341
Ann. Hum. Uenel. (1978), 41, 341 Printed in Great Britain
Polymorphism of red cell enzymes in Alaskan ethnic groups BY EDWARD M. SCOTT AND RITA C . WRIGHT Alaska Activity, Center for Disease Control, U.S. Dept of Health, Education and Welfare, 225 Eagle Street, Anchorage, Alaska 99501, U.S.A. The frequencies of some polymorphic genes in Alaskan Eskimos, Indians and Aleuts have been reported (Blumberg, Allison & Garry, 1959;Scott et al. 1966; Duncan, Scott & Wright, 1973). With one exception, heterogeneity within the ethnic groups was not evident and the gene frequencies were assumed to be characteristic. The exception was a deficiency of heterozygotes when the Hardy-Weinberg expectation for phosphoglucomutase (PGM,) was compared with the results found in Yupik (southern) Eskimos (x2= 6.5, 1 df). A linguistic classification of the major ethnic groups which divides Yupik into 3 dialects and Athabaskan into 11 dialects is now available. Using this classiiication, far more diversity in gene frequency has been disclosed in these people than was previously suspected. I n this report, gene frequencies of the red cell enzymes esterase D (EsD), glyoxalase I (GLO) and uridine monophosphate kinase (UMPK) are estimated. Three rare variants of adenylate kinase (AK,), one of them new, and two variants of NADH diaphorase (DIA) are described. Improved methods for demonstration of phenotypes of uridine monophosphate kinase and glyoxalase I after electrophoresis are presented.
METHODS AND COMMENTS
Red cell samples were obtained as previously noted (Duncan et al. 1973). Esterase D - the method of Hopkinson et al. (1973)was used. Glyoxalase I - the method of Kompf et al. (1975),which is a two step procedure, was simplified as follows: After horizontal electrophoresis in starch gel in 0.04 M imidazole phosphate, pH 7.8, for 5 h at 7 V cm-l, a solution containing 0.75 yoagarose, 0.13 M-methylglyoxal, 8 mM 2,6-dichlorophenolindophenoland 0.1 Mreduced glutathione, 2 mM MTT tetrazolium, 3 Tris was brought to pH 6.0 with solid sodium bicarbonate and poured onto the gel. Clear white bands appeared in a purple background in about 1 h at 37 "C. The pH of the reaction mixture is critical. Histidine buffer was not used because methylglyoxal and primary amines react to form a Schiff base that reduces tetrazolium. Uridine monophosphate b a s e - the method of Fildes & Harris (1966)for demonstrating adenylate kinase on gels was modified by replacing half the adenine diphosphate with uridine diphosphate and by including 1 mM sodium cyanide. Purple adenylate kinase bands appeared within 15 min and UMPK bands after 1 h. Cyanide was added to inactive superoxide dismutase, which interferes with slow variants of UMPK. NADH diaphorase - the method of Hopkinson et al. (1970)was used routinely. To avoid possible interference from haemoglobin, which migrates more rapidly than diaphorase in Tris borate, the electrophoresis was also performed in 15 mM Tris brought to pH 8.0 with citric acid. In this system, haemoglobin migrates more slowly than diaphorase. A weakly stained 23
HGE
41
E. M. SCOTT AND RITAC. WRIGHT
342
Table 1. Esterase D frequency in Alaska Ethnic group Iiiupiat Aleuts Tlingits Yupilc Siberian Contra1 Sugpiaq Athabaskans Ahtna Ingalik Koyukon Tanaina
Total population
EsD2
Total tested
II000
223
2000 9 000
I22
EsD
I 000
17000 3 000 7 000 600 450
EsD I
34 42 45 59 4
I37 324 31 243
21000
2-1
3
8 4 0
2 2
41
14 75 16 9
50
I75 46 18
2 200
29
I2
900
60
26
16 3 4 5 4
2
frequoncy and
S.E.
0.081 f 0.013 0.197 f 0'025 0-223f 0.025 0.103 f0.012 0.065 f 0.03 I 0.092 f 0.013 0.I 80 f 0.038 0.306 f 0.025 0'239 f 0.044 0.472 f 0.083 0'379 f 0.064 0.283 f 0.041
Table 2. Glyoxalase I frequency in Alaska QLO2 Frequency and Ethnic group Iiiupiat Aleuts Tlingits Yupik Siberian Central Sugpiaq Athabaskans Ahtna Ingalik Koyukon Tanaina
Total tested
303 92 I02
421 33 338 50
242 50 34 34 69
GLO 2
S.E.
I 18
29
37 39 I79 I7 146 16
3 7 40
93
I0
20 I0
0
0.290 f 0.018 0.234 f 0.031 0.260 f 0.03 I 0.308 f 0.016 0.318 ko.057 0.310 fo.018 0.280 f 0.045 0.233 f 0.019 0.280 f 0.044 0.147 f 0.043 0.324 & 0.057 0.217 5 0.035
GLO
2-1
16 26
2
32 6 4 3 2
band, anodal to the DIA 1 band, is revealed ; it is masked by haemoglobin in buffers other than citrate. RESULTS
Esterase D frequencies in Alaskan ethnic groups and an estimate of the total populations of the groups are shown in Table 1. Esterase D was heterogeneous in Yupik (x2= 8.0,2 D.F.), and EsD2 frequency in Sugpiaq was significantlyhigher than in all Yupik. EsD2 frequency was higher in Ingalik than in all Athabaskans. Glyoxalase I (Table 2) appeared to be homogenoous in Yupik but not in Athabaskans. Six phenotypes of uridine monophosphate kinase were found in Yupik (Table 3 ) . UMPK3, which is rare in whites but frequent in Cree Indians (Giblett et al. 1974), was found in all Alaskan groups including Tsimshians (2 of 4 tested) and Haidas (1 of 5). Since it is rare or absent in Asiatics and Africans, UMPK3 may be a typically American gene. The higher UJilPK3 and the lower UMPK2 frequency in Iiiupiat (Iiiupiaq-speaking or northern Eskimos) compared with that of Yupik (Yupik-speaking or southern Eskimos) is the first significant difference in gene frequency demonstrated between these groups. U M P K ais rare in Athabaskans and 2 of the 3 Indians shown to have this gene lived near Eskimos. U M P K a frequencies were heterogeneous in both Yupik (x2= 19.9, 2 D.F.)and Athabaskans (x2= 23.8, 4 D.F.).
Polymorphism of red cell enzymes in Alaskan ethnic groups
343
Table 3. Uridine monophosphate kinase polymorphism in Alaska Gene frequency and S.E. Ethnic group Ifiupiat Aleuts Tlingits Yupik Siberian Central Sugpiaq Athabaskans Athna Ingalik Koyukon Tanaina
Total UMPK UMPK UMPK UMPK UMPK tested 2-1 3-1 2 3 3-2 228
9
78
0
56 55 296
7
10
3 27
I8 55
0 0
37
I
I0
0
210
20
3
2
6 3
32 13
I
49
0
6 8 6
0 0 0
213
45
I
0.022
2
0.080 f 0.026
0.232 f 0.040
0
o
0.027 f 0.017
0.I 64f 0.040
I1 2
2
o o
0.052 f 0.009 0.014 0.057f 0'01 I 0.061f 0.024
o
0.007k 0.004
0.133 f 0.014 0.189 0.046 0.095f 0.014 0.255 f 0.044 0.131f0.016 0.267f 0.047
6
0
0 0
I4
0
2
20 41
59
0
UMPK8
I
3
I2
>
h
U M P Ka
6 7
0 0 0
40
0 2 0
r
0
f 0.007
0
0.050 f 0.034 0 0
0.200 f 0.019
0.075 f 0'042
0.073f 0.029 0.153 k 0.033
Table 4. Adenylate kinme variants in Alaska Gene frequency Ethnic group Yupik Athabaskans Aleuts
Total tested 5 2-1 AK, 3-1
297
L
I
\
AK,5-1
AK;
AK:
AK:
2 I
0
4
0.003
0'0
212
4
3
0002
0.009
0.007 0.007
58
I
0
0
0.009
0'0
0'0
Previously, it was concluded that variants of adenylate kinase did not occur in Alaskan ethnic groups (Duncan et al. 1973) but 3 rare variants have since been found (Table 4). The frequency of A K ? in whites is 0.047 (Fildes & Harris, 1966; Bowman et al. 1967) and it is possible that it was introduced by whites into Alaska. If it was introduced, about a 5 yowhite admixture in the sample is implied. A K : is very rare (Bowman et al. 1967). The 4 examples of AK, 3-1 listed in Table 4 represent 2 unrelated mother and son combinations that were independently selected by a random procedure. One of the mothers was subsequently found to be part (one-eighth or less) Russian. On the assumption that A K ! was more apt to be of Athabaskan than of Russian origin, she and her son were included in the calculation of gene frequency. The A K , 5-1 phenotype shown in Fig. l has not been previously described. Eight examples were found, but one of these could not be classified ethnically. Three unrelated Eskimos wit.h this gene were from one Sugpiaq village of less than 200 people, but the fourth was a Siberian Yupik who lived 1300 km away. Two of the 3 Athabaskans with this phenotype were a brother and sister selected at random. No AK, variants were found in 232 Iiiupiat. The DIA 0 phenotype is associated with methaemoglobinemia (Scott, 1960) and 35 examples in 14 families have been found in Alaska. Twenty DIA 0 people from 12 families were tested by electrophoresisbut aside from the M u s e anodal band only one faint band of activity was found. This band was slightly cathodal to the DIA 1 band in citrate buffer (Pig. 2) and vaned in intensity in different families, but was never present in amount sufficient to be detected by assay for diaphorase. Since it was not found in 2 DIA 4 people, it may be related to deficiency, but its significance is uncertain because it would be masked if it occurred in DIA 1 or DIA 1-0 people. The 6 affected Eskimo families are widely scattered, unrelated Central Yupik, and the DIAO 23-2
E. M. SCOTT AND RITAC. WRIGHT
344
Fig. 1
Fig. 2
Fig. 3
Fig. 1. Adenylate kinase (AK,) variants. Fig. 2. Diaphorase deficiency in an Eskimo family. The parents' samples are at the right. Elcctrophoresis waa in Tris citrate buffer. Fig. 3. Diaphorase variants after electrophoresisin Tris borate buffer.
frequency is estimated at about 0.03. The 8 Athabaskan families are of Ingalik, Holikachuk or Koyukon origin, and the DIAO frequency is about 0.08.The DIA 0 phenotype does not correspond exactly to any of the other variants that have been associated with methaemoglobinemia (Hsieh & Jaffe, 1971) but it could be the same as case 5 of Bloom & Zarkowsky (1969). A band with the mobility of DIA4 (Hopkinson et al. 1970) was found in 32 Iiiupiat; 30 were DIA 4-1 and 2 were DIA 4 (Fig. 3). All of these cases could be ascribed to DIA4 in 6 family lines - 5 from a single village on the Chukchi Sea and the sixth from the nearest village to the west, 125 km away. Thus the variant could be the result of a single mutation. Although the 6 affected families believe themselves unrelated, any relationship prior to about 1890 would be unknown now. The DIAd frequency in a sample of 104 people from the village with the 5 affected families was 0.077 & 0.019. The DIA 4 individuals had normal levels of diaphorase and were not methaemoglobinemic. The diffuse band anodal to the DIA 1 band was the same in DIA 1, DIA 0, and DIA 4 haemolysates and thus unrelated to the DIA locus.
DISCUSSION
Alaskans lived in small villages ranging in size from a single household to as many as 200 people. They were isolated by distance, geographic barriers, and language differences, but isolation was never complete. Relatives could be found in nearby locations, and it was often necessary to go outside the village to find an unrelated person to marry. People travelled locally quite often, but they were not nomads and a change of residence to a location more than 80 km away was a rare event. Thus local dialects developed and local variability in gene frequencies might be expected. Variability was found in a study of the E, cholinesterase locus in Eskimos (Scott, Weaver &
Polymorphism of red cell enzymes in Alaskan ethnic groups
345
Wright, 1970), b u t the full extent of genetic diversity was not realized until too few heterozygotes for UMPK3 were found in Yupik (x2= 8.3, 1 D.F.), Athabaskans ( x 2 = 6.7), and Aleuts (x2 = 8.9). Further tests for heterogeneity showed that diversity was commonplace. When earlier results (Scott et al. 1966) were re-examined, the gene frequency of acid phosphatase (ACP) in Athabaskans and the gene frequency of haptoglobin (Hp) in both Athabaskans and Yupik were found to be heterogeneous. Since Central Yupik, Ifiupiat, Aleuts, and Tlingits were also spread over large areas, additional diversity might be found if objective geographic criteria for classification of these people were available. Gene frequency is usually assumed to be characteristic of a population if the observed data agree with the Hardy-Weinberg expectations even though this criterion is inefficient in detecting heterogeneity. Thus, haptoglobin in Athabaskans was homogeneous by the Hardy-Weinberg criterion (x2= 0-1), but in 5 linguistic subgroups heterogeneity was found (x2= 15.0, 4 D.F.) and Hpl frequency varied from 0.28 to 0.73. The variability in gene frequency that was found when major Alaskan ethnic groups were subdivided indicates that factors tending to stabilize the polymorphisms were not of local importance. Environmental influences, if they exist, are unknown, and chance seems to be sufficient to account for the diversity observed. I n any case this diversity shows that Alaskan populations consisted of small groups that did not intermix appreciably - a population structure that is consistent with linguistic evidence. SUMMARY
1. Gene frequencies of esterase D, glyoxalase I and uridine monophosphate kinase in Alaskan
populations were determined. 2. Improved methods for demonstrating phenotypes of glyoxalase I and uridine monophosphate kinase are presented. 3. Rare variants of adenylate kinase (AK,) and diaphorase (DIA) were found. One of the AK, variants is new. 4. Gene frequencies were notably diverse within major ethnic groups. This variability was consistent with a population structure composed of small groups that were relatively isolated from one another. REFERENCES
BLOOM, G. E. & ZARKOWSKY,H. S. (1969). Heterogeneity of the enzymatic defect in congenital methemoglobinemia. New Eng. J . Med. 281, 919-22. BLUMBERO. B. S., ALLISON,A. C. & GARRY,B. (1959). Haptoglobins and haemoglobins of Alaskan Eskimos and Indians. Ann. Hum. Genet, Lond. 23, 349-56. BOWMAN, J. E., FRISCHER, H., AJAMAR, F., CARSON, P. E. & GOWER,M. K. (1967). Population, family and biochemical investigation of human adenylate kinase polymorphism. Nature, Lond. 214, 1156-8. DUNCAN, I. W., SCOTT, E. M. & WRIam, R. C. (1973). Gene frequencies of erythrocytic enzymes of Alaskan Eskimos and Athabaskan Indians. Am. J . Hum. Genet. 26, 244-6. FILDES,R. A. t HAFCRIS, H. (1966). Genetically determined variation of adenylate kinase in man. Nature, Lond. 209, 261-3. GIBLETT,E. R., ANDERSON, J. E., CHEN, S.-H., TENG,Y.-S. & COHEN, F. (1974). Uridine monophosphate kinase: A new genetic polymorphism with possible clinical implications. Am. J . Hum. Genet. 26, 627-35. HOPKINSON, D. A., CORNEY, G., COOK,P. G. L., ROBSON, E. B. &HARRIS,H. (1970). Genetically determined variants of human red cell NADH diaphorase. Ann. Hum. Genet. 34, 1-10. HOPKINSON, D. A., MESTRINER,M. A., CORTNER, J. & HARRIS, H. (1973). Esterase D : a new human polymorphism. Ann. Hum. Genet., Lond. 37, 119-37.
E. M. SCOTTAND RITAC. WRIGHT HSIEH,H.-S. & JAFFI~, E. R. (1971). Electrophoretic and functional variants of NADH-methemoglobin reductase in hereditary methemoglobinemia. J . Clin. Invmt. 50, 196-202. KOMPF,J., BISSBORT, S., GUSSMAN, S. & RITTER,H. (1978). Polymorphism of red cell glyoxalaae I (E.C. 4 . 4 . 1 . 1 ) . A new genetic marker in man. Humangenetik 27, 141-3. SCOTT, E. M. (1960). The relation of diaphorase of human erythrocytes t o inheritance of methemoglobinemia. J . Clin. Inveat. 39, 1176-9. SCOTT, E. M., DTJNOAN, I. W., EKSTRAND, V. & WRIGHT, R. C. (1966). Frequency of polymorphic types of red cell enzymes and serum factors in Alaskan Eskimos and Indians. Am. J . Genet. 18, 408-11. SCOTT,E. M., WEAVER,D. D. & WRIGHT,R. C. (1970). Discrimination of phenotypes in human serum cholinesterase deficiency. Am. J. Hum. Genet. 22, 363-9.
Ann. Hum. Uenel. (1978), 41, 341 Printed in Great Britain
Polymorphism of red cell enzymes in Alaskan ethnic groups BY EDWARD M. SCOTT AND RITA C . WRIGHT Alaska Activity, Center for Disease Control, U.S. Dept of Health, Education and Welfare, 225 Eagle Street, Anchorage, Alaska 99501, U.S.A. The frequencies of some polymorphic genes in Alaskan Eskimos, Indians and Aleuts have been reported (Blumberg, Allison & Garry, 1959;Scott et al. 1966; Duncan, Scott & Wright, 1973). With one exception, heterogeneity within the ethnic groups was not evident and the gene frequencies were assumed to be characteristic. The exception was a deficiency of heterozygotes when the Hardy-Weinberg expectation for phosphoglucomutase (PGM,) was compared with the results found in Yupik (southern) Eskimos (x2= 6.5, 1 df). A linguistic classification of the major ethnic groups which divides Yupik into 3 dialects and Athabaskan into 11 dialects is now available. Using this classiiication, far more diversity in gene frequency has been disclosed in these people than was previously suspected. I n this report, gene frequencies of the red cell enzymes esterase D (EsD), glyoxalase I (GLO) and uridine monophosphate kinase (UMPK) are estimated. Three rare variants of adenylate kinase (AK,), one of them new, and two variants of NADH diaphorase (DIA) are described. Improved methods for demonstration of phenotypes of uridine monophosphate kinase and glyoxalase I after electrophoresis are presented.
METHODS AND COMMENTS
Red cell samples were obtained as previously noted (Duncan et al. 1973). Esterase D - the method of Hopkinson et al. (1973)was used. Glyoxalase I - the method of Kompf et al. (1975),which is a two step procedure, was simplified as follows: After horizontal electrophoresis in starch gel in 0.04 M imidazole phosphate, pH 7.8, for 5 h at 7 V cm-l, a solution containing 0.75 yoagarose, 0.13 M-methylglyoxal, 8 mM 2,6-dichlorophenolindophenoland 0.1 Mreduced glutathione, 2 mM MTT tetrazolium, 3 Tris was brought to pH 6.0 with solid sodium bicarbonate and poured onto the gel. Clear white bands appeared in a purple background in about 1 h at 37 "C. The pH of the reaction mixture is critical. Histidine buffer was not used because methylglyoxal and primary amines react to form a Schiff base that reduces tetrazolium. Uridine monophosphate b a s e - the method of Fildes & Harris (1966)for demonstrating adenylate kinase on gels was modified by replacing half the adenine diphosphate with uridine diphosphate and by including 1 mM sodium cyanide. Purple adenylate kinase bands appeared within 15 min and UMPK bands after 1 h. Cyanide was added to inactive superoxide dismutase, which interferes with slow variants of UMPK. NADH diaphorase - the method of Hopkinson et al. (1970)was used routinely. To avoid possible interference from haemoglobin, which migrates more rapidly than diaphorase in Tris borate, the electrophoresis was also performed in 15 mM Tris brought to pH 8.0 with citric acid. In this system, haemoglobin migrates more slowly than diaphorase. A weakly stained 23
HGE
41
E. M. SCOTT AND RITAC. WRIGHT
342
Table 1. Esterase D frequency in Alaska Ethnic group Iiiupiat Aleuts Tlingits Yupilc Siberian Contra1 Sugpiaq Athabaskans Ahtna Ingalik Koyukon Tanaina
Total population
EsD2
Total tested
II000
223
2000 9 000
I22
EsD
I 000
17000 3 000 7 000 600 450
EsD I
34 42 45 59 4
I37 324 31 243
21000
2-1
3
8 4 0
2 2
41
14 75 16 9
50
I75 46 18
2 200
29
I2
900
60
26
16 3 4 5 4
2
frequoncy and
S.E.
0.081 f 0.013 0.197 f 0'025 0-223f 0.025 0.103 f0.012 0.065 f 0.03 I 0.092 f 0.013 0.I 80 f 0.038 0.306 f 0.025 0'239 f 0.044 0.472 f 0.083 0'379 f 0.064 0.283 f 0.041
Table 2. Glyoxalase I frequency in Alaska QLO2 Frequency and Ethnic group Iiiupiat Aleuts Tlingits Yupik Siberian Central Sugpiaq Athabaskans Ahtna Ingalik Koyukon Tanaina
Total tested
303 92 I02
421 33 338 50
242 50 34 34 69
GLO 2
S.E.
I 18
29
37 39 I79 I7 146 16
3 7 40
93
I0
20 I0
0
0.290 f 0.018 0.234 f 0.031 0.260 f 0.03 I 0.308 f 0.016 0.318 ko.057 0.310 fo.018 0.280 f 0.045 0.233 f 0.019 0.280 f 0.044 0.147 f 0.043 0.324 & 0.057 0.217 5 0.035
GLO
2-1
16 26
2
32 6 4 3 2
band, anodal to the DIA 1 band, is revealed ; it is masked by haemoglobin in buffers other than citrate. RESULTS
Esterase D frequencies in Alaskan ethnic groups and an estimate of the total populations of the groups are shown in Table 1. Esterase D was heterogeneous in Yupik (x2= 8.0,2 D.F.), and EsD2 frequency in Sugpiaq was significantlyhigher than in all Yupik. EsD2 frequency was higher in Ingalik than in all Athabaskans. Glyoxalase I (Table 2) appeared to be homogenoous in Yupik but not in Athabaskans. Six phenotypes of uridine monophosphate kinase were found in Yupik (Table 3 ) . UMPK3, which is rare in whites but frequent in Cree Indians (Giblett et al. 1974), was found in all Alaskan groups including Tsimshians (2 of 4 tested) and Haidas (1 of 5). Since it is rare or absent in Asiatics and Africans, UMPK3 may be a typically American gene. The higher UJilPK3 and the lower UMPK2 frequency in Iiiupiat (Iiiupiaq-speaking or northern Eskimos) compared with that of Yupik (Yupik-speaking or southern Eskimos) is the first significant difference in gene frequency demonstrated between these groups. U M P K ais rare in Athabaskans and 2 of the 3 Indians shown to have this gene lived near Eskimos. U M P K a frequencies were heterogeneous in both Yupik (x2= 19.9, 2 D.F.)and Athabaskans (x2= 23.8, 4 D.F.).
Polymorphism of red cell enzymes in Alaskan ethnic groups
343
Table 3. Uridine monophosphate kinase polymorphism in Alaska Gene frequency and S.E. Ethnic group Ifiupiat Aleuts Tlingits Yupik Siberian Central Sugpiaq Athabaskans Athna Ingalik Koyukon Tanaina
Total UMPK UMPK UMPK UMPK UMPK tested 2-1 3-1 2 3 3-2 228
9
78
0
56 55 296
7
10
3 27
I8 55
0 0
37
I
I0
0
210
20
3
2
6 3
32 13
I
49
0
6 8 6
0 0 0
213
45
I
0.022
2
0.080 f 0.026
0.232 f 0.040
0
o
0.027 f 0.017
0.I 64f 0.040
I1 2
2
o o
0.052 f 0.009 0.014 0.057f 0'01 I 0.061f 0.024
o
0.007k 0.004
0.133 f 0.014 0.189 0.046 0.095f 0.014 0.255 f 0.044 0.131f0.016 0.267f 0.047
6
0
0 0
I4
0
2
20 41
59
0
UMPK8
I
3
I2
>
h
U M P Ka
6 7
0 0 0
40
0 2 0
r
0
f 0.007
0
0.050 f 0.034 0 0
0.200 f 0.019
0.075 f 0'042
0.073f 0.029 0.153 k 0.033
Table 4. Adenylate kinme variants in Alaska Gene frequency Ethnic group Yupik Athabaskans Aleuts
Total tested 5 2-1 AK, 3-1
297
L
I
\
AK,5-1
AK;
AK:
AK:
2 I
0
4
0.003
0'0
212
4
3
0002
0.009
0.007 0.007
58
I
0
0
0.009
0'0
0'0
Previously, it was concluded that variants of adenylate kinase did not occur in Alaskan ethnic groups (Duncan et al. 1973) but 3 rare variants have since been found (Table 4). The frequency of A K ? in whites is 0.047 (Fildes & Harris, 1966; Bowman et al. 1967) and it is possible that it was introduced by whites into Alaska. If it was introduced, about a 5 yowhite admixture in the sample is implied. A K : is very rare (Bowman et al. 1967). The 4 examples of AK, 3-1 listed in Table 4 represent 2 unrelated mother and son combinations that were independently selected by a random procedure. One of the mothers was subsequently found to be part (one-eighth or less) Russian. On the assumption that A K ! was more apt to be of Athabaskan than of Russian origin, she and her son were included in the calculation of gene frequency. The A K , 5-1 phenotype shown in Fig. l has not been previously described. Eight examples were found, but one of these could not be classified ethnically. Three unrelated Eskimos wit.h this gene were from one Sugpiaq village of less than 200 people, but the fourth was a Siberian Yupik who lived 1300 km away. Two of the 3 Athabaskans with this phenotype were a brother and sister selected at random. No AK, variants were found in 232 Iiiupiat. The DIA 0 phenotype is associated with methaemoglobinemia (Scott, 1960) and 35 examples in 14 families have been found in Alaska. Twenty DIA 0 people from 12 families were tested by electrophoresisbut aside from the M u s e anodal band only one faint band of activity was found. This band was slightly cathodal to the DIA 1 band in citrate buffer (Pig. 2) and vaned in intensity in different families, but was never present in amount sufficient to be detected by assay for diaphorase. Since it was not found in 2 DIA 4 people, it may be related to deficiency, but its significance is uncertain because it would be masked if it occurred in DIA 1 or DIA 1-0 people. The 6 affected Eskimo families are widely scattered, unrelated Central Yupik, and the DIAO 23-2
E. M. SCOTT AND RITAC. WRIGHT
344
Fig. 1
Fig. 2
Fig. 3
Fig. 1. Adenylate kinase (AK,) variants. Fig. 2. Diaphorase deficiency in an Eskimo family. The parents' samples are at the right. Elcctrophoresis waa in Tris citrate buffer. Fig. 3. Diaphorase variants after electrophoresisin Tris borate buffer.
frequency is estimated at about 0.03. The 8 Athabaskan families are of Ingalik, Holikachuk or Koyukon origin, and the DIAO frequency is about 0.08.The DIA 0 phenotype does not correspond exactly to any of the other variants that have been associated with methaemoglobinemia (Hsieh & Jaffe, 1971) but it could be the same as case 5 of Bloom & Zarkowsky (1969). A band with the mobility of DIA4 (Hopkinson et al. 1970) was found in 32 Iiiupiat; 30 were DIA 4-1 and 2 were DIA 4 (Fig. 3). All of these cases could be ascribed to DIA4 in 6 family lines - 5 from a single village on the Chukchi Sea and the sixth from the nearest village to the west, 125 km away. Thus the variant could be the result of a single mutation. Although the 6 affected families believe themselves unrelated, any relationship prior to about 1890 would be unknown now. The DIAd frequency in a sample of 104 people from the village with the 5 affected families was 0.077 & 0.019. The DIA 4 individuals had normal levels of diaphorase and were not methaemoglobinemic. The diffuse band anodal to the DIA 1 band was the same in DIA 1, DIA 0, and DIA 4 haemolysates and thus unrelated to the DIA locus.
DISCUSSION
Alaskans lived in small villages ranging in size from a single household to as many as 200 people. They were isolated by distance, geographic barriers, and language differences, but isolation was never complete. Relatives could be found in nearby locations, and it was often necessary to go outside the village to find an unrelated person to marry. People travelled locally quite often, but they were not nomads and a change of residence to a location more than 80 km away was a rare event. Thus local dialects developed and local variability in gene frequencies might be expected. Variability was found in a study of the E, cholinesterase locus in Eskimos (Scott, Weaver &
Polymorphism of red cell enzymes in Alaskan ethnic groups
345
Wright, 1970), b u t the full extent of genetic diversity was not realized until too few heterozygotes for UMPK3 were found in Yupik (x2= 8.3, 1 D.F.), Athabaskans ( x 2 = 6.7), and Aleuts (x2 = 8.9). Further tests for heterogeneity showed that diversity was commonplace. When earlier results (Scott et al. 1966) were re-examined, the gene frequency of acid phosphatase (ACP) in Athabaskans and the gene frequency of haptoglobin (Hp) in both Athabaskans and Yupik were found to be heterogeneous. Since Central Yupik, Ifiupiat, Aleuts, and Tlingits were also spread over large areas, additional diversity might be found if objective geographic criteria for classification of these people were available. Gene frequency is usually assumed to be characteristic of a population if the observed data agree with the Hardy-Weinberg expectations even though this criterion is inefficient in detecting heterogeneity. Thus, haptoglobin in Athabaskans was homogeneous by the Hardy-Weinberg criterion (x2= 0-1), but in 5 linguistic subgroups heterogeneity was found (x2= 15.0, 4 D.F.) and Hpl frequency varied from 0.28 to 0.73. The variability in gene frequency that was found when major Alaskan ethnic groups were subdivided indicates that factors tending to stabilize the polymorphisms were not of local importance. Environmental influences, if they exist, are unknown, and chance seems to be sufficient to account for the diversity observed. I n any case this diversity shows that Alaskan populations consisted of small groups that did not intermix appreciably - a population structure that is consistent with linguistic evidence. SUMMARY
1. Gene frequencies of esterase D, glyoxalase I and uridine monophosphate kinase in Alaskan
populations were determined. 2. Improved methods for demonstrating phenotypes of glyoxalase I and uridine monophosphate kinase are presented. 3. Rare variants of adenylate kinase (AK,) and diaphorase (DIA) were found. One of the AK, variants is new. 4. Gene frequencies were notably diverse within major ethnic groups. This variability was consistent with a population structure composed of small groups that were relatively isolated from one another. REFERENCES
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