Vox Sang. 29: 217-220 (1975)
ADA7- A New Allele K. BERG,H. CLEVE, G . HOFMANN, F. SCHWARZFISCHER, G. G. WENDT and H. WISCHERATH Institute for Antropology and Human Genetics, University of Munich, Munich, and Institute for Human Genetics, University of Marburg, Marburg
A h t r a c t . A new ADA phenotype which is particularly susceptible to phenotypic changes following storage was observed. Family studies indicate that this type may be due to a new allele, ADA’. The phenotype observed was classified as ADA 7-1.
Inherited variation of the human erythrocyte adenosine deaminase (ADA) was described first by SPENCER et al. [8]. They observed three phenotypes by starch gel electrophoresis: ADA 1, ADA 2-1, and ADA 2. Through family studies it was established that the three phenotypes were determined by two co-dominant autosomal alleles, ADA1 and ADA2. Subsequently, additional alleles have been observed which occur at very low frequencies. These include the allele ADA3 of HOPKINSON et a / . [ 5 ] , the phenotype reported by DISSINC and KNUDSEN [4], the allele ADA5postulated by DETTER et a / . [3] which was confirmed by RADAM e t a / . [7], the allele ADA6 of RADAM et al. [6],and the ‘silent’ allele ADAo of BRINKMANN et al. [I] and CHENet al. [2]. CHENet al. [2] demonstrated furthermore the association of a ‘silent’ gene at the ADA locus with combined immunodeficiency disease. In this report a previously unknown ADA phenotype is described. This phenotype was observed first by one of us (G. H.) in a family from Regensburg, Bavaria. Family studies indicate that the phenotype is due to an additional allele at the ADA locus called ADA7. Materials and Methods Eight members of four generations of a family from Regensburg, Bavaria, were examined. Hemolysates were prepared from blood samples obtained by venous puncture. ADA et al. [8]. phenotypes were examined by starch gel electrophoresis according to SPENCER Received: December 2, 1974; accepted: December 12, 1974.
BERGet al.
218
-I
ADA1
ADA 2-1
ADA 7-1
ADA 7-1
ADA 7-1
ADA 7-1
A
B
C
D
E
F
ADA2
Start G
Fig. 1. Starch gel electrophoreticpattern of human erythrocyte adenosine deaminase. Common phenotypes ADA 1 (A), ADA 2-1 (B), and ADA 2 (G) are compared with a new phenotype ADA 7-1 (C). Changes of pattern during storage are shown after 1 (D), 2 (E), and 3 weeks (F).
A
B
C
D
E
F
G
Fig. 2. Starch gel photograph of the above-mentioned isozyme patterns in the variant phenotype. The figure has been composed from gel strips from different electrophoretic runs.
Results and Discussion
In four family members from four generations a new ADA phenotype was observed (fig. 1). The new phenotype resembled the ADA 2-1 type: the fresh hemolysate (fig. 1,C) showed four bands of decreasing intensity. However, the slowest and most intense band was located more towards the starting point than the slower band of the 2-1 type. The band appeared,
ADA'
-
A New Allele
112 ADA 7-1
111 Not t e s t e d
1112 ADA 7-1
1111 Not t e s t e d 11111 ADA 1
11112 ADA 7-1
IVI1 ADAl
IV12 ADAl
219
Not t e s t e d
11113 ADA 1
IV13 ADA7-1
Fig. 3. ADA phenotypes in a family from Regensburg, Bavaria.
furthermore, to be more intensely stained than the corresponding band of the 2-1 type. It was also observed that the cathodal edge of the second slower band was blurred. Characteristic changes of pattern were observed after storage of hemolysates at 4°C (fig. 1, D-F). The changes included decreasing intensity of the slower band and increasing intensity of the two faster bands. The second slower band was converted into a double-banded pattern, until after 3 weeks of storage a single, but slower moving second band was noted (fig. 1 m . ADA phenotypes were determined also after storage of hemolysates for 3 weeks and preincubation with 10 mM 2-mercaptoethanol for 30 min. prior to electrophoresis. As observed by SPENCER et al. [8] in the ADA 1 and ADA 2-1 phenotypes, the changes of pattern occuring after storage of samples with the new ADA phenotype were completely reversed by preincubation with 2-mercaptoethanol. It may therefore be concluded that the new ADA phenotype does not directly involve the free -SH group of the isozymes demonstrated by HOPKINSON and HARRIS[5]. The particular susceptibility to phenotypic changes after storage indicates perhaps anincreased reactivity of the free -SH group in the mutant type. The results of the family studies are presented in figure 3. The new phenotype was observed in members of four generations: I/2, 11/2, III/2 and IV/3. The results are in agreement with the assumption of an additional allele at the ADA locus which we propose to call ADA'. The observed phenotype can thus be classified as ADA 7-1.
220
BERGet al.
Rejerences 1 BRINKMANN, B. ; BRINKMANN, M., and MARTIN,H.: A new allele in red cell adenosine deaminase polymorphism: ADA'. Hum. Hered. 23 :603-607 (1973). E. R.: Adenosine deaminase: demonstration 2 CHEN,S. H.; SCOTT,C. R., and GIBLETT, of a 'silent' gene associated with combined immunodeficiency disease. Am. J. hum. Genet. 26:103-107 (1974). 3 DETTER,J. C.; STAMATOYANNOPOULOS, G.; GIBLETT, E. R., and MOTULSKY, A. G.: Adenosine deaminase: racial distribution and report of a new phenotype. J. med. Genet. 7: 356 (1970). 4 DISSING,J. and KNUDSEN, J. B.: A new red cell adenosine deaminase phenotype in man. Hum. Hered. I9:375-377 (1969). 5 HOPKINSON, D. A. and HARRIS, H. : The investigation of reactive sulphydryls in enzymes and their variants by starch gel electrophoresis. Studies on red cell adenosine deaminase. Ann. hum. Genet. 3.3~81-87(1969). G. ; STRAUCH, H. und PROKOP,0.: Ein seltener Phanotyp im Adenosindesam6 RADAM, inase-Polymorphismus: Hinweis auf die Existenz eines neuen Allels. Humangenetik 25 :247-250 (1974). 7 RADAM, G.; STRAUCH, H. und VAVRUSA, B.: Zur Differenzierung der Varianten 5-1 und 6-1 im Adenosindesaminase-Polymorphismus. Nachweis des neuen Phanotyps ADA 5 2 in der CSSR. (in press). 8 SPENCER, N. ; HOPKINSON, D. A., and HARRIS, H. : Adenosine deaminase polymorphism in man. Ann. hum. Genet. 32:9-14 (1968).
Dr. Dr. HANSWISCHERATH, Institut fur Anthropologie und Humangenetik der Universitit Miinchen, Richard-Wagner-Str. lo1, 0-8000Munchen 2 (FRG)
ADA7- A New Allele K. BERG,H. CLEVE, G . HOFMANN, F. SCHWARZFISCHER, G. G. WENDT and H. WISCHERATH Institute for Antropology and Human Genetics, University of Munich, Munich, and Institute for Human Genetics, University of Marburg, Marburg
A h t r a c t . A new ADA phenotype which is particularly susceptible to phenotypic changes following storage was observed. Family studies indicate that this type may be due to a new allele, ADA’. The phenotype observed was classified as ADA 7-1.
Inherited variation of the human erythrocyte adenosine deaminase (ADA) was described first by SPENCER et al. [8]. They observed three phenotypes by starch gel electrophoresis: ADA 1, ADA 2-1, and ADA 2. Through family studies it was established that the three phenotypes were determined by two co-dominant autosomal alleles, ADA1 and ADA2. Subsequently, additional alleles have been observed which occur at very low frequencies. These include the allele ADA3 of HOPKINSON et a / . [ 5 ] , the phenotype reported by DISSINC and KNUDSEN [4], the allele ADA5postulated by DETTER et a / . [3] which was confirmed by RADAM e t a / . [7], the allele ADA6 of RADAM et al. [6],and the ‘silent’ allele ADAo of BRINKMANN et al. [I] and CHENet al. [2]. CHENet al. [2] demonstrated furthermore the association of a ‘silent’ gene at the ADA locus with combined immunodeficiency disease. In this report a previously unknown ADA phenotype is described. This phenotype was observed first by one of us (G. H.) in a family from Regensburg, Bavaria. Family studies indicate that the phenotype is due to an additional allele at the ADA locus called ADA7. Materials and Methods Eight members of four generations of a family from Regensburg, Bavaria, were examined. Hemolysates were prepared from blood samples obtained by venous puncture. ADA et al. [8]. phenotypes were examined by starch gel electrophoresis according to SPENCER Received: December 2, 1974; accepted: December 12, 1974.
BERGet al.
218
-I
ADA1
ADA 2-1
ADA 7-1
ADA 7-1
ADA 7-1
ADA 7-1
A
B
C
D
E
F
ADA2
Start G
Fig. 1. Starch gel electrophoreticpattern of human erythrocyte adenosine deaminase. Common phenotypes ADA 1 (A), ADA 2-1 (B), and ADA 2 (G) are compared with a new phenotype ADA 7-1 (C). Changes of pattern during storage are shown after 1 (D), 2 (E), and 3 weeks (F).
A
B
C
D
E
F
G
Fig. 2. Starch gel photograph of the above-mentioned isozyme patterns in the variant phenotype. The figure has been composed from gel strips from different electrophoretic runs.
Results and Discussion
In four family members from four generations a new ADA phenotype was observed (fig. 1). The new phenotype resembled the ADA 2-1 type: the fresh hemolysate (fig. 1,C) showed four bands of decreasing intensity. However, the slowest and most intense band was located more towards the starting point than the slower band of the 2-1 type. The band appeared,
ADA'
-
A New Allele
112 ADA 7-1
111 Not t e s t e d
1112 ADA 7-1
1111 Not t e s t e d 11111 ADA 1
11112 ADA 7-1
IVI1 ADAl
IV12 ADAl
219
Not t e s t e d
11113 ADA 1
IV13 ADA7-1
Fig. 3. ADA phenotypes in a family from Regensburg, Bavaria.
furthermore, to be more intensely stained than the corresponding band of the 2-1 type. It was also observed that the cathodal edge of the second slower band was blurred. Characteristic changes of pattern were observed after storage of hemolysates at 4°C (fig. 1, D-F). The changes included decreasing intensity of the slower band and increasing intensity of the two faster bands. The second slower band was converted into a double-banded pattern, until after 3 weeks of storage a single, but slower moving second band was noted (fig. 1 m . ADA phenotypes were determined also after storage of hemolysates for 3 weeks and preincubation with 10 mM 2-mercaptoethanol for 30 min. prior to electrophoresis. As observed by SPENCER et al. [8] in the ADA 1 and ADA 2-1 phenotypes, the changes of pattern occuring after storage of samples with the new ADA phenotype were completely reversed by preincubation with 2-mercaptoethanol. It may therefore be concluded that the new ADA phenotype does not directly involve the free -SH group of the isozymes demonstrated by HOPKINSON and HARRIS[5]. The particular susceptibility to phenotypic changes after storage indicates perhaps anincreased reactivity of the free -SH group in the mutant type. The results of the family studies are presented in figure 3. The new phenotype was observed in members of four generations: I/2, 11/2, III/2 and IV/3. The results are in agreement with the assumption of an additional allele at the ADA locus which we propose to call ADA'. The observed phenotype can thus be classified as ADA 7-1.
220
BERGet al.
Rejerences 1 BRINKMANN, B. ; BRINKMANN, M., and MARTIN,H.: A new allele in red cell adenosine deaminase polymorphism: ADA'. Hum. Hered. 23 :603-607 (1973). E. R.: Adenosine deaminase: demonstration 2 CHEN,S. H.; SCOTT,C. R., and GIBLETT, of a 'silent' gene associated with combined immunodeficiency disease. Am. J. hum. Genet. 26:103-107 (1974). 3 DETTER,J. C.; STAMATOYANNOPOULOS, G.; GIBLETT, E. R., and MOTULSKY, A. G.: Adenosine deaminase: racial distribution and report of a new phenotype. J. med. Genet. 7: 356 (1970). 4 DISSING,J. and KNUDSEN, J. B.: A new red cell adenosine deaminase phenotype in man. Hum. Hered. I9:375-377 (1969). 5 HOPKINSON, D. A. and HARRIS, H. : The investigation of reactive sulphydryls in enzymes and their variants by starch gel electrophoresis. Studies on red cell adenosine deaminase. Ann. hum. Genet. 3.3~81-87(1969). G. ; STRAUCH, H. und PROKOP,0.: Ein seltener Phanotyp im Adenosindesam6 RADAM, inase-Polymorphismus: Hinweis auf die Existenz eines neuen Allels. Humangenetik 25 :247-250 (1974). 7 RADAM, G.; STRAUCH, H. und VAVRUSA, B.: Zur Differenzierung der Varianten 5-1 und 6-1 im Adenosindesaminase-Polymorphismus. Nachweis des neuen Phanotyps ADA 5 2 in der CSSR. (in press). 8 SPENCER, N. ; HOPKINSON, D. A., and HARRIS, H. : Adenosine deaminase polymorphism in man. Ann. hum. Genet. 32:9-14 (1968).
Dr. Dr. HANSWISCHERATH, Institut fur Anthropologie und Humangenetik der Universitit Miinchen, Richard-Wagner-Str. lo1, 0-8000Munchen 2 (FRG)
