy. 2 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. 3Istituto di Genetica, Facolta' di Scienze dell'Universita, Roma, Italia. @ 1976 by the American Society of Human Genetics. All rights reserved.
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IMMUNOGLOBULIN ALLOTYPES IN SARDINIA
79
SUBJECTS AND METHODS
Serum samples were collected during field expeditions between 1970-1971 from unrelated school boys (mean age about 14 years) from the north, southeast, and south sides of Sardinia as part of the International Biological Program, Human Adaptability Section; and between 1971-1972 from unrelated adults of the northeast lowland and central highland as part of the 5th International Histocompatibility Workshop [9]. Control samples were collected from unrelated adults from Ferrara, a town on the mainland. Samples have not been collected from families. Area sampling was based on geographic and genetic considerations. Figure 1 shows the
FIG. 1.-Villages sampled
PIAZZA ET AL.
80
locations of the sampled villages. The following list groups the villages according to region and gives the number of individuals tested in each village: (1) north: Thiesi (54), Sorso (85), Ittiri (71), Ozieri (113), Tempio Pausania (118); (2) northeast: Galtelli (99), Orosei (99); (3) southeast: Villaputzu (67), Muravera (48), San Vito (41), Ballao (34); (4) south: Pula (91), Santadi (85); (5) mountain: Desulo (100), Tonara (103). Most of the important regions of the island are well represented. Groups 1-4 are located in different lowland regions. Group 5 is formed by two villages in the Gennargentu mountains at 900 m above the sea level and for this reason deserves special attention. There is a striking negative correlation between incidence of thalassemia, G6PD deficiency (and hence most likely malaria), and altitude in Sardinia [10-12]. The mountain villages, Desulo and Tonara, are and have been malaria and thalassemia trait free [10, 13]. The other villages in the lowlands have been exposed to endemic malaria from Plasmodium falciparum until 1946-1948 [15]. They also show a high incidence of thalassemia and G6PD deficiency [16]. The Sardinian population by itself is a fascinating puzzle for anthropologists. Sardinians probably descend from neolithic and bronze age colonizers with subsequent infusion of Phoenician, Carthaginian, and perhaps Arabic blood. Notwithstanding invasions and conquests, the gene flow into the aboriginal population has probably been quite limited with the exception of some well identified foreign settlements on the coast. The isolation of Sardinians is proved by the uniqueness of gene frequency for some polymorphisms [17]. They are, in general, distinguished by a very low frequency of Rh-negative genes (cde = .19) which is not matched by any other population in the Mediterranean region and by a very high frequency, one of the highest in the world, of the gene M (.78) [11, 13, 16]. allele has a frequency in Sardinia unique among populations yet studied [18]. The DOa2 frequencies of HL-A antigens [13] show very interesting features: HL-A 3, HL-A 7, HL-A 12 share figures similar to those of Middle Easterners; W30, W17 show typically African frequencies; the W18 gene frequency (.31) has not been found in other populations. All the other HL-A antigens are distributed as they are in whites. Sardinians seem to have been subjected to a long independent evolution with high drift because of a relatively strong isolation between the villages [19]. Their highest, but in absolute value not very high, association is with Italians, probably because of a common neolithic origin and to some extent, of more recent hybridization. Other associations are with Yemenite Jews and Lebanese probably due to contributions from Phoenicians and Carthaginians. They also correlate more with Africans than the average white individual, probably due to their association with the Middle East. However, there is no historical or archeological evidence of African contact, except for the Carthaginian colonizers who were originally Phoenician but not from Africa. Today there are about 1.5 million Sardinians. There is an increasing net rate of emigration to continental Italy 1.3%), which is not balanced by immigration due to the poor economy. Migration and frequencies of consanguineous marriages have been documented and will be published soon by the same authors. Gm, Am, and Km (formerly Inv) Typing Each sample was tested for the markers listed in table 2 except for A2m(1), A2m(2), Km(2), and Km(3). The latter markers were determined in the samples of groups 2 and 5, as well as in the control group from Ferrara. Typing for the markers Gm, Am, and Km was carried out with the conventional agglutination-inhibition techniques. Samples were tested in two dilutions, 1:10 and 1:30. When disturbing antibodies were present, the samples were absorbed and/or retested in a series of twofold dilutions, starting with undiluted serum. The Gm haplotype frequencies were estimated by the maximum-likelihood method using a very general FORTRAN IV program (LIKTYP) written for the IBM 360 system. This computer program has special options to carry genetic systems with high number of
(a
IMMUNOGLOBULIN ALLOTYPES IN SARDINIA
81
TABLE 2 IMMUNOGLOBULMN ALLOTYPE REAGENTS Chain
Genetic marker*
yl .......... Glm(a) Glm(x) Glm(f) Glm(z)
y2
..........
G2m(n)
y3 .......... G3m(g) G3m(bO) G3m(bl) G3m(b3) G3m(b5) G3m(s) G3m(t) G3m(c3) G3m(c5) c.. A2m(1) A2m(2) K .Km(l) Km(2) Km(3)
or
1 2 3 17 23 21 11 5 13 10 15 16 6 24
Antiglobulin
3294 human 2984 human 2871 human 3272 human 9547 rabbit 5306 rabbit 2357 human 2933 human 2277 human 2123 human 2624 human 2639 human 2646 human 3271 human Jelle human Taylor humant 2151 human Vir humanjl Nee human
Dilution
10
15 60 30 30
Coating antigen
Anti-Rh Anti-Rh Anti-Rh Anti-Rh
3417 2880 3097 3417
Myeloma protein, Jasp
5 15 8 60 10 15 10 15
Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh
40
Myeloma protein, Menst Myeloma protein, Ford§
100
80
3359 2127 2127 2127 2127 3068 3068 2781 2781
20
Anti-Rh 2290
100 8
Anti-Rh 2290 Anti-Rh BKO
* Nomenclature according to proposals of World Health Organization meeting on Human Immunoglobulin Allotypes (Rouen, France, 1974). f Gift of Dr. B. Zegers (Utrecht, The Netherlands). $ Gift of Dr. J. Shuster (Montreal, Canada). § Gift of Dr. A. C. Wang (San Francisco, California). 1 Gift of Dr. C. Ropartz (Rouen, France).
alleles, such as Gm or HL-A, and to choose either analytical or numerical solutions for solving the derivatives of the maximum-likelihood scores. RESULTS AND DISCUSSION
Gm Allotypes The Gm phenotypes and the estimated haplotype frequencies of the Sardinians divided over the groups described above are presented in tables 3 and 4, respectively. Goodness of fit with Hardy-Weinberg proportions as well as the degrees of freedom resulting from the number of allotypes used in estimating the expected frequencies of the phenotypes is given in the last columns of table 4. The f;n;b and f; - ;b haplotypes, characteristic of whites, are clearly present in Sardinia with a frequency not significantly different from that of continental Italy (group 7). The za;-;g haplotype varies in whites from . .15 in southern .795 in the Inari Lapps [20], showing a and eastern European populations to dine from southeast to northwest first noted by Grubb [21]. The data for continental Italy and for Sardinia conform to this pattern. The zax;@ ;g haplotype shows low frequency both in continental Italy and Sardinia; it is significantly lower for Sardinia (X21 = 19.99, P < .001) and probably the lowest one yet recorded in Europe. A striking feature of the Sardinian population is the presence
PIAZZA ET AL.
82
TABLE 3 OBSERVED GM PHENOTYPES IN SARDINIANS AND CONTINENTAL ITALIANS (FERRARA) CONTINENTAL ITALIANS
SARDINIANS GM PHENOTYPES
za;.. ;g
North
....... .
zax;..;g
6
za;.. ;gb
....... .
3
... 2 1
zafIn bsc5 ........ Total
........
441
1
Mountain
5
Total
Ferrara
14
1
2
2
14 3
... ... ...
24 6 1 1 1
1 ... 4
155 72 5 6 12
...
...
...
...
3
129
124
120
119
763
2 12 9 10 2
...
...
9 5 5
2 2 21 1
...
... ... ... ... ... ...
2
... ... ...
3 62 34 61 11 2 I 2
190
176
1,208
86
...
19 3 9 ...
.I
. ...
South
.....
22 7
.........
..........
... ..
19 12
...........
;ib ;bst. .........
2
62 27
3 zaf;n;gbsc5 ...... 6 zaxf;n;gb ........ 3 zaxf; gb f;n b ............ 271 zaf;n;bst 1......... f;n bsc5 .......... 20 zaf n;b .......... 15 16 f;.. ;b .5 zaf;.. ;b za; .. za; ..
Southeast
...
...
zaf;n;gb ......... zaf; ;gb .......
Northeast
...
... 198
3
28 20
... ...
... 203
... ...
5 1 60
.
of the za; * * ;b, f ;n;bsc5, and za;- ;bOstb3b5 haplotypes. The za; * * ;b haplotype, typically African, is uniformly distributed in every Sardinian group and its frequency (.023) is significantly different from zero. The low frequency of za;* *;bOstb3b5 haplotype may reflect the Eastern contact of old Sardinians with Carthaginian colonizers. The haplotype f;n;bsc5, interesting from an evolutionary standpoint, has not been described before. The yl marker f is characteristic for whites though it is commonly coupled to the y2 marker n. Although the y3 markers bsc5 in this combination have not been observed in human populations, it is found in all apes, except Pan paniscus and in many species of old world monkeys [22]. The difference between Sardinian and continental Italian (Ferrara) Gm haplotypes is another proof of the essential demographic isolation of the island. The heterogeneity of these two ethnic groups as measured by differences in phenotype frequencies gives a X2 of 46.75 (with 16 df), which is highly significant (P < .001). On the other hand, the homogeneity of the Sardinian population must be checked by testing possible differences of phenotype frequencies in the above mentioned geographical subdivisions (groups 1-5). This gives a X2 of 103.25 (with 64 df) which is highly significant for heterogeneity. A reasonable hypothesis for this heterogeneity may be the different ecological environments of highland and lowland Sardinia. As a matter of fact, the previous X2 can be partitioned as follows: (1) between groups 1, 2, 3, 4, 5, X2- 103.25, 64 df, P < .01; (2) groups 1, 2, 3, 4, vs. 5, X2= 42.7, 16 df, P < .001; (3) between groups 1, 2, 3, 4, X2 = 61.18, 48 df, -
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P > .05 showing that the heterogeneity may be ascribed to the different environment of mountains and lowlands. What genes contribute most to these differences remains an important point to be elucidated. A simple inspection of tables 3 and 4 points to G2m(n) as the allotype primarily responsible: its frequency is .655 in lowland and .518 in highland (X21 - 17.29, P < .001). G2m(n) is an allele of the subclass IgG2 genes. It has not yet been established which amino acid sequence is coded by the presence of this allele. The antigenic determinant G2m(n) is located in the CH2 homology region [23]. Recently increasing attention has been given to the biological activities in which the Fc part of the heavy chains is mainly involved. It has been observed that each subclass exerts selective distribution. The antibody activity of the IgG2 molecule has been reported to be related to carbohydrate antigens [24]. Our data on heterogeneity between highland and lowland Sardinia, as expressed by G2m(n) suggest an adaptive value for this marker or for closely linked determinants of the CH2 region. This hypothesis is not contradicted by the Wahlund variance between the two areas calculated for the G2m(n) allotype, being .0274, a value of the same order of magnitude as that found previously [13] for thalassemia (.0465) and G6PD (.0612) genes, the adaptive meaning of which does not require further comments. A2m Allotypes A2m(1) is the most common allotype in all human populations. A2m(2) is very rare in whites, but occurs with varying frequency in blacks and orientals. Groups 2 and 5 were all found to be A2m(1+2-), except for four samples that were A2m(1+2+). Three of those samples were heterozygous for the haplotype Gm za;v ;b. This indicates admixture with the Negroid haplotype Gm za; .;b; A2m(2). All samples from Ferrara, the control group, were homozygous for A2m(1), except for one which was found to be deficient in IgA and contained anti-a antibodies.
Km Allotypes The Km phenotypes and the gene frequencies estimated for Sardinians and continental Italians are presented in table 5. All 1,218 individuals in the sample were tested for both Gm and Km markers except for 10 individuals only tested for Km. The gene frequency of Km(1) is typically white. No heterogeneity has been found within the Sardinian groups nor between Sardinians and continental Italians. SUMMARY
1218 individuals from Sardinia island (Italy) were tested for Gm and Km markers; 10 were not tested for Gm and only 401 were typed for Am markers. The peculiar genetic makeup of the Sardinian population is confirmed by their Gm allotypes. Their differences from those found in a control population of continental Italy (Ferrara), suggest ancient contacts with the Middle East and Africa. An indication for such contacts may also be found in the striking presence of the
IMMUNOGLOBULIN ALLOTYPES IN SARDINIA
85
TABLE 5
OBSERVED KM PHENOTYPES AND ESTIMATED GENE FREQUENCIES FOR SARDINIANS AND CONTINENTAL ITALIANS Km' REGION
North .............................. Northeast .198* Southeast .191 South .182 Mountain .203* Total Sardinia .1218 Continental Italy .86* *
Tested
No. Tested
444
Km(l)
(W)
(± SE)
82 24 33 43 44 226 14
.097 .063 .090 .126 .115 .098 .085
.010 .012 .015 .018 .016 .006 .022
also for Km(2) and Km(3).
haplotype Gm f;n;bsc5, a haplotype not previously found in a human population. A significant difference of G2m(n) allotype was observed between highland and lowland regions. If confirmed, it may suggest an adaptive pressure related to the CH2 region of the y2 chain, possibly due to endemic malaria in the past. REFERENCES 1. LOGHEM E VAN, NATVIG JB, MATSUMOTO H: Genetic markers on immunoglobulins in Japanese families. Inheritance of associated markers belonging to three IgG and IgA subclasses. Ann Hum Genet 33 :351-359, 1970 2. KUNKEL HG, JOSLIN FG, PENN GM, NATVIG JB: Genetic variants of yG4 globulin. A unique relationship to other classes of yG globulin. J Exp Med 132 :508-520, 1970 3. GRUBB R: The Genetic Markers of Human Immunoglobulins. New York, SpringerVerlag, 1970 4. GEDDE-DAHL T, FAGERHOLD MK, COOK PGL, NOADES J: Autosomal linkage between the Gm and R loci in man. Ann Hum Genet 35:393-399, 1972 5. FISHER R: The Mathematical Theory of Natural Selection. New York, Dover, 1959 6. STEINBERG AG: Globulin polymorphisms in man. Annu Rev Genet 3:25-52, 1969 7. LOGHEM E VAN: Stability of Gm polymorphism, in Human AntiHuman Gammaglobulins, edited by GRUBB R, SAMMELSON G, Oxford, Pergamon, 1971, pp 29-37 8. NATVIG JB, KUNKEL HG: Human immunoglobulins: classes, subclasses, genetic variants, and idiotypes. Adv Immunol 16:1-59, 1973 9. BODMER J, RoQuEs P, BODMER WF, DAUSSET J, DEGOS L, PIAZZA A: Joint report of the 5th International Histocompatibility Workshop, in Histocompatibility Testing 1972, edited by DAUSSET J, COLOMBANI J, Copenhagen, Munksgaard, 1973, pp 621667 10. CEPPELLINI R: Negative correlation between altitude above sea level and incidence of thalassemia in four Sardinian villages. Cold Spring Harbor Symp Quant Biol 26:252-255, 1955 11. CARCASSI U, CEPPELLINI R, PITZu E: Frequenza della talassemia in quattro popolazioni sarde e suoi rapporti con la distribuzione dei gruppi sanguigni e della malaria. Boll Ist Sieroter Milan 36:206-214, 1957 12. SINISCALCO M, BERNINI L, FILIPPI G, LATTE B, MEERA KHAN P, PIOMELLI S, RATTAZZI M: Population genetics of haemoglobin variants, thalassemia and glucose6-phosphate dehydrogenase deficiency, with particular reference to the malaria hypothesis. Bull WHO 34:378-393, 1966
86
PIAZZA ET AL.
13. PIAZZA A, BELVEDERE MC, BERNOCO D, CONIGHI C, CONTU L, CURTONI ES, MATTIUZ PL, MAYR W, RICHIARDI P, SCUDELLER G, CEPPELLINI R: HL-A variation in four Sardinian villages under differential selective pressure by malaria, in Histocompatibility Testing 1972, edited by DAUSSET J, COLOMBANI J, Copenhagen, Munksgaard, 1973, pp 73-84 14. FERMI E: La Malaria in Sardegna. Sassari, Stamperia Libreria Italiana e Straniera, 1938 15. LOGAN JA: The Sardinian Project. Baltimore, Johns Hopkins Press, 1953 16. TERRENATO L, VAN LOGHEM E, BERNINI L, SANTACHIARA-BENERECETTI AS, MODIANO G, SANTOLAMAZZA C, SCOZZARI R, ULIZZI L, BERRETTA M: Preliminary data on the genetic heterogeneity among the Sardinian isolates. Rend Acad Naz Lincei, Serie VIII, 51:249-253, 1971 17. TERRACINI A: Saggio di un Atlante Linguistico delta Sardegna. Torino, Stamperia Editoriale Rattero, 1964 18. SANTACHIARA-BENERECETTI AS, BERRETTA M, ULIzZI L, TERRENATO L: The frequency of the red cell NADH diaphorase Dia2 allele in Sardinia. Human Hered 21:290-293, 1972 19. PIAZZA A, SGARAMELLA-ZONATA L, GLUCKMANN P, CAVALLI-SFORZA LL: Analysis of human evolution. Phylogenetic analysis of a sample of 35 human populations tested for HL-A and other markers. Submitted for publication 20. STEINBERG AG, TIILIKAINEM A, ESKOLA M, ERIKSSON AW: Gammaglobulin allotypes in Finnish Lapps, Finns, Aland Islanders, Maris (Cheremis) and Greenland Eskimos. Am J Hum Genet 26:223-243, 1974 21. GRUBB R: Hereditary gamma globulin groups in man. Ann Hum Genet 13:171-174, 1961 22. LOGHEM E VAN, LITWIN SD: Antigenic determinants on Immunoglobulins of non human primates. Transplant Proc 4:129-135, 1972 23. NATVIG JB, TURNER MW: Localization of Gm markers to different molecular regions of the Fc fragment. Clin Exp Immunol 8:685-700, 1971 24. YOUNT WJ, DORNER MM, KUNKEL HG, KABAT EA: Studies on human antibodies. VI. Selective variations in subgroup composition and genetic markers. J Exp Med 127:633-646, 1968
Immunoglobulin Allotypes in Sardinia A. PIAZZA,1 E. VAN LOGHEM,2 G. DE LANGE,2 E. S. CURTONI, L. ULIZZI,3 AND L. TERRENATO3 INTRODUCTION
The presence of inherited, serologically detectable antigens on immunoglobulin molecules provides a useful tool for studying the genetic control, structural characteristics, and biological activity of human immunoglobulins. It is also a powerful index of discrimination between different ethnic groups. The genes specifying Y1, y3, and y2 chains appear to be closely linked to one another and to the gene specifying a2 chains [1]. Linkage data are minimal for markers controlled by the y4 gene [2]. No evidence of linkage has been found between the heavy chain or K light chain genetic regions and the genetic regions controlling red and white cell antigens [3]. Only recently has linkage been found between the genes for heavy chains and genes controlling al-antitrypsin [4]. Allotypic and isoallotypic antigens are expressed on the heavy chain of the four IgG subclasses as sets of markers specified by several regions of the homologous chromosomes and inherited as supergenes or "epistatic blocks" (Fisher, [5]), now referred to as haplotypes. Haplotypes vary among racial groups. Their distribution is roughly described in table 1. (See Steinberg [6], Grubb [3], van Loghem [7], and Natvig and Kunkel [8] for review.) A problem of major interest for the student of human biology is the distinction between adaptive and nonadaptive differentiation of the various ethnic groups. Study of microgeographic variation of Gm and Km (formerly Inv) allotypes can be of great relevance to this approach. The island of Sardinia seemed well suited to this kind of analysis because of its geography and sociology. The following is a report on the immunoglobulin allotype results which were part of a more extensive project related to the genetic and demographic structure of the Sardinian population. Received February 11, 1975; revised June 27, 1975. This investigation was supported in part by CNR, International Biological Program (Istituto di Genetica, Facolth di Scienze, Roma) and by CNR, Centro di Studio per l'Immunogenetica e l'Istocompatibilita, Torino, Italy. 1 Istituto di Genetica Medica, Torino, Italy. 2 Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. 3Istituto di Genetica, Facolta' di Scienze dell'Universita, Roma, Italia. @ 1976 by the American Society of Human Genetics. All rights reserved.
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IMMUNOGLOBULIN ALLOTYPES IN SARDINIA
79
SUBJECTS AND METHODS
Serum samples were collected during field expeditions between 1970-1971 from unrelated school boys (mean age about 14 years) from the north, southeast, and south sides of Sardinia as part of the International Biological Program, Human Adaptability Section; and between 1971-1972 from unrelated adults of the northeast lowland and central highland as part of the 5th International Histocompatibility Workshop [9]. Control samples were collected from unrelated adults from Ferrara, a town on the mainland. Samples have not been collected from families. Area sampling was based on geographic and genetic considerations. Figure 1 shows the
FIG. 1.-Villages sampled
PIAZZA ET AL.
80
locations of the sampled villages. The following list groups the villages according to region and gives the number of individuals tested in each village: (1) north: Thiesi (54), Sorso (85), Ittiri (71), Ozieri (113), Tempio Pausania (118); (2) northeast: Galtelli (99), Orosei (99); (3) southeast: Villaputzu (67), Muravera (48), San Vito (41), Ballao (34); (4) south: Pula (91), Santadi (85); (5) mountain: Desulo (100), Tonara (103). Most of the important regions of the island are well represented. Groups 1-4 are located in different lowland regions. Group 5 is formed by two villages in the Gennargentu mountains at 900 m above the sea level and for this reason deserves special attention. There is a striking negative correlation between incidence of thalassemia, G6PD deficiency (and hence most likely malaria), and altitude in Sardinia [10-12]. The mountain villages, Desulo and Tonara, are and have been malaria and thalassemia trait free [10, 13]. The other villages in the lowlands have been exposed to endemic malaria from Plasmodium falciparum until 1946-1948 [15]. They also show a high incidence of thalassemia and G6PD deficiency [16]. The Sardinian population by itself is a fascinating puzzle for anthropologists. Sardinians probably descend from neolithic and bronze age colonizers with subsequent infusion of Phoenician, Carthaginian, and perhaps Arabic blood. Notwithstanding invasions and conquests, the gene flow into the aboriginal population has probably been quite limited with the exception of some well identified foreign settlements on the coast. The isolation of Sardinians is proved by the uniqueness of gene frequency for some polymorphisms [17]. They are, in general, distinguished by a very low frequency of Rh-negative genes (cde = .19) which is not matched by any other population in the Mediterranean region and by a very high frequency, one of the highest in the world, of the gene M (.78) [11, 13, 16]. allele has a frequency in Sardinia unique among populations yet studied [18]. The DOa2 frequencies of HL-A antigens [13] show very interesting features: HL-A 3, HL-A 7, HL-A 12 share figures similar to those of Middle Easterners; W30, W17 show typically African frequencies; the W18 gene frequency (.31) has not been found in other populations. All the other HL-A antigens are distributed as they are in whites. Sardinians seem to have been subjected to a long independent evolution with high drift because of a relatively strong isolation between the villages [19]. Their highest, but in absolute value not very high, association is with Italians, probably because of a common neolithic origin and to some extent, of more recent hybridization. Other associations are with Yemenite Jews and Lebanese probably due to contributions from Phoenicians and Carthaginians. They also correlate more with Africans than the average white individual, probably due to their association with the Middle East. However, there is no historical or archeological evidence of African contact, except for the Carthaginian colonizers who were originally Phoenician but not from Africa. Today there are about 1.5 million Sardinians. There is an increasing net rate of emigration to continental Italy 1.3%), which is not balanced by immigration due to the poor economy. Migration and frequencies of consanguineous marriages have been documented and will be published soon by the same authors. Gm, Am, and Km (formerly Inv) Typing Each sample was tested for the markers listed in table 2 except for A2m(1), A2m(2), Km(2), and Km(3). The latter markers were determined in the samples of groups 2 and 5, as well as in the control group from Ferrara. Typing for the markers Gm, Am, and Km was carried out with the conventional agglutination-inhibition techniques. Samples were tested in two dilutions, 1:10 and 1:30. When disturbing antibodies were present, the samples were absorbed and/or retested in a series of twofold dilutions, starting with undiluted serum. The Gm haplotype frequencies were estimated by the maximum-likelihood method using a very general FORTRAN IV program (LIKTYP) written for the IBM 360 system. This computer program has special options to carry genetic systems with high number of
(a
IMMUNOGLOBULIN ALLOTYPES IN SARDINIA
81
TABLE 2 IMMUNOGLOBULMN ALLOTYPE REAGENTS Chain
Genetic marker*
yl .......... Glm(a) Glm(x) Glm(f) Glm(z)
y2
..........
G2m(n)
y3 .......... G3m(g) G3m(bO) G3m(bl) G3m(b3) G3m(b5) G3m(s) G3m(t) G3m(c3) G3m(c5) c.. A2m(1) A2m(2) K .Km(l) Km(2) Km(3)
or
1 2 3 17 23 21 11 5 13 10 15 16 6 24
Antiglobulin
3294 human 2984 human 2871 human 3272 human 9547 rabbit 5306 rabbit 2357 human 2933 human 2277 human 2123 human 2624 human 2639 human 2646 human 3271 human Jelle human Taylor humant 2151 human Vir humanjl Nee human
Dilution
10
15 60 30 30
Coating antigen
Anti-Rh Anti-Rh Anti-Rh Anti-Rh
3417 2880 3097 3417
Myeloma protein, Jasp
5 15 8 60 10 15 10 15
Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh Anti-Rh
40
Myeloma protein, Menst Myeloma protein, Ford§
100
80
3359 2127 2127 2127 2127 3068 3068 2781 2781
20
Anti-Rh 2290
100 8
Anti-Rh 2290 Anti-Rh BKO
* Nomenclature according to proposals of World Health Organization meeting on Human Immunoglobulin Allotypes (Rouen, France, 1974). f Gift of Dr. B. Zegers (Utrecht, The Netherlands). $ Gift of Dr. J. Shuster (Montreal, Canada). § Gift of Dr. A. C. Wang (San Francisco, California). 1 Gift of Dr. C. Ropartz (Rouen, France).
alleles, such as Gm or HL-A, and to choose either analytical or numerical solutions for solving the derivatives of the maximum-likelihood scores. RESULTS AND DISCUSSION
Gm Allotypes The Gm phenotypes and the estimated haplotype frequencies of the Sardinians divided over the groups described above are presented in tables 3 and 4, respectively. Goodness of fit with Hardy-Weinberg proportions as well as the degrees of freedom resulting from the number of allotypes used in estimating the expected frequencies of the phenotypes is given in the last columns of table 4. The f;n;b and f; - ;b haplotypes, characteristic of whites, are clearly present in Sardinia with a frequency not significantly different from that of continental Italy (group 7). The za;-;g haplotype varies in whites from . .15 in southern .795 in the Inari Lapps [20], showing a and eastern European populations to dine from southeast to northwest first noted by Grubb [21]. The data for continental Italy and for Sardinia conform to this pattern. The zax;@ ;g haplotype shows low frequency both in continental Italy and Sardinia; it is significantly lower for Sardinia (X21 = 19.99, P < .001) and probably the lowest one yet recorded in Europe. A striking feature of the Sardinian population is the presence
PIAZZA ET AL.
82
TABLE 3 OBSERVED GM PHENOTYPES IN SARDINIANS AND CONTINENTAL ITALIANS (FERRARA) CONTINENTAL ITALIANS
SARDINIANS GM PHENOTYPES
za;.. ;g
North
....... .
zax;..;g
6
za;.. ;gb
....... .
3
... 2 1
zafIn bsc5 ........ Total
........
441
1
Mountain
5
Total
Ferrara
14
1
2
2
14 3
... ... ...
24 6 1 1 1
1 ... 4
155 72 5 6 12
...
...
...
...
3
129
124
120
119
763
2 12 9 10 2
...
...
9 5 5
2 2 21 1
...
... ... ... ... ... ...
2
... ... ...
3 62 34 61 11 2 I 2
190
176
1,208
86
...
19 3 9 ...
.I
. ...
South
.....
22 7
.........
..........
... ..
19 12
...........
;ib ;bst. .........
2
62 27
3 zaf;n;gbsc5 ...... 6 zaxf;n;gb ........ 3 zaxf; gb f;n b ............ 271 zaf;n;bst 1......... f;n bsc5 .......... 20 zaf n;b .......... 15 16 f;.. ;b .5 zaf;.. ;b za; .. za; ..
Southeast
...
...
zaf;n;gb ......... zaf; ;gb .......
Northeast
...
... 198
3
28 20
... ...
... 203
... ...
5 1 60
.
of the za; * * ;b, f ;n;bsc5, and za;- ;bOstb3b5 haplotypes. The za; * * ;b haplotype, typically African, is uniformly distributed in every Sardinian group and its frequency (.023) is significantly different from zero. The low frequency of za;* *;bOstb3b5 haplotype may reflect the Eastern contact of old Sardinians with Carthaginian colonizers. The haplotype f;n;bsc5, interesting from an evolutionary standpoint, has not been described before. The yl marker f is characteristic for whites though it is commonly coupled to the y2 marker n. Although the y3 markers bsc5 in this combination have not been observed in human populations, it is found in all apes, except Pan paniscus and in many species of old world monkeys [22]. The difference between Sardinian and continental Italian (Ferrara) Gm haplotypes is another proof of the essential demographic isolation of the island. The heterogeneity of these two ethnic groups as measured by differences in phenotype frequencies gives a X2 of 46.75 (with 16 df), which is highly significant (P < .001). On the other hand, the homogeneity of the Sardinian population must be checked by testing possible differences of phenotype frequencies in the above mentioned geographical subdivisions (groups 1-5). This gives a X2 of 103.25 (with 64 df) which is highly significant for heterogeneity. A reasonable hypothesis for this heterogeneity may be the different ecological environments of highland and lowland Sardinia. As a matter of fact, the previous X2 can be partitioned as follows: (1) between groups 1, 2, 3, 4, 5, X2- 103.25, 64 df, P < .01; (2) groups 1, 2, 3, 4, vs. 5, X2= 42.7, 16 df, P < .001; (3) between groups 1, 2, 3, 4, X2 = 61.18, 48 df, -
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00
+
0
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t +
+1 +1 +1 +1+1+1+1
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PIAZZA ET AL.
P > .05 showing that the heterogeneity may be ascribed to the different environment of mountains and lowlands. What genes contribute most to these differences remains an important point to be elucidated. A simple inspection of tables 3 and 4 points to G2m(n) as the allotype primarily responsible: its frequency is .655 in lowland and .518 in highland (X21 - 17.29, P < .001). G2m(n) is an allele of the subclass IgG2 genes. It has not yet been established which amino acid sequence is coded by the presence of this allele. The antigenic determinant G2m(n) is located in the CH2 homology region [23]. Recently increasing attention has been given to the biological activities in which the Fc part of the heavy chains is mainly involved. It has been observed that each subclass exerts selective distribution. The antibody activity of the IgG2 molecule has been reported to be related to carbohydrate antigens [24]. Our data on heterogeneity between highland and lowland Sardinia, as expressed by G2m(n) suggest an adaptive value for this marker or for closely linked determinants of the CH2 region. This hypothesis is not contradicted by the Wahlund variance between the two areas calculated for the G2m(n) allotype, being .0274, a value of the same order of magnitude as that found previously [13] for thalassemia (.0465) and G6PD (.0612) genes, the adaptive meaning of which does not require further comments. A2m Allotypes A2m(1) is the most common allotype in all human populations. A2m(2) is very rare in whites, but occurs with varying frequency in blacks and orientals. Groups 2 and 5 were all found to be A2m(1+2-), except for four samples that were A2m(1+2+). Three of those samples were heterozygous for the haplotype Gm za;v ;b. This indicates admixture with the Negroid haplotype Gm za; .;b; A2m(2). All samples from Ferrara, the control group, were homozygous for A2m(1), except for one which was found to be deficient in IgA and contained anti-a antibodies.
Km Allotypes The Km phenotypes and the gene frequencies estimated for Sardinians and continental Italians are presented in table 5. All 1,218 individuals in the sample were tested for both Gm and Km markers except for 10 individuals only tested for Km. The gene frequency of Km(1) is typically white. No heterogeneity has been found within the Sardinian groups nor between Sardinians and continental Italians. SUMMARY
1218 individuals from Sardinia island (Italy) were tested for Gm and Km markers; 10 were not tested for Gm and only 401 were typed for Am markers. The peculiar genetic makeup of the Sardinian population is confirmed by their Gm allotypes. Their differences from those found in a control population of continental Italy (Ferrara), suggest ancient contacts with the Middle East and Africa. An indication for such contacts may also be found in the striking presence of the
IMMUNOGLOBULIN ALLOTYPES IN SARDINIA
85
TABLE 5
OBSERVED KM PHENOTYPES AND ESTIMATED GENE FREQUENCIES FOR SARDINIANS AND CONTINENTAL ITALIANS Km' REGION
North .............................. Northeast .198* Southeast .191 South .182 Mountain .203* Total Sardinia .1218 Continental Italy .86* *
Tested
No. Tested
444
Km(l)
(W)
(± SE)
82 24 33 43 44 226 14
.097 .063 .090 .126 .115 .098 .085
.010 .012 .015 .018 .016 .006 .022
also for Km(2) and Km(3).
haplotype Gm f;n;bsc5, a haplotype not previously found in a human population. A significant difference of G2m(n) allotype was observed between highland and lowland regions. If confirmed, it may suggest an adaptive pressure related to the CH2 region of the y2 chain, possibly due to endemic malaria in the past. REFERENCES 1. LOGHEM E VAN, NATVIG JB, MATSUMOTO H: Genetic markers on immunoglobulins in Japanese families. Inheritance of associated markers belonging to three IgG and IgA subclasses. Ann Hum Genet 33 :351-359, 1970 2. KUNKEL HG, JOSLIN FG, PENN GM, NATVIG JB: Genetic variants of yG4 globulin. A unique relationship to other classes of yG globulin. J Exp Med 132 :508-520, 1970 3. GRUBB R: The Genetic Markers of Human Immunoglobulins. New York, SpringerVerlag, 1970 4. GEDDE-DAHL T, FAGERHOLD MK, COOK PGL, NOADES J: Autosomal linkage between the Gm and R loci in man. Ann Hum Genet 35:393-399, 1972 5. FISHER R: The Mathematical Theory of Natural Selection. New York, Dover, 1959 6. STEINBERG AG: Globulin polymorphisms in man. Annu Rev Genet 3:25-52, 1969 7. LOGHEM E VAN: Stability of Gm polymorphism, in Human AntiHuman Gammaglobulins, edited by GRUBB R, SAMMELSON G, Oxford, Pergamon, 1971, pp 29-37 8. NATVIG JB, KUNKEL HG: Human immunoglobulins: classes, subclasses, genetic variants, and idiotypes. Adv Immunol 16:1-59, 1973 9. BODMER J, RoQuEs P, BODMER WF, DAUSSET J, DEGOS L, PIAZZA A: Joint report of the 5th International Histocompatibility Workshop, in Histocompatibility Testing 1972, edited by DAUSSET J, COLOMBANI J, Copenhagen, Munksgaard, 1973, pp 621667 10. CEPPELLINI R: Negative correlation between altitude above sea level and incidence of thalassemia in four Sardinian villages. Cold Spring Harbor Symp Quant Biol 26:252-255, 1955 11. CARCASSI U, CEPPELLINI R, PITZu E: Frequenza della talassemia in quattro popolazioni sarde e suoi rapporti con la distribuzione dei gruppi sanguigni e della malaria. Boll Ist Sieroter Milan 36:206-214, 1957 12. SINISCALCO M, BERNINI L, FILIPPI G, LATTE B, MEERA KHAN P, PIOMELLI S, RATTAZZI M: Population genetics of haemoglobin variants, thalassemia and glucose6-phosphate dehydrogenase deficiency, with particular reference to the malaria hypothesis. Bull WHO 34:378-393, 1966
86
PIAZZA ET AL.
13. PIAZZA A, BELVEDERE MC, BERNOCO D, CONIGHI C, CONTU L, CURTONI ES, MATTIUZ PL, MAYR W, RICHIARDI P, SCUDELLER G, CEPPELLINI R: HL-A variation in four Sardinian villages under differential selective pressure by malaria, in Histocompatibility Testing 1972, edited by DAUSSET J, COLOMBANI J, Copenhagen, Munksgaard, 1973, pp 73-84 14. FERMI E: La Malaria in Sardegna. Sassari, Stamperia Libreria Italiana e Straniera, 1938 15. LOGAN JA: The Sardinian Project. Baltimore, Johns Hopkins Press, 1953 16. TERRENATO L, VAN LOGHEM E, BERNINI L, SANTACHIARA-BENERECETTI AS, MODIANO G, SANTOLAMAZZA C, SCOZZARI R, ULIZZI L, BERRETTA M: Preliminary data on the genetic heterogeneity among the Sardinian isolates. Rend Acad Naz Lincei, Serie VIII, 51:249-253, 1971 17. TERRACINI A: Saggio di un Atlante Linguistico delta Sardegna. Torino, Stamperia Editoriale Rattero, 1964 18. SANTACHIARA-BENERECETTI AS, BERRETTA M, ULIzZI L, TERRENATO L: The frequency of the red cell NADH diaphorase Dia2 allele in Sardinia. Human Hered 21:290-293, 1972 19. PIAZZA A, SGARAMELLA-ZONATA L, GLUCKMANN P, CAVALLI-SFORZA LL: Analysis of human evolution. Phylogenetic analysis of a sample of 35 human populations tested for HL-A and other markers. Submitted for publication 20. STEINBERG AG, TIILIKAINEM A, ESKOLA M, ERIKSSON AW: Gammaglobulin allotypes in Finnish Lapps, Finns, Aland Islanders, Maris (Cheremis) and Greenland Eskimos. Am J Hum Genet 26:223-243, 1974 21. GRUBB R: Hereditary gamma globulin groups in man. Ann Hum Genet 13:171-174, 1961 22. LOGHEM E VAN, LITWIN SD: Antigenic determinants on Immunoglobulins of non human primates. Transplant Proc 4:129-135, 1972 23. NATVIG JB, TURNER MW: Localization of Gm markers to different molecular regions of the Fc fragment. Clin Exp Immunol 8:685-700, 1971 24. YOUNT WJ, DORNER MM, KUNKEL HG, KABAT EA: Studies on human antibodies. VI. Selective variations in subgroup composition and genetic markers. J Exp Med 127:633-646, 1968
