Humangenetik 28, 75--78 (1975) © by Springer-Verlag 1975
Esterase D Polymorphism: High-Voltage Agarose-Gel Electrophoresis and Distribution of Phenotypes in Different European Populations B. K 6 s t e r , H a n n a Leupold, a n d G. Mauff Hygiene-Institut der Universit~it K61n Received December 27, 1974 Summary. Esterase D phenotypes were determined in 1082 non-related individuals from the western region of Germany by agarose-gel electrophoresis. Gene frequencies were compared with previous data and all European populations studied so far agreed with the HardyWeinberg equilibrium. Mean gene frequencies for Europeans are: E s D 1 0.8888, E s D 2 0.1112.
P o p u l a t i o n studies of t h e r e c e n t l y discovered esterase D (EsD) p o l y m o r p h i s m in h u m a n red cells ( H o p k i n s o n et al., 1973) h a v e been r e p o r t e d for different racial groups ( H o p k i n s o n et al., 1973; Welch, 1974; W e l c h a n d Lee, 1974; B e n d e r a n d F r a n k , 1974; B e n k m a n n a n d Goedde, 1974). Two a u t o s o m a l c o m m o n alleles, E s D 1 a n d E s D 2, as well as one rare allele, E s D 3 (Bender a n d F r a n k , 1974), are known. All E s D d e t e r m i n a t i o n s r e p o r t e d u n t i l now were p e r f o r m e d in starch-gel electrophoresis. The p r e s e n t s t u d y is concerned with a more simplified m e t h o d for t h e d e t e c t i o n of E s D p h e n o t y p e s which has r e s u l t e d from a c o m p a r i s o n of several different electrophoretic carrier media. I n a d d i t i o n , gene frequencies are r e p o r t e d for a W e s t G e r m a n p o p u l a t i o n a n d c o m p a r e d w i t h o t h e r E u r o p e a n populations. Material and Methods Blood samples were obtained from 1082 healthy non-related individuals from the Cologne area. Hemolysates--prepared by freezing and thawing--were kept at --80°C until determination. For agarose-gel, polyacrylamide-gel, and cellulose-acetate electrophoresis they were separated from stromata according to Marti (1963). Electrophoretic separations were carried out: a) in starch-gel according to Hopkinson et al. (1973), b) in agarose-gel (1°/0 Agarose Behring), 5 ~1 samples applied to 8 mm slots formed with filter paper Whatman No. 3, separation on water-cooled glass plates (200 × 200 mm) for 135 rain at 22.5 V/cm (total input 16 W). Bridge buffer: 62 mM Tris, 16.5 mM citric acid, 18 mM boric acid, 1.65 mM lithium hydroxyde, p i t 7.5. Gel buffer: 1:5 diluted bridge buffer, e) in polyaerylamide-gel according to Allen et al. (1969), electrophoretic apparatus ORTEC GmbH, Miinchen. Buffer systems: 1 tris-citrate-borate-LiOIt, 2 tris-borate, tris-eitrate as a discontinuous buffer system. Gel concentrations: 3, 5, 7.5, and 12% in different runs, d) on cellulose-acetate, electrophoretic apparatus BOSKAMP GmbH, Hersel/Bonn. Buffer system : tris-citrate-borate-LiOH. All electrophoretic separations were stained with 4-methyl-umbelliferyl acetate as a substrate (Hopkinson et al., 1973). UV-fluorescent esterase A bands were identified by staining with ~-naphthyl-acetate and fast blue RR (Tashian, 1969).
76
B. KSster et al.
Fig. 1. Esterase D phenotypes in high-voltage agarose-gel electrophoresis; start at bottom, anode at top
Discussion of Results In starch-gel electrophoresis satisfactory separation was seen with the buffer systems originally described by Hopkinson and coworkers, the best results were achieved with the tris-citrate-borate-LiOH buffer. This buffer also was applicable to polyacrylamide-gel electrophoresis in a continuous 7.5% gel. Discontinuous systems and gel concentrations below or above 7.5% resulted in unsatisfactory separation of EsD bands. Poor separations could be observed on cellulose-acetate electrophoresis and studies were discontinued with this method. To the present authors the most suitable procedure for the determination of EsD polymorphism seems to be agarose-gel electrophoresis. Less material is required, separation time is short, and phenotypic bands are remarkably more distinct than in any other medium tested (Fig. 1). At room temperature bands could be observed for approximately 45 min. In methanol/water/acetic acid (5:5:1) agarose-gels were kept at - - 8 0 ° C for 24 hrs and bands were well distinguishable. Hemolysates and whole blood stored at room temperature and at 4°C were stable for over 2 weeks, and hemolysates were stable at - - 8 0 ° C for a period of more than 21/2 years. Phenotypes and gene frequencies in a West German population can be seen in Table 1. Values correspond well to frequencies in Europeans reported in previous studies. Although consistency is not equally well distributed the frequencies of phenotypes seem to be comparable as seen in the Z 2 test. By further evaluation the data proves to be in agreement with the Hardy-Weinberg equilibrium. Mean
Esterase D in Agarose-Gel Electrophoresis
77
Table 1. Distribution of phenotypes and gent frequencies of Esterase D in different European populations Author
Phenotypes EsD 1
t EsD 2
Gene frequencies
1 EsD 2
EsD 1
Hopkinson etal., 1973
n
obs. exp.
705 705.22
154 153.46
8 8.32
867 867
Europeans
~o
obs. exp.
81.31 81.34
17.76 17.70
0.93 0.96
100 100
Welch and Lee, 1974
n
obs. exp.
312 314.13
84 79.80
3 5.07
399 399
English
o~
obs. exp.
78.20 78.73
21.05 20.00
0.75 1.27
100 100
Irish
n
obs. exp.
144 142.87
38 40.29
4 2.84
186 186
o~
obs. exp.
77.42 76.81
20.43 21.66
2.15 1.53
100 100
Bender and Frank, 1974
n
obs. exp.
147 146.17
34 35.66
3 2.17
184 a 184
Southwest Germans
°h
obs. exp.
79.89 79.44
18.48 19.38
1.63 1.18
100 100
Benkmann and Goedde, 1974
n
obs. exp.
318 317.67
84 84.66
6 5.67
408 408
North Germans
o~
obs. exp.
77.14 77.86
20.58 20.75
1.48 1.39
100 100
This publication
n
obs. exp.
844 843.85
223 223.33
15 14.82
1082 1082
West Germans
~o
obs. exp.
78.00 77.99
20.61 20.64
1.39 1.37
100 100
EsD ~
0.9019 0.0981 X~=0.0143
0.8873 0.1127 Z ~ : 1.0806
0.8764 0.1236 Xe~0.6129
0.8913 0.1087 Z 2 --0.3995
0.8824 0.1176 22=0.0247
0.8831 0.1169 Z ~ = 0.0027
E X e = 2.1347 z0.0~ ~ = 11.070 (5 d/)
Total Europeans
n
obs. exp.
°/o
obs. exp.
2470 2469.2 79.09 78.99
617 618.0 19.74 19.77
39 38.8 1.25 1.24
3126 3126 100 100
0.8888 0.1112 Z~= 0.0034 P > 0.95 (1 d])
a Frequency without EsD 3--1. g e n e f r e q u e n c i e s in all E u r o p e a n s t e s t e d so far are for E s D 1 0.8888, for E s D 2 0.1112. I n 608 r e p o r t e d m o t h e r / c h i l d c o m b i n a t i o n s ( H o p k i n s o n et al., 1973 ; B e n d e r a n d F r a n k , 1974; B e n k m a n n a n d G o e d d e , 1974) n o e x c e p t i o n f r o m t h e e x p e c t e d m o d e of i n h e r i t a n c e was seen. I n a d d i t i o n t o t h e u n c o m p l i c a t e d t e c h n i q u e of p h e n o t y p e d e t e r m i n a t i o n it m a y t h e r e f o r e be a s s u m e d t h a t E s D p o l y m o r p h i s m will s o o n be a p p l i c a b l e in p a t e r n i t y eases. Single e x c l u s i o n c h a n c e f r o m t h e m e a n g e n e freq u e n c i e s was c a l c u l a t e d t o be 0.089 ( p q ( 1 - - p q ) , g i e d w y l , 1970). We wish to thank Mrs. S. Glansehneider for her very skillful photographic assistance.
78
B. K6ster et al. References
Allen, R. C., moore, I). J., Dilworth, R. H. : A new rapid electrophoresis procedure employing pulsed power in gradient gels at a continuous pH: the effect of various discontinuous buffer systems on esterase zymograms. J. I-Iistochem. Cytochem. 17, 189 (1969) Bender, K., Frank, R.: Esterase D-Polymorphismus: Darstellung in der Hochspannungselektrophorese und Mitteilung yon Allelh~ufigkeiten. I.iumangenetik 23, 315 (1974) Benkmann, H., Goedde, H. W. : Esterase D polymorphism: gene frequencies and family data. Humangenetik 24, 325 (1974) Hopkinson, D. A., Mestriner, M. A., Cortner, J., Harris, H. : Esterase I): a new human polymorphism. Ann. hum. Genet. 37, 119 (1973) Marti, H. : Normale und anomale menschliche HI~moglobine, S. 51. Berlin-G6ttingen-Heidelberg: Springer 1963 Riedwyl, H. : Wahrscheinlichkeitsberechnung bei Blutgruppengutachten. Arztl. Lab. 16, 86 (1970) Tashian, R. E. : In: Biochemical methods in red cell genetics (ed. J. J. Yunis). New York-London: Academic Press 1969 Welch, S. G. : Red cell esterase D in Gambia. Humangenetik 21, 365 (1974) Welch, S. G., Lee, J. : The population distribution of genetic variants of human esterase D. Humangenetik 24, 329 (1974) Bernd K6ster Dr. med. Hanna Leupold Dr. reed. Gottfried Mauff (for reprints) Hygiene-Institut der Universit~i~t D-5000 K6ln 41, Goldenfelsstral3e 21 Federal Republic of Germany
Esterase D Polymorphism: High-Voltage Agarose-Gel Electrophoresis and Distribution of Phenotypes in Different European Populations B. K 6 s t e r , H a n n a Leupold, a n d G. Mauff Hygiene-Institut der Universit~it K61n Received December 27, 1974 Summary. Esterase D phenotypes were determined in 1082 non-related individuals from the western region of Germany by agarose-gel electrophoresis. Gene frequencies were compared with previous data and all European populations studied so far agreed with the HardyWeinberg equilibrium. Mean gene frequencies for Europeans are: E s D 1 0.8888, E s D 2 0.1112.
P o p u l a t i o n studies of t h e r e c e n t l y discovered esterase D (EsD) p o l y m o r p h i s m in h u m a n red cells ( H o p k i n s o n et al., 1973) h a v e been r e p o r t e d for different racial groups ( H o p k i n s o n et al., 1973; Welch, 1974; W e l c h a n d Lee, 1974; B e n d e r a n d F r a n k , 1974; B e n k m a n n a n d Goedde, 1974). Two a u t o s o m a l c o m m o n alleles, E s D 1 a n d E s D 2, as well as one rare allele, E s D 3 (Bender a n d F r a n k , 1974), are known. All E s D d e t e r m i n a t i o n s r e p o r t e d u n t i l now were p e r f o r m e d in starch-gel electrophoresis. The p r e s e n t s t u d y is concerned with a more simplified m e t h o d for t h e d e t e c t i o n of E s D p h e n o t y p e s which has r e s u l t e d from a c o m p a r i s o n of several different electrophoretic carrier media. I n a d d i t i o n , gene frequencies are r e p o r t e d for a W e s t G e r m a n p o p u l a t i o n a n d c o m p a r e d w i t h o t h e r E u r o p e a n populations. Material and Methods Blood samples were obtained from 1082 healthy non-related individuals from the Cologne area. Hemolysates--prepared by freezing and thawing--were kept at --80°C until determination. For agarose-gel, polyacrylamide-gel, and cellulose-acetate electrophoresis they were separated from stromata according to Marti (1963). Electrophoretic separations were carried out: a) in starch-gel according to Hopkinson et al. (1973), b) in agarose-gel (1°/0 Agarose Behring), 5 ~1 samples applied to 8 mm slots formed with filter paper Whatman No. 3, separation on water-cooled glass plates (200 × 200 mm) for 135 rain at 22.5 V/cm (total input 16 W). Bridge buffer: 62 mM Tris, 16.5 mM citric acid, 18 mM boric acid, 1.65 mM lithium hydroxyde, p i t 7.5. Gel buffer: 1:5 diluted bridge buffer, e) in polyaerylamide-gel according to Allen et al. (1969), electrophoretic apparatus ORTEC GmbH, Miinchen. Buffer systems: 1 tris-citrate-borate-LiOIt, 2 tris-borate, tris-eitrate as a discontinuous buffer system. Gel concentrations: 3, 5, 7.5, and 12% in different runs, d) on cellulose-acetate, electrophoretic apparatus BOSKAMP GmbH, Hersel/Bonn. Buffer system : tris-citrate-borate-LiOH. All electrophoretic separations were stained with 4-methyl-umbelliferyl acetate as a substrate (Hopkinson et al., 1973). UV-fluorescent esterase A bands were identified by staining with ~-naphthyl-acetate and fast blue RR (Tashian, 1969).
76
B. KSster et al.
Fig. 1. Esterase D phenotypes in high-voltage agarose-gel electrophoresis; start at bottom, anode at top
Discussion of Results In starch-gel electrophoresis satisfactory separation was seen with the buffer systems originally described by Hopkinson and coworkers, the best results were achieved with the tris-citrate-borate-LiOH buffer. This buffer also was applicable to polyacrylamide-gel electrophoresis in a continuous 7.5% gel. Discontinuous systems and gel concentrations below or above 7.5% resulted in unsatisfactory separation of EsD bands. Poor separations could be observed on cellulose-acetate electrophoresis and studies were discontinued with this method. To the present authors the most suitable procedure for the determination of EsD polymorphism seems to be agarose-gel electrophoresis. Less material is required, separation time is short, and phenotypic bands are remarkably more distinct than in any other medium tested (Fig. 1). At room temperature bands could be observed for approximately 45 min. In methanol/water/acetic acid (5:5:1) agarose-gels were kept at - - 8 0 ° C for 24 hrs and bands were well distinguishable. Hemolysates and whole blood stored at room temperature and at 4°C were stable for over 2 weeks, and hemolysates were stable at - - 8 0 ° C for a period of more than 21/2 years. Phenotypes and gene frequencies in a West German population can be seen in Table 1. Values correspond well to frequencies in Europeans reported in previous studies. Although consistency is not equally well distributed the frequencies of phenotypes seem to be comparable as seen in the Z 2 test. By further evaluation the data proves to be in agreement with the Hardy-Weinberg equilibrium. Mean
Esterase D in Agarose-Gel Electrophoresis
77
Table 1. Distribution of phenotypes and gent frequencies of Esterase D in different European populations Author
Phenotypes EsD 1
t EsD 2
Gene frequencies
1 EsD 2
EsD 1
Hopkinson etal., 1973
n
obs. exp.
705 705.22
154 153.46
8 8.32
867 867
Europeans
~o
obs. exp.
81.31 81.34
17.76 17.70
0.93 0.96
100 100
Welch and Lee, 1974
n
obs. exp.
312 314.13
84 79.80
3 5.07
399 399
English
o~
obs. exp.
78.20 78.73
21.05 20.00
0.75 1.27
100 100
Irish
n
obs. exp.
144 142.87
38 40.29
4 2.84
186 186
o~
obs. exp.
77.42 76.81
20.43 21.66
2.15 1.53
100 100
Bender and Frank, 1974
n
obs. exp.
147 146.17
34 35.66
3 2.17
184 a 184
Southwest Germans
°h
obs. exp.
79.89 79.44
18.48 19.38
1.63 1.18
100 100
Benkmann and Goedde, 1974
n
obs. exp.
318 317.67
84 84.66
6 5.67
408 408
North Germans
o~
obs. exp.
77.14 77.86
20.58 20.75
1.48 1.39
100 100
This publication
n
obs. exp.
844 843.85
223 223.33
15 14.82
1082 1082
West Germans
~o
obs. exp.
78.00 77.99
20.61 20.64
1.39 1.37
100 100
EsD ~
0.9019 0.0981 X~=0.0143
0.8873 0.1127 Z ~ : 1.0806
0.8764 0.1236 Xe~0.6129
0.8913 0.1087 Z 2 --0.3995
0.8824 0.1176 22=0.0247
0.8831 0.1169 Z ~ = 0.0027
E X e = 2.1347 z0.0~ ~ = 11.070 (5 d/)
Total Europeans
n
obs. exp.
°/o
obs. exp.
2470 2469.2 79.09 78.99
617 618.0 19.74 19.77
39 38.8 1.25 1.24
3126 3126 100 100
0.8888 0.1112 Z~= 0.0034 P > 0.95 (1 d])
a Frequency without EsD 3--1. g e n e f r e q u e n c i e s in all E u r o p e a n s t e s t e d so far are for E s D 1 0.8888, for E s D 2 0.1112. I n 608 r e p o r t e d m o t h e r / c h i l d c o m b i n a t i o n s ( H o p k i n s o n et al., 1973 ; B e n d e r a n d F r a n k , 1974; B e n k m a n n a n d G o e d d e , 1974) n o e x c e p t i o n f r o m t h e e x p e c t e d m o d e of i n h e r i t a n c e was seen. I n a d d i t i o n t o t h e u n c o m p l i c a t e d t e c h n i q u e of p h e n o t y p e d e t e r m i n a t i o n it m a y t h e r e f o r e be a s s u m e d t h a t E s D p o l y m o r p h i s m will s o o n be a p p l i c a b l e in p a t e r n i t y eases. Single e x c l u s i o n c h a n c e f r o m t h e m e a n g e n e freq u e n c i e s was c a l c u l a t e d t o be 0.089 ( p q ( 1 - - p q ) , g i e d w y l , 1970). We wish to thank Mrs. S. Glansehneider for her very skillful photographic assistance.
78
B. K6ster et al. References
Allen, R. C., moore, I). J., Dilworth, R. H. : A new rapid electrophoresis procedure employing pulsed power in gradient gels at a continuous pH: the effect of various discontinuous buffer systems on esterase zymograms. J. I-Iistochem. Cytochem. 17, 189 (1969) Bender, K., Frank, R.: Esterase D-Polymorphismus: Darstellung in der Hochspannungselektrophorese und Mitteilung yon Allelh~ufigkeiten. I.iumangenetik 23, 315 (1974) Benkmann, H., Goedde, H. W. : Esterase D polymorphism: gene frequencies and family data. Humangenetik 24, 325 (1974) Hopkinson, D. A., Mestriner, M. A., Cortner, J., Harris, H. : Esterase I): a new human polymorphism. Ann. hum. Genet. 37, 119 (1973) Marti, H. : Normale und anomale menschliche HI~moglobine, S. 51. Berlin-G6ttingen-Heidelberg: Springer 1963 Riedwyl, H. : Wahrscheinlichkeitsberechnung bei Blutgruppengutachten. Arztl. Lab. 16, 86 (1970) Tashian, R. E. : In: Biochemical methods in red cell genetics (ed. J. J. Yunis). New York-London: Academic Press 1969 Welch, S. G. : Red cell esterase D in Gambia. Humangenetik 21, 365 (1974) Welch, S. G., Lee, J. : The population distribution of genetic variants of human esterase D. Humangenetik 24, 329 (1974) Bernd K6ster Dr. med. Hanna Leupold Dr. reed. Gottfried Mauff (for reprints) Hygiene-Institut der Universit~i~t D-5000 K6ln 41, Goldenfelsstral3e 21 Federal Republic of Germany
