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Typing of the Common Phosphoglucomutase Variants Using Isoelectric Focusing—A New Interpretation of the Phosphoglucomutase System
J . Forens. Sci. Sac. (1976), 16, 115
Typing of the Common Phosphoglucomutase Variants Using Isoelectric Focusing-A New Interpretation of the Phosphoglucomutase System J. E. BARK, M. J. HARRIS and M. FIRTH Home Ofice Forensic Science Laboratory, Washington Hall, Euxton, Charley, Lancashire, England PR7 6HJ. Ten new variants have been demonstrated in erythrocyte phosphoglucomutase using isoelectric focusing over a pH range 5-7 in polyacrylamide gel. A genetic explanation for thesejindings is proposed. A survey has been carried out on 123 blood samples and the frequencies of these variants are given. Bloodstains prepared from liquid blood samples have been typed successfully in the same way.
Introduction Polymorphism in enzymes and other proteins has generally been observed by using standard electrophoretic techniques to separate structurally related variants in the enzyme or protein. Genetic information on these enzymes and proteins has then been determined on the premise that resolution of these variants was complete. Methods of blood typing in the polymorphic phosphoglucomutase (PGM) system have employed both starch-gel electrophoresis and isoelectric focusing. The currently accepted first locus phenotypes PGM,l, PGM,2 and PGM12-1 were first separated by Spencer et al. (1964) using starch-gel electrophoresis. A sensitive thin layer technique suitable for typing bloodstains was subsequently developed for forensic purposes by Culliford (1967) and Wraxall and Culliford (1968). Since the development of isoelectric focusing using thin layer polyacrylamide gels, largely resulting from the system devised by Awdeh et al. (1968) and refined by Vesterberg (1973), there have been very few investigations of the PGM system using this technique. Ishimoto and Kuwata (1972) used isoelectric focusing at low voltage and confirmed the three PGM, phenotypes although they obtained more bands than are found with starch gel methods. They suggested that extra bands produced by isoelectric focusing could be the result of a complex interaction between the enzyme and the ampholytes in the gel. Additional bands were also observed by Burdett and Whitehead (in press). This present investigation was aimed at determining the advantage to be gained by employing isoelectric focusing techniques in forensic science, and exploring the possibility of achieving further discrimination within the PGM system.
Method The gel is prepared as follows: 60ml of a solution 5.25% acrylamide 0.25% bisacrylamide (BDH) in distilled water. 3ml Arnpholine pH5-7 range 40°/, w/v (LKB). 0-5ml riboflavin lOmg% (BDH) in distilled water. 7.5g sucrose. The mixture is degassed under vacuum and transferred to the mould by pipette. The gel is formed in a mould constructed from two glass plates separated at
the edges by a silicone rubber gasket. The first plate which bears the gel measures 230mm x 140mm x lmm and the second plate 230mm x 140mm x 2mm acts as a cover. The gasket, which is 5mm wide and 2mm thick, is cut across at one point so that it can be opened sufficiently to allow the introduction of the acrylamide mixture. A further plate 230mm x 140mm x 2mm may be used behind the Imm plate to strengthen the mould. The whole mould is held together with large "bulldog" spring clips. After filling the mould the gasket is pushed into place excluding any air. The mould is placed on a cold light viewer and pl~otopolymerisationof the gel is achieved in 2 hours. The gels may be conveniently stored in the mould. When the gel is required for use, the clips are released and the gasket, backing and cover plates carefully removed, leaving the gel on the lmm thick plate. The isoelectric focusing apparatus consists of a glass cooling plate in a perspex tank, the lid of which has two platinum electrodes 100mm apart. These electrodes are so positioned that they can make contact across the gel when the lid is in place. The lmm glass plate bearing the gel rests on the cooling plate. Good thermal contact can be achieved with a layer of paraffin between the cooling plate and gel plate. Blood samples-lysed red cells or stains dissolved in a minimum of distilled water-are applied to the surface of the gel on Whatman 3MM paper strips 5mm x 3mm. Larger strips up to lOmm x 5mm may be used for weak extracts. The sample strips are applied between the centre of the plate and the anodic end with the 5mm dimension placed parallel to thc electrodes. Four layers of 1Omm wide Whatman 3MM paper soaked in 1% aqueous ethanolamine are applied at the cathodic edge of the gel and cut to the same length as the gel. 1% aqueous acetic acid is applied in the same manner to the anodic edge of the gel. With the lid in place the electrodes make contact with these solutions. Initial current is restricted to 18mA, the voltage being increased to a maximum of 1250 volts as the current falls. The sample strips can be removed approximately 90 minutes after the start of a run. Final focusing occurs some 2-3 hours after this. An indication of completion of a run can be obtained by applying a sample at both anodic and cathodic ends of a gel-when stained the bands of each should be aligned. Sufficient reaction mixture (Culliford 1967) is made up in 0.3M Tris/HCl buffer at pH8.0. The gel is incubated at 37OC until bands appear-normally in 15-60 minutes. Results and Discussion One hundred and fifty liquid blood samples of the three common PGM, types as determined by starch electrophoresis were typed by the above method. Our results show that from the three types ten different patterns emerge. These patterns are reproducible over a period of time for each particular blood sample and for blood samples taken at different times from the same individual. Their appearance is not altered by prior treatment of the sample with 2-mercaptoethanol and dithiothreitol. Seventy laboratory prepared bloodstains, from two to four weeks old, gave patterns corresponding to those exhibited by their respective liquid blood samples. Combining blood samples of different PGM types yielded a mixed pattern in which the isozyme bands from each sample focused independently of each other. This enabled a control sample to be prepared containing all of the bands so far observed. The three main phenotypes of erythrocyte PGM observed by starch-gel electrophoresis PGM,l , PGM, 2 - 1 and PGM, 2 have been attributed to the occurrence of two common alleles PGM: and PGM: at the PGM, locus (Spencer et al. 1964). The homozygous genotypes PGM; PGM: and PGM: PGM: were considered to givc rise to phenotypes PGM, 1 and PGMl 2
respectively. The heterozygous genotype PGM? PGM: was considered to give rise to the phenotype PGM, 2-1. Using the technique of isoelectric focusing it is apparent that the PGM polymorphism is different from that observed by starch-gel electrophoresis. This difference is seen within the four isozyme bands which we have called 1-, 1 2 - and 2 focusing at high pH in the gel (Figures 1 and 2). Ten new t v ~ e sare identified which have either one or two of these four desi~nated " bands. For each of these four isozyme bands there are further characteristic bands focusing at lower pH, assisting in their correct assignment. All the evidence is consistent with this new polymorphism having genetic origin. The PGM, 1 phenotype as determined by starch-gel electrophoresis is observed to occur as three phenotypes by isoelectric focusing which are now designated PGM, 1 PGM, 1 - and PGM, 1 1 -. The PGM, 2 phenotype is observed to occur also as three phenotypes which are now designated PGM, 2 PGM, 2- and PGM, 2 +2 -. The PGM, 2-1 phenotype is observed to occur as four phenotypes which are now - designated PGM, 2 1 PGM, 2 1 -, PGM, 2- 1 and PGM, 2-1-. These ten new phenotypes of PGM can be explained on the basis of the occurrence of four common alleles PGM:+, PGM:-, PGM:+ and PGM:- at the PGM, locus. If the a, b, c and d bands seen on starch-gel electrophoresis at pH7.4 represent a series of diffcrently charged isozyrrles as suggested for rabbit muscle PGM (Green and Dawson 1973), it is probable that the 1 and 1- bands a n d the 2+ and 2- bands by isoelectric focusing result from splitting the a and b bands respectively. Some of the bands focusing at lower pH would then result from the c and d bands of starch-gel electrophoresis. This is consistent with observations made so far. We intend to clarify this situation by further investigation. --.--. The frequency of occurrence of the ten new phenotypes of PGM based on a survey of 123 blood samples is given in Table 1. Although this survey is relatively
+,
+
/ 1
+
+,
+, + +,
+
+
+
Figure 1. Five PGM isozyme patterns from haemolysates after isoelectric focusing. a and f PGM 2 + I + , b PGM 2 + 2 + , c and i control mixture (2+ l+/2- I-), d and g PGM 1+ I + , e PGM 1+ 1-, h PGM 2- 1-. 117
TABLE 1 FREQUENCY OF PGM PHENOTYPES Old Phenotype
Observed Frequency % *
1
55
* Spencer et al.,
New PhenotyPe 1+ 1+ 1-
1+ 11-
Observed Number 48 17 2
Observed Expected Frequency % Frequency % 39.0 13.8 1.6
40.2 14.3 1.3
1964.
small good agreement is obtained between expected and observed frequencies. The phenotype PGM, 2 .-, although observed previously, was not encountered in the survey. Additional support for the genetic explanation of the new polymorphism has been provided by several family studies, one of which is given in Figure 3. I t can be seen from this example that whereas three members of this family would be typed as PGM, 2-1 by starch electrophoresis, isoelectric focusing gives complete discrimination between all members of the family. The discriminating power of the PGhl system in blood has been increased from 0.56 for three phenotypes observed by starch electrophoresis to 0.74 for the ten phenotypes observed by isoelectric focusing. This means that out of 200 blood samples randomly paired, 44 pairs match and 56 are discriminated using starch electrophoresis whilst 26 pairs match and 74 are discriminated by isoelectric focusing. Furthermore, approximately 20% of the population now possess one of seven relatively rare phenotypes (frequency 7%). This is of great statistical value when these phenotypes are encountered in bloodstains. Using isoelectric focusing, phenotyping of PGM and other systems can be accomplished in three to four hours. The technique concentrates as it focuses the components of a bloodstain and therefore some stains that are too diffuse for starch electrophoresis may be successfully typed. Simultaneous separations of several polymorphic systems have been accomplished using isoelectric focusing (Burdett and Whitehead, in press) and promise to reduce the real cost of the
<
Figure 3. Diagram of the PGM phenotypes observed in one family.
system whilst economising in time. I n our opinion, the finding of additional PGM phenotypes and the other advantages over starch electrophoresis render isoelectric focusing an invaluable aid to the forensic biologist. References AWDEH,Z. L., WILLIAMSON, A. R., and ASKONAS, B. A., 1968, Nature, 219, 66. P. E., and WHITEHEAD, P. H., Anal. Biochem., (in press). BURDETT, CULLIFORD, B. J., 1967, J. Forens. Sci. Soc., 7, 131. GREEN, J. M., and DAWSON, D. M., 1973, Ann. Hum. Genet. Lond., 36, 355. ISHIMOTO, G., and KUWATA, M., 1972, Reports of the National Research Imtitute of Police Science, Japan, 25, No. 4, 13. SPENCER, N., HOPKINSON, D. A., and HARRIS,H., 1964, Nature, 204, 742. B. G. D., and CULLIFORD, B. J., 1968, J. Forens. Sci. Soc., 8, 81. WRAXALL, VESTERBERG, O., 1973, Science Tools, 20, 22.
Typing of the Common Phosphoglucomutase Variants Using Isoelectric Focusing-A New Interpretation of the Phosphoglucomutase System J. E. BARK, M. J. HARRIS and M. FIRTH Home Ofice Forensic Science Laboratory, Washington Hall, Euxton, Charley, Lancashire, England PR7 6HJ. Ten new variants have been demonstrated in erythrocyte phosphoglucomutase using isoelectric focusing over a pH range 5-7 in polyacrylamide gel. A genetic explanation for thesejindings is proposed. A survey has been carried out on 123 blood samples and the frequencies of these variants are given. Bloodstains prepared from liquid blood samples have been typed successfully in the same way.
Introduction Polymorphism in enzymes and other proteins has generally been observed by using standard electrophoretic techniques to separate structurally related variants in the enzyme or protein. Genetic information on these enzymes and proteins has then been determined on the premise that resolution of these variants was complete. Methods of blood typing in the polymorphic phosphoglucomutase (PGM) system have employed both starch-gel electrophoresis and isoelectric focusing. The currently accepted first locus phenotypes PGM,l, PGM,2 and PGM12-1 were first separated by Spencer et al. (1964) using starch-gel electrophoresis. A sensitive thin layer technique suitable for typing bloodstains was subsequently developed for forensic purposes by Culliford (1967) and Wraxall and Culliford (1968). Since the development of isoelectric focusing using thin layer polyacrylamide gels, largely resulting from the system devised by Awdeh et al. (1968) and refined by Vesterberg (1973), there have been very few investigations of the PGM system using this technique. Ishimoto and Kuwata (1972) used isoelectric focusing at low voltage and confirmed the three PGM, phenotypes although they obtained more bands than are found with starch gel methods. They suggested that extra bands produced by isoelectric focusing could be the result of a complex interaction between the enzyme and the ampholytes in the gel. Additional bands were also observed by Burdett and Whitehead (in press). This present investigation was aimed at determining the advantage to be gained by employing isoelectric focusing techniques in forensic science, and exploring the possibility of achieving further discrimination within the PGM system.
Method The gel is prepared as follows: 60ml of a solution 5.25% acrylamide 0.25% bisacrylamide (BDH) in distilled water. 3ml Arnpholine pH5-7 range 40°/, w/v (LKB). 0-5ml riboflavin lOmg% (BDH) in distilled water. 7.5g sucrose. The mixture is degassed under vacuum and transferred to the mould by pipette. The gel is formed in a mould constructed from two glass plates separated at
the edges by a silicone rubber gasket. The first plate which bears the gel measures 230mm x 140mm x lmm and the second plate 230mm x 140mm x 2mm acts as a cover. The gasket, which is 5mm wide and 2mm thick, is cut across at one point so that it can be opened sufficiently to allow the introduction of the acrylamide mixture. A further plate 230mm x 140mm x 2mm may be used behind the Imm plate to strengthen the mould. The whole mould is held together with large "bulldog" spring clips. After filling the mould the gasket is pushed into place excluding any air. The mould is placed on a cold light viewer and pl~otopolymerisationof the gel is achieved in 2 hours. The gels may be conveniently stored in the mould. When the gel is required for use, the clips are released and the gasket, backing and cover plates carefully removed, leaving the gel on the lmm thick plate. The isoelectric focusing apparatus consists of a glass cooling plate in a perspex tank, the lid of which has two platinum electrodes 100mm apart. These electrodes are so positioned that they can make contact across the gel when the lid is in place. The lmm glass plate bearing the gel rests on the cooling plate. Good thermal contact can be achieved with a layer of paraffin between the cooling plate and gel plate. Blood samples-lysed red cells or stains dissolved in a minimum of distilled water-are applied to the surface of the gel on Whatman 3MM paper strips 5mm x 3mm. Larger strips up to lOmm x 5mm may be used for weak extracts. The sample strips are applied between the centre of the plate and the anodic end with the 5mm dimension placed parallel to thc electrodes. Four layers of 1Omm wide Whatman 3MM paper soaked in 1% aqueous ethanolamine are applied at the cathodic edge of the gel and cut to the same length as the gel. 1% aqueous acetic acid is applied in the same manner to the anodic edge of the gel. With the lid in place the electrodes make contact with these solutions. Initial current is restricted to 18mA, the voltage being increased to a maximum of 1250 volts as the current falls. The sample strips can be removed approximately 90 minutes after the start of a run. Final focusing occurs some 2-3 hours after this. An indication of completion of a run can be obtained by applying a sample at both anodic and cathodic ends of a gel-when stained the bands of each should be aligned. Sufficient reaction mixture (Culliford 1967) is made up in 0.3M Tris/HCl buffer at pH8.0. The gel is incubated at 37OC until bands appear-normally in 15-60 minutes. Results and Discussion One hundred and fifty liquid blood samples of the three common PGM, types as determined by starch electrophoresis were typed by the above method. Our results show that from the three types ten different patterns emerge. These patterns are reproducible over a period of time for each particular blood sample and for blood samples taken at different times from the same individual. Their appearance is not altered by prior treatment of the sample with 2-mercaptoethanol and dithiothreitol. Seventy laboratory prepared bloodstains, from two to four weeks old, gave patterns corresponding to those exhibited by their respective liquid blood samples. Combining blood samples of different PGM types yielded a mixed pattern in which the isozyme bands from each sample focused independently of each other. This enabled a control sample to be prepared containing all of the bands so far observed. The three main phenotypes of erythrocyte PGM observed by starch-gel electrophoresis PGM,l , PGM, 2 - 1 and PGM, 2 have been attributed to the occurrence of two common alleles PGM: and PGM: at the PGM, locus (Spencer et al. 1964). The homozygous genotypes PGM; PGM: and PGM: PGM: were considered to givc rise to phenotypes PGM, 1 and PGMl 2
respectively. The heterozygous genotype PGM? PGM: was considered to give rise to the phenotype PGM, 2-1. Using the technique of isoelectric focusing it is apparent that the PGM polymorphism is different from that observed by starch-gel electrophoresis. This difference is seen within the four isozyme bands which we have called 1-, 1 2 - and 2 focusing at high pH in the gel (Figures 1 and 2). Ten new t v ~ e sare identified which have either one or two of these four desi~nated " bands. For each of these four isozyme bands there are further characteristic bands focusing at lower pH, assisting in their correct assignment. All the evidence is consistent with this new polymorphism having genetic origin. The PGM, 1 phenotype as determined by starch-gel electrophoresis is observed to occur as three phenotypes by isoelectric focusing which are now designated PGM, 1 PGM, 1 - and PGM, 1 1 -. The PGM, 2 phenotype is observed to occur also as three phenotypes which are now designated PGM, 2 PGM, 2- and PGM, 2 +2 -. The PGM, 2-1 phenotype is observed to occur as four phenotypes which are now - designated PGM, 2 1 PGM, 2 1 -, PGM, 2- 1 and PGM, 2-1-. These ten new phenotypes of PGM can be explained on the basis of the occurrence of four common alleles PGM:+, PGM:-, PGM:+ and PGM:- at the PGM, locus. If the a, b, c and d bands seen on starch-gel electrophoresis at pH7.4 represent a series of diffcrently charged isozyrrles as suggested for rabbit muscle PGM (Green and Dawson 1973), it is probable that the 1 and 1- bands a n d the 2+ and 2- bands by isoelectric focusing result from splitting the a and b bands respectively. Some of the bands focusing at lower pH would then result from the c and d bands of starch-gel electrophoresis. This is consistent with observations made so far. We intend to clarify this situation by further investigation. --.--. The frequency of occurrence of the ten new phenotypes of PGM based on a survey of 123 blood samples is given in Table 1. Although this survey is relatively
+,
+
/ 1
+
+,
+, + +,
+
+
+
Figure 1. Five PGM isozyme patterns from haemolysates after isoelectric focusing. a and f PGM 2 + I + , b PGM 2 + 2 + , c and i control mixture (2+ l+/2- I-), d and g PGM 1+ I + , e PGM 1+ 1-, h PGM 2- 1-. 117
TABLE 1 FREQUENCY OF PGM PHENOTYPES Old Phenotype
Observed Frequency % *
1
55
* Spencer et al.,
New PhenotyPe 1+ 1+ 1-
1+ 11-
Observed Number 48 17 2
Observed Expected Frequency % Frequency % 39.0 13.8 1.6
40.2 14.3 1.3
1964.
small good agreement is obtained between expected and observed frequencies. The phenotype PGM, 2 .-, although observed previously, was not encountered in the survey. Additional support for the genetic explanation of the new polymorphism has been provided by several family studies, one of which is given in Figure 3. I t can be seen from this example that whereas three members of this family would be typed as PGM, 2-1 by starch electrophoresis, isoelectric focusing gives complete discrimination between all members of the family. The discriminating power of the PGhl system in blood has been increased from 0.56 for three phenotypes observed by starch electrophoresis to 0.74 for the ten phenotypes observed by isoelectric focusing. This means that out of 200 blood samples randomly paired, 44 pairs match and 56 are discriminated using starch electrophoresis whilst 26 pairs match and 74 are discriminated by isoelectric focusing. Furthermore, approximately 20% of the population now possess one of seven relatively rare phenotypes (frequency 7%). This is of great statistical value when these phenotypes are encountered in bloodstains. Using isoelectric focusing, phenotyping of PGM and other systems can be accomplished in three to four hours. The technique concentrates as it focuses the components of a bloodstain and therefore some stains that are too diffuse for starch electrophoresis may be successfully typed. Simultaneous separations of several polymorphic systems have been accomplished using isoelectric focusing (Burdett and Whitehead, in press) and promise to reduce the real cost of the
<
Figure 3. Diagram of the PGM phenotypes observed in one family.
system whilst economising in time. I n our opinion, the finding of additional PGM phenotypes and the other advantages over starch electrophoresis render isoelectric focusing an invaluable aid to the forensic biologist. References AWDEH,Z. L., WILLIAMSON, A. R., and ASKONAS, B. A., 1968, Nature, 219, 66. P. E., and WHITEHEAD, P. H., Anal. Biochem., (in press). BURDETT, CULLIFORD, B. J., 1967, J. Forens. Sci. Soc., 7, 131. GREEN, J. M., and DAWSON, D. M., 1973, Ann. Hum. Genet. Lond., 36, 355. ISHIMOTO, G., and KUWATA, M., 1972, Reports of the National Research Imtitute of Police Science, Japan, 25, No. 4, 13. SPENCER, N., HOPKINSON, D. A., and HARRIS,H., 1964, Nature, 204, 742. B. G. D., and CULLIFORD, B. J., 1968, J. Forens. Sci. Soc., 8, 81. WRAXALL, VESTERBERG, O., 1973, Science Tools, 20, 22.