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The Determination of Carbonic Anhydrase II (CA-II) Types in Human Bloodstains
The Determination of Carbonic Anhydrase I1 JCA-II) Types in Human Bloodstains K. M. HUGHES .\letropolitan Police Forensic Science Laboratory, 109 Lambeth Road, London S E I 7LP T h i s paper describes a method of typing carbonic anhydrase II (CA-II) isozymes iri human bloodstains by electrophoresis using a thin layer of starch gel. Stains up to four weeks old can be typed successfully and the method is now used routinely in the Metropolitan Police Forensic Science Laboratory. T h e variant allele C A - I I h a s found to occur with a frequency o f 0 . 0 6 7 in a population sample of negroes in South-East England. No variants were found in white skinned Europeans. A new, previously unreported variant has been found in two of the blood samples analysed. T h i s has been designated CA-II 3-1. (Rccrived 18,Junr 1977; Accepted 27 .July 1977)
Introduction Carbonic Anhydrase is known to occur in llurnan erythrocytes in two forms, CA-I and CA-11. Genetic variants of CA-I have been identified (Shaw et al., 1962, Tashian et al., 1963) but their frequency of occurrence is too low for forensic purposes. Moore, Funakoslii and Deutsch (1971) using a specific antiserum, differentiated CA-I1 from CA-I after electrophoresis. They discovered a polymorphism of CA-I1 in USA Blacks, but because of the high cost of the specific antiserum no extensive work was carried out. Recently an inexpensive method wa\ described (Hopkinson, Coppock, Muhlemann and Edwards, 1974) using fluorescein diacetate which is hydrolysed rapidly by CA-I1 hut very slowly by CA-I. This method has been adapted so that CA-I1 can be typed from the small amounts of Ijloodstained material associated with forcnsic work. Method A 10%)starch gel, Imm thick, was prepared using the method of Wraxall and Culliford ( 1968). Buffers Stock solutior~ 0.9M l'ris, O.5M Boric Acid, 0 4 2 M EDTA, pH8.5. Bridge Buffer 1 :I4 dilution of stock solution with distilled water. Gel Buffcl 1 :40 dilution of stock solution with distilled water. Cotton thrcads 0.8cm long were soaked in lysate and inserted into the gel 6cin from the cathode. Threads of bloodstained material were cut, soaked in thr minimum quantity of gel buffer and applied in the same way. Bloodstained material that could not be cut was swabbed onto cotton threads soaked in gel buffer. Electrophoresis was carried out at 12V/cm for 4 hours on cooling plates at 6-8'6. I,ocnlion of Carbonic Anhydrase 11
Figure 1 .
The electrophoretic patterns of four CA-I1 phenotypes in lysates.
Results The three commonest phenotypes of carbonic anhydrase I1 are CA-I1 1, CA-I1 2 and the heterozygote CA-I1 2-1 (Figure 1 ) . Of these CA-I1 1 consists of a single fluorescent band on the cathode side of the origin, CA-I1 2 is distinguished by a single band on the anode side of the origin, and CA-I1 2-1 has both bands present in equal intensity. If the plate was incubated for longer than one hour another weakly fluorescing band could be seen which had an electrophoretic mobility slightly more anodal than the CA-I1 2 band (Figure 2). This was shown to be the CA-I 1 isozyme, as development with 4-Me-umbelliferyl acetate gave very strong fluorescence in the same position (Hopkinson et al., 1974). The presence of this band however should not cause serious problems in confusing a CA-I1 1 with a type 2-1 if it
-
I I
0 0 0 I Hb
n II II II
U
I
I II I I I I I I 0 Figure 2 .
2-1 2 I+ CAI band
1
3-1 1
is remembered that the 2-1 type has bands of equal intensity. Normally the CA-I1 isozymes can be read before the CA-I 1 band starts to appear. A previously unreported variant has been found in two of the blood samples analysed. This consists of a band in the 1 position and another band in a more cathodal position. These components were still present after treatment with either dithiothreitol, mercaptoethanol or oxidised glutathione. Bloodstains made from these samples gave the same band pattern as the liquid blood. Although follow up family studies were not possible, this variant was tentatively designated CA-I1 3-1. Bloodstains The same band patterns were observed in corresponding dried stains (Figure 3). The intensities of these bands decreased with age although it was
Figure 3.
CA-I1 types in bloodstains.
found that stains up to four weeks old could be typed successfully. So far the technique has been used to type stains prepared in the laboratory using a variety of substrates commonly met in casework. The stains were made using liquid blood of known CA-I1 type by one person and then grouped by another with no knowledge of the bloods involved. This was followed by typing of bloodstains in case material where there could be little doubt, from other grouping systems, that the blood involved was that of the victim and no one else (Table 1 ) . TABLE 1 TABLE OF LABORATORY TRIAL RESULTS Total Laboratory Trials Casework Trials
75 50
Correct 75
32
Incorrect
0 0
Negative 0 18
No additional bands were formed on storage of the bloodstains and in a type 2-1 both bands disappeared at the same rate. T o date, 706 blood samples have been typed for CA-11. The bloods are those from suspects and victims of violent crime in South-East England. The frequencies of the CA-I1 phenotypes in various racial types are shown in Table 2.
FREQUENCY OF CA-I1 PHENOTYPES AND ESTIMATES OF CA-11' AND CA-ll' GENE FREQIJENCIES IN A POPULATION OF DIFFERENT RACIAL TYPES IN SOUTII-EAST ENGI.ANI) Estimated Popidlation Sample
CA-ZI 2
Gene Frpquenq) (:A-111 (,'A- I12
White Skinned European (Total = 128) Ob~ervedFrequency E.xpected Frequency Dark Skinned European (Total = 72) Obserued Frequency Expected Frequency Negroid (Total = 3 15) 0baen.erl Frequency Expected Frequency Indian (Total = 138) Observed Frequency Expected Frequency Oriental (Total = 20) Obsvrr ed Frequenry Exf~ectedFrequenry nrab and Egyptian (Tuial = 35) Jbserixd Frequenry Expected Frequency
References
HOPKINSON, D. A., COPPOCK, J. S., MUHLEMANN, M. F. and EDWARDS, Y. H . , 1974, Ann. Hum. Genet., 38, 155-162. M. J., FUNAKOSHI, S. and D E U T S ~H. H ,F., 1971, Riochern. Genet., 5,497. MOORE, SHAW,C:. K., SYNER,F. N. and TASHIAN, K. E., 1962, ,Sci~nce,138, 31. TASHIAN, R. E., PLATO,C. C. and SHOWS, T. B., 1963, Science, 140, 53. TASHIAN, K. E., 1969, In Biochemical ivfethods in Red Cell Genetics, ed. J . .J. Yunis, pp. 307-336. New Yor-k, London: Academic Press. WRAXALL, B. G. D. and CULLIFORD, B. J., 1968, J . Foretu. Sci. Soc., 8 , 81-82.
Introduction Carbonic Anhydrase is known to occur in llurnan erythrocytes in two forms, CA-I and CA-11. Genetic variants of CA-I have been identified (Shaw et al., 1962, Tashian et al., 1963) but their frequency of occurrence is too low for forensic purposes. Moore, Funakoslii and Deutsch (1971) using a specific antiserum, differentiated CA-I1 from CA-I after electrophoresis. They discovered a polymorphism of CA-I1 in USA Blacks, but because of the high cost of the specific antiserum no extensive work was carried out. Recently an inexpensive method wa\ described (Hopkinson, Coppock, Muhlemann and Edwards, 1974) using fluorescein diacetate which is hydrolysed rapidly by CA-I1 hut very slowly by CA-I. This method has been adapted so that CA-I1 can be typed from the small amounts of Ijloodstained material associated with forcnsic work. Method A 10%)starch gel, Imm thick, was prepared using the method of Wraxall and Culliford ( 1968). Buffers Stock solutior~ 0.9M l'ris, O.5M Boric Acid, 0 4 2 M EDTA, pH8.5. Bridge Buffer 1 :I4 dilution of stock solution with distilled water. Gel Buffcl 1 :40 dilution of stock solution with distilled water. Cotton thrcads 0.8cm long were soaked in lysate and inserted into the gel 6cin from the cathode. Threads of bloodstained material were cut, soaked in thr minimum quantity of gel buffer and applied in the same way. Bloodstained material that could not be cut was swabbed onto cotton threads soaked in gel buffer. Electrophoresis was carried out at 12V/cm for 4 hours on cooling plates at 6-8'6. I,ocnlion of Carbonic Anhydrase 11
Figure 1 .
The electrophoretic patterns of four CA-I1 phenotypes in lysates.
Results The three commonest phenotypes of carbonic anhydrase I1 are CA-I1 1, CA-I1 2 and the heterozygote CA-I1 2-1 (Figure 1 ) . Of these CA-I1 1 consists of a single fluorescent band on the cathode side of the origin, CA-I1 2 is distinguished by a single band on the anode side of the origin, and CA-I1 2-1 has both bands present in equal intensity. If the plate was incubated for longer than one hour another weakly fluorescing band could be seen which had an electrophoretic mobility slightly more anodal than the CA-I1 2 band (Figure 2). This was shown to be the CA-I 1 isozyme, as development with 4-Me-umbelliferyl acetate gave very strong fluorescence in the same position (Hopkinson et al., 1974). The presence of this band however should not cause serious problems in confusing a CA-I1 1 with a type 2-1 if it
-
I I
0 0 0 I Hb
n II II II
U
I
I II I I I I I I 0 Figure 2 .
2-1 2 I+ CAI band
1
3-1 1
is remembered that the 2-1 type has bands of equal intensity. Normally the CA-I1 isozymes can be read before the CA-I 1 band starts to appear. A previously unreported variant has been found in two of the blood samples analysed. This consists of a band in the 1 position and another band in a more cathodal position. These components were still present after treatment with either dithiothreitol, mercaptoethanol or oxidised glutathione. Bloodstains made from these samples gave the same band pattern as the liquid blood. Although follow up family studies were not possible, this variant was tentatively designated CA-I1 3-1. Bloodstains The same band patterns were observed in corresponding dried stains (Figure 3). The intensities of these bands decreased with age although it was
Figure 3.
CA-I1 types in bloodstains.
found that stains up to four weeks old could be typed successfully. So far the technique has been used to type stains prepared in the laboratory using a variety of substrates commonly met in casework. The stains were made using liquid blood of known CA-I1 type by one person and then grouped by another with no knowledge of the bloods involved. This was followed by typing of bloodstains in case material where there could be little doubt, from other grouping systems, that the blood involved was that of the victim and no one else (Table 1 ) . TABLE 1 TABLE OF LABORATORY TRIAL RESULTS Total Laboratory Trials Casework Trials
75 50
Correct 75
32
Incorrect
0 0
Negative 0 18
No additional bands were formed on storage of the bloodstains and in a type 2-1 both bands disappeared at the same rate. T o date, 706 blood samples have been typed for CA-11. The bloods are those from suspects and victims of violent crime in South-East England. The frequencies of the CA-I1 phenotypes in various racial types are shown in Table 2.
FREQUENCY OF CA-I1 PHENOTYPES AND ESTIMATES OF CA-11' AND CA-ll' GENE FREQIJENCIES IN A POPULATION OF DIFFERENT RACIAL TYPES IN SOUTII-EAST ENGI.ANI) Estimated Popidlation Sample
CA-ZI 2
Gene Frpquenq) (:A-111 (,'A- I12
White Skinned European (Total = 128) Ob~ervedFrequency E.xpected Frequency Dark Skinned European (Total = 72) Obserued Frequency Expected Frequency Negroid (Total = 3 15) 0baen.erl Frequency Expected Frequency Indian (Total = 138) Observed Frequency Expected Frequency Oriental (Total = 20) Obsvrr ed Frequenry Exf~ectedFrequenry nrab and Egyptian (Tuial = 35) Jbserixd Frequenry Expected Frequency
References
HOPKINSON, D. A., COPPOCK, J. S., MUHLEMANN, M. F. and EDWARDS, Y. H . , 1974, Ann. Hum. Genet., 38, 155-162. M. J., FUNAKOSHI, S. and D E U T S ~H. H ,F., 1971, Riochern. Genet., 5,497. MOORE, SHAW,C:. K., SYNER,F. N. and TASHIAN, K. E., 1962, ,Sci~nce,138, 31. TASHIAN, R. E., PLATO,C. C. and SHOWS, T. B., 1963, Science, 140, 53. TASHIAN, K. E., 1969, In Biochemical ivfethods in Red Cell Genetics, ed. J . .J. Yunis, pp. 307-336. New Yor-k, London: Academic Press. WRAXALL, B. G. D. and CULLIFORD, B. J., 1968, J . Foretu. Sci. Soc., 8 , 81-82.