ANNALS OF HUMAN BIOLOGY, 1979, VOL. 6, NO. 3, 221--230
Phosphoglucomutase polymorphism detected by isoelectric focusing: gene frequencies, evolution and linkage N. D. CARTER and C. M. WEST Department of Child Health, St George's Hospital Medical School, London
E. E~aEs and B. PARKIN Ann Hum Biol Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
The Metropolitan Police Forensic Science Laboratory, London
and W. H. MARSHALL Faculty of Medicine, Memorial University of Newfoundland, Canada
Receit, ed 13 November 1978; revised 15 January 1979
Summary. Four alleles at the phosphoglucomutase (PGM 1) locus in man have been recently demonstrated by isoelectric focusing, and in this study gene frequencies have been estimated for four populations, and significant differences have been indicated between the results. Pedigree analysis of the PGM~ and rhesus loci confirm a weak linkage in males, but none in females. An examination of PGM~ phenotypes in a range of primates shows that a proportion possess a band focusing at the same isoelectric point as human PGM~ +. We propose that all the human and primate alleles may have evolved from a common ancestral PGM 11+ locus. 1.
Introduction Recent investigations of phosphoglucomutase (PGM1) phenotypes by isoelectric focusing have shown four common alleles at this locus (Bark, Harris and Firth 1976, Kiihnl, Schmidtmann and Spielmann 1977). Sutton and Burgess (1978) demonstrate tlSat these alleles are determined by one locus and are inherited in a Mendelian fashion. The present study compares frequencies of these four P G M 1 alleles in several human populations, confirming that they are related to the two common alleles previously described at the locus (Spencer, Hopkinson and Harris 1964). One of the populations examined was a community o f 1500 people in Newfoundland, from which a sample of 296 members of extended family groups was taken for P G M 1 phenotyping by isoelectric focusing. It was therefore possible to examine the inheritance of the four alleles. The information provided was also used to investigate linkage with the locus for the rhesus complex on chromosome 1. While a number of human lysates have now been typed for P G M by isoelectric focusing, the related non-human primate patterns have not been reported. Since this has a bearing on the evolution of this locus, we examined a number oflysates, from a range of primate species, on isoelectric focusing and starch gels for P G M zymograms. 2.
Materials and methods Red cell lysates were run on isoelectric focusing gels with an LKB Multiphor 2117 and LKB 2103 power supply, over a pH range of 5 7 formed by LKB 'Ampholytes', as described by Bark et al. (1976). Starch gel electrophoresis was run using the method of Spencer et al. (1964). Linkage analysis was as described by Maynard-Smith, Penrose and Smith (1961). A.H.B.
0301 4460/79/0(703 0221 $02"00 ~'~ I979 Taylor & Francis Ltd
Q
222
N . D . Carter et al.
Of the four populations studied, three were different ethnic groups living in London (Northern European, Negro, and Asian), from which blood samples were routinely obtained by the Metropolitan Police Forensic Laboratory. The fourth population was a genetically isolated group of about 1500 people in Newfoundland, being studied as part of an extensive health survey (Buehler, Firme, Fodor, Fraser, Marshall and Vase 1975). Primate bloods were supplied by the Wellcome Institute of the Zoological Society of London.
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3.
Results
Figure 1 shows diagrammatically the ten phenotypes of P G M 1 on isoelectric focusing and indicates how they are derived from the three phenotypes described for starch gel electrophoresis; the nomenclature is that introduced by Bark et al. (1976). Examples of all 10 phenotypes have been observed in the populations studied, with different frequencies for each of the four populations. Table 1 gives allele frequencies for the four populations; significant differences may be observed between these frequenAllele frequency Population N. European Newfoundland Negroes in London Asians in London
1-
1+
0.132 0.153 0-t26 0.096
0.627 0.537 0.672 0.537
2 0.058 0.076 0-027 0-121
2+
No.
0.182 0.233 0-173 0-246
2773 296 384 120
Table 1. Frequencies of the four human P G M ~ alleles determined by isoelectric focusing. 'Africans' include a random selection of people of African origin living in London; 'Asians' include a random selection of people of Indian origin who have immigrated to London from Asia and Africa.
i
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d
m
c
a
b
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m
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~-
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m mmm
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m
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m
m
n
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I
bands
m
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Figure 1. P G M 1 phenotypes on starch gel electrophoresis and isoelectric focusing. For clarity the isoelectric focusing diagram shows the separation of the 'a' and 'b' isozymes delineated on starch gel, ie. the primary P G M 1 gene products. The 'c' and 'd' bands on starch gel are thought to represent secondary post-translational components.
223
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lsoelectric Jocusing o f P G M 1
cies. Chi z tests comparing each of the populations show P < 0.001 for Europeans and Asians, Europeans and Negroes and Asians and Negroes; the Newfoundland population is included for comparison, although, as will be discussed below, it is not a randomly selected population. Kfihnl and Spielmann (1978) have quoted gene frequencies for the four P G M 1 alleles in a German population, and these agree closely with those given in table 1 for a Northern European population in London. Although allele frequencies are significantly different for each of the populations studied, all have a highest frequency of P G M I + and all show lower frequencies of the 1 - and 2 alleles. To compare the frequency data produced by isoelectric foc.using with information already published from starch gel electrophoresis (Spencer et al. 1964, Hopkinson and Harris 1966, Hopkinson and Harris 1968, Hopkinson and Harris 1969), the allele frequencies for P G M I + and P G M I - were combined (table 2). This gave a frequency for the P G M I allele very similar to frequencies ah'eady quoted for the populations under investigation. The isoelectric point of each P G M 1 isozyme band was measured by a surface pH electrode in 250 samples, the average result for each band was as follows: P G M 1 1 - pH 6.127, P G M 1 l + p H 6'075, PGM1 2 - p H 5"975 and PGM1 2 + p H 5-9.
Population
N. European Newfoundland Negroes in London Asians in London
PGMI~ gene t?equency detected by 1.E.F.
Published frequency(starch gel) 0'738 0"764
Spenceret al. (1964) Hopkinsonand Harris (1966)
0"74 0.767
Hopkinsonand Harris (1968) Hopkinsonand Harris (1969)
0.782 0.72
Hopkinsonand Harris (1966) Hopkinsonand Harris (1969)
0.759 0"69 0-798 0.633
Table 2. Summedhequenciesofthc PGM{ ~ and PGM{ allelescompared with published PGMI allele frequencies.
Family groups from the Newfoundland community were examined to confirm the Mendelian inheritance of the four alleles, and to provide linkage data with other gene loci on chromosome 1. Figure 2 gives one pedigree from the family showing autosomal codominant inheritance of the alleles: data from other family groups examined confirm this. Linkage has been reported (Robson, Cook, Corney, Hopkinson, Noades and Cleghorn 1973) between the loci for P G M 1 and the rhesus complex on Chromosome 1. Studies using isoelectric focusing to type individuals for P G M 1 show from L O D scores that linkage is present but weak in males; it could not however be confirmed in females. Table 3 gives LOD scores for all informative families, and recombination frequencies for phase known families. A male LOD peak of0-138 occurred at 0 = 0.3; in females the peak was 0.0 at 0 = 0.5. In four families the phase was known for female meioses, giving a recombination frequency of 53"8~o; in only one family was the phase known for male meioses, two of the three children showed recombination of the paternal haplotype. Q2
N.D. Carter et al.
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2-1-
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1-1-
2-1- ~ . . J
, .-,
2-1-
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Figure 2.
1-1-
Male Female
1 6
47 32
Table 3.
1+1-
2-1-
,, 2-1+
1+1-
L O D scores at recombination frequency 0
Total RecomNoninformative binants recombinants offspring 2 7
,-
Pedigree of a Newfoundland family.
Phase known Informative meioses
1+1-1+1+1+1+
0.05
0.1
0-15
0.2
0.25
0.3
0.35
0.4
-5,2 -2-35 -1-02 -0-33 0.01 0.14 0.13 0.06 -8.16 -3.96 -3.56 -2.13 -1.39 -0.86 -0.5 -0.24
0.45 -0,001 -0.08
Linkage data for P G M 1 and Rhesus alleles.
A range of primate species was screened for PGM 1 phenotypes, to determine whether isozyme patterns resembling one or more of the human allelic products occurred among them. Primate lysates were examined for PGM1 by both starch gel electrophoresis and isoelectric focusing, and isozyme patterns were compared with those from human lysates. All the Hominoidea studied (Pan(7), Gorilla(4) and Pongo(3)) showed an isozyme pattern identical to the human PGM ~pattern on Starch gel electrophoresis (figures 3(a) and 3(b)) and to human PGMI + on isoelectric focusing (figure 3(c)). Among old world monkeys, Presbytis(1), Cercocebus(1) and Macaca mulatta(8) all showed the same pattern, identical to human PGMI +, but Papio anubis(1) and Papio papio(1) gave different patterns with cathodal isozyme bands. Among new world monkeys Callithrix(4) showed an isozyme pattern identical to human PGMI +, but Saimiri(1) and Aotus(2) gave completely different patterns. The isozyme pattern in the lemur showed a band cathodal to human PGMI -. (Figures in brackets indicate the number of each species examined.) Where more than one sample from a species was available, PGM isozymes observed were identical; however intraspecific variation has been reported for Pan, Gorilla and Macaca iris (Schmitt, Lichte and Fuhrrnann 1970, Goodman and Tashian 1969, Barnicot and Cohen 1970); each was found to possess additional alleles, with different electrophoretic mobilities from common human phenotypes. 4.
Discussion The results from electrophoresis and isoelectric focusing of primate bloods suggest that the PGM~ + allele, or one identical to it in electrophoretic mobility and isoelectric point, is common to man and a number of higher primates.
Isoelectric Jocusing of PGM 1
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Figure 3 (a)
Origin
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N . D . Carter et al.
+ m m
m mm
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Figure 3 (b)
lsoelectric focusing
of PGM~
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Figure 3 (e)
~'igure 3. Primate P G M phenotypes. (a) Starch gel electrophoresis, l, H u m a n 2 1; 2, Presbytis; 3, H u m a n 2 - 1; 4, Aotus; 5, Lemur; 6, H u m a n 2-1; 7, Pongo; 8, Pongo; 9, H u m a n 2 1; 10, Gorilla; 11, Gorilla,• 12, H u m a n :2 1. (a') Block d i a g r a m of figure 3(a). (b) Starch gel electrophoresis. 1, t t u m a n 2 -- 1, 2, Papio pupio: 3, CalIithrix, 4, H u m a n 2 - 1; 5, Saimiri sciureus: 6, Papio anubis; 7, H u m a n 2 1; 8, Pan; 9, Macaca mulat tel; 10, H u m a n 2 1. (h') Block diagram of figure 3(b). (c) isoelectric focusing• 1, H u m a n 2 + 1 .- ; 2, Papio anubis; 3, Saimiri seiureus: 4, H u m a n 2 - 1 + ; 5, Callithrix; 6, Papio papio; 7, H u m a n 2 + 1 - ; 8, Gorilla; 9, Gorilla; 10, H u m a n 2 ,- I + ; 11, Ponqo; 12, Pongo; 13, t l u m a n 2 + 1 - ; 14 Lemur; 15, Aotus;16, H m n a n 2 1 + . (c') Block d i a g r a m of m a i n bands in figure 3 (c), for ease of interpretation only b a n d s in the zone between h u m a n P G M 1z ~ and P G M ] and cathodal to this zone are indicated in all 'lanes' of this gel. K n o w n h u m a n P G M 1 allelic products are indicated with a white spot.
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Despite the differences in gene frequencies between the four human populations in this study, all show similarity, having P G M ~+ as the most frequent allele, and all except the Asian population have PGM~z- as the least frequent allele. The high frequency of the P G M ~+ allele could relate to evidence from primate studies that this is an ancestral allele in man, from which the other three common alleles, as well as rarer alleles have evolved. I f P G M I + is taken as the ancestral P G M 1 gene there are several different methods by which the other alleles could have evolved; these may be considered in two categories, (a) three independent mutations, and (b) two independent mutations and a cross-over between variant allelic genes to form the fourth PGM1 allele. There is no direct evidence for either hypothesis, but it is interesting to speculate on the mechanism if (b) is correct. Starch gel electrophoresis of P G M 1 gives three phenotypes suggesting two alleles (see figure 1), but when the same phenotypes are subjected to isoelectric focusing, four distinct allelic products can be recognized giving a total of ten phenotypes (see figure 1). This latter observation leads to the hypothesis that an original PGM1 gene, for example PGMI + mutated by one charged amino acid substitution to form a second allele, for example PGM~ +, which is clearly seen as a faster moving band on starch gel electrophoresis, and differs in isoelectric point by about 0.13 pH units. At some other time another amino acid substitution occurred in the P G M I + cistron, forming a gene coding for a protein with a slightly more basic isoelectric point, differing from the original gene by about 0.05 pH units. At this point there are three PGM1 alleles, the original PGM~ + and the two mutated alleles, PGM~ + and PGM~ - ; if an intracistronic cross-over then occurred between these two mutated genes forming a new cistron containing both mutations, its enzyme product would be expected to differ in isoelectric point from the original PGM~ + by the large positive charge that formed the P G M 2 + isozyme plus the additional smaller negative charge that resulted in the P G M ~ - alMe (see figure 4), and would focus in approximately the same position as the PGM~ isozyme, which has an isoelectric point that is more basic than P G M 2 + by about 0.08 pH units. Point mutations as the source of new alleles have been well documented (c.f. Lehmann and Kynoch 1976), and intracistronic crossing over has also been implicated for example, in the production of new alleles at the haptoglobin locus (Smithies, Connell and Dixon 1962). P G M is a relatively large monomeric enzyme, molecular weight approximately 60000 (i.e. 180000 coding nucleotides) and there is therefore an increased likelihood for an intracistronic cross-over to take place at this locus. It would be very interesting to undertake a peptide analysis of the products of these four P G M 1 alleles to verify this mutational hypothesis, but since the amount of enzyme protein in red cells is extremely small, it may be necessary to investigate such tissues as muscle, which have markedly higher glycolytic activity. The increase in the number of alleles available for study through the technique of isoelectric focusing gives more variability and is particularly useful for linkage analysis. A greater number of informative families may now be recognized than was previously possible using starch gel electrophoresis. For example, if one parent was typed homozygous P G M 1 1-1 on starch, and the other homozygous P G M 1 2-2, no recombinants with the other locus being investigated would be recognized among the offspring; however, ifisoelectric focusing showed that one parent was heterozygous, e.g. PGM1 1 + 1 - , recombinants could be recognized among the offspring. The LOD scores for PGMa/rhesus linkage give a recombination fraction of 30~ between the loci in males and this is in close agreement with the data provided by
229
Isoelectric focusing o f P G M 1 Chromosome 1
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PGM 1 locus
1 + Allele
Mu,ad/ t,=
Mutation
2-P Allele
1 - Allele
2 - Allele
Figure 4. Hypothesis for the evolution of three PGM 1 alleles from an ancestral PGMI allele.
Robson et al. (1973), who also show that the recombination fraction in females is higher than in males. O u r results indicate no clear linkage association between these two loci in females.
Acknowledgements This work was supported by grants from the U.S. National Institutes of Health (1 ROI CA19821~)1) the Canadian Medical Research Council and the Canadian National Cancer Institute. We are also grateful for the continued support from the Newfoundland T.B. and R.D. Association and the Newfoundland Cancer Treatment and Research Foundation. The pedigrees were collected and organized by Sharon Buehler and Joyce Crumley and the rhesus typing was performed by Michael Newton using antisera that was a gift from the Canadian Red Cross Blood Transfusion Service, courtesy of Dr P. B. L. Moore. Primate bloods were kindly supplied by Dr Christine Hawkey of the Zoological Society of London.
230
Isoelectric f o c u s i n g o f P G M a
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References BARK, J. E., HARRIS, M. J., and FmTlq, M., 1976, Typing of the common PGM variants using isoelectric focusing--a new interpretation of the P G M system. Journal of the Forensic Science Society, 16, 115 120. BARNICOT,N. A., and COHEN,P., 1970, Red cell enzymes of primates (Anthropoidea). Biochemical Genetics, 4, 41:-57. BUEHLER, S. K., FraME, F., FOOOR, G., FRASER, G. R., MARSHALL, W. H., and VAZE, P., 1975, Common Variable Immunodeficiency, Hodgkin's Disease, and other malignancies in a Newfoundland family, The lancet, i, 195-197. GOODMAN, M., and TASHIAN, R. E., 1969, Geographic variation in the serum transferrin and red cell phosphoglucomutase polymorphisms of chimpanzees. Human Biology, 41, 237-249. HOPK1NSON,D. A., and HARRlS,H., 1966, Rare phosphoglucomutase phenotypes. Annals ofiluman Genetics, 30, 167 168. HOPKINSON, D. A., and HARRIS, H., 1968, A third phosphoglucomutase locus in man. Annals of Human Genetics, 31,359 367. HOPIONSON, D. A., and HARRIS, H., 1969, Red cell acid phosphatase, phosphoglucomutase and adenylate kinase. In Biochemical Methods in Red Cell Genetics (New York: Academic Press), 337-374. KOHNL, P., SCUMIDTMANN,U., and SPIELMANN,W., 1977, Evidence for two additional common alleles at the PGM~ locus (phosphoglucomutase--E.C.: 2.7.5.1.). Human Genetics, 35, 219-223. KOrrNL, P., and SPEILMANN,W., 1978, Investigations on the PGM] polymorphism (phosphoglucomutase EC 2.7.5.1) by isoelectric focusing. Human Genetics, 43, 57-67. LEHMANN, H., and K~q~OCH, P. A. M., 1976, Human Ilaemoglobin Variants and their Characteristics (Amsterdam, New York, Oxford: North Holland Publishing Company). MAYNARD-SMITH,S., PENROSE,L. S., and SMm~, C. A. B., 1961, Mathematical Tablesfor Research Workers in Human Genetics (Edinburgh: Churchill Livingstone). ROBSON,E. B., COOK,P. J. L., CORNEY,G., HOPKINSON,D. A., NOADES,J., and CLEGHORN,T. E., 1973, Linkage data on Rh., PGM, PGD, Peptidase C and Fy from family studies. Annals of Human Genetics, 36, 393 399. SCHMITT,J., LICHTE, K. H., and FUHRMANN,W., 1970, Red cell enzymes of the Pongidae. Humangenetik, 10, 138-144. SMI1:mES,O., CONNELL, G. E., and DIxoN, G. H., 1962, Chromosomal rearrangements and the evolution of haptoglobin genes. Nature (Lond.), 196, 232 236. SPENCER,N., HOPK1NSON,D. A., and HARRIS,H., 1964, Phosphoglucomutase polymorphism in man. Nature, 204, 742-745. SUTTON,J. G., and BURGESS,R., 1978, Genetic evidence for four common alleles at the phosphoglucomutase1 locus (PGM 0 detectable by isoelectric focusing. Vox Sanguinis, 34, 97 103. Address correspondence to: Dr. N. D. Carter, Department of Child Health, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE. Zusammenfassung. Jiingst wurden durch Isoelektrofokusierung vier Allele des PhosphoglukomutaseLokus des MensChen (PGM 1) dargestellt. In dieser Arbeit werden ffir vier Bev61kerungen Genfrequenzen gesch/itzt, die Ergebnisse zeigen signifikante Unterschiede. Die Stammbaumanalyse des PG M t- und RhesusLokus bestg.tigen eine schwache Koppelung bei Miinnern, keine jedoch bei Frauen. Eine Prtifung der PGM lPh~inotypen bei einer Reihe yon Primaten zeigt, dab ein Teil von ihnen ein Band besitzt, das beim selben isoelektrischen Punkt wie menschliches PGM~ + fokusiert. Wir schliegen daraus, dab alle Allele des Menschen und der iibrigen Primaten aus einem gemeinsam,' PGM~ +-Lokus evoluiert haben k6nnen. R6sum~. Quatre alleles du locus de la phosphoglucomutase ( P G M 0 chez l'homme ont 6t6 r~cemment mis en 6vidence par focalisation iso~lectrique; dans la pr6sente 6tude les fr6quences g6niques en ont ~t~ estim~es dans quatre populations, et des differences significatives sont apparues entre les r~sultats. L'analyse du p6digr~e des locus PGM1 et Rhesus confirment un faible linkage dans le sexe masculin, mais aucun dans le sexe f~minin. Un examen des ph6notypes P G M t dans une s~rie de primates montre qu'une certaine proportion poss~dent une bande focalis~e au m~me point iso~lectrique que le PGMI + humain. Nous proposons que tousles alleles de l'homme et des primates peuvent avoir d~riv~ d'un locus ancestral commun PGM ~+.
Phosphoglucomutase polymorphism detected by isoelectric focusing: gene frequencies, evolution and linkage N. D. CARTER and C. M. WEST Department of Child Health, St George's Hospital Medical School, London
E. E~aEs and B. PARKIN Ann Hum Biol Downloaded from informahealthcare.com by Nyu Medical Center on 01/07/15 For personal use only.
The Metropolitan Police Forensic Science Laboratory, London
and W. H. MARSHALL Faculty of Medicine, Memorial University of Newfoundland, Canada
Receit, ed 13 November 1978; revised 15 January 1979
Summary. Four alleles at the phosphoglucomutase (PGM 1) locus in man have been recently demonstrated by isoelectric focusing, and in this study gene frequencies have been estimated for four populations, and significant differences have been indicated between the results. Pedigree analysis of the PGM~ and rhesus loci confirm a weak linkage in males, but none in females. An examination of PGM~ phenotypes in a range of primates shows that a proportion possess a band focusing at the same isoelectric point as human PGM~ +. We propose that all the human and primate alleles may have evolved from a common ancestral PGM 11+ locus. 1.
Introduction Recent investigations of phosphoglucomutase (PGM1) phenotypes by isoelectric focusing have shown four common alleles at this locus (Bark, Harris and Firth 1976, Kiihnl, Schmidtmann and Spielmann 1977). Sutton and Burgess (1978) demonstrate tlSat these alleles are determined by one locus and are inherited in a Mendelian fashion. The present study compares frequencies of these four P G M 1 alleles in several human populations, confirming that they are related to the two common alleles previously described at the locus (Spencer, Hopkinson and Harris 1964). One of the populations examined was a community o f 1500 people in Newfoundland, from which a sample of 296 members of extended family groups was taken for P G M 1 phenotyping by isoelectric focusing. It was therefore possible to examine the inheritance of the four alleles. The information provided was also used to investigate linkage with the locus for the rhesus complex on chromosome 1. While a number of human lysates have now been typed for P G M by isoelectric focusing, the related non-human primate patterns have not been reported. Since this has a bearing on the evolution of this locus, we examined a number oflysates, from a range of primate species, on isoelectric focusing and starch gels for P G M zymograms. 2.
Materials and methods Red cell lysates were run on isoelectric focusing gels with an LKB Multiphor 2117 and LKB 2103 power supply, over a pH range of 5 7 formed by LKB 'Ampholytes', as described by Bark et al. (1976). Starch gel electrophoresis was run using the method of Spencer et al. (1964). Linkage analysis was as described by Maynard-Smith, Penrose and Smith (1961). A.H.B.
0301 4460/79/0(703 0221 $02"00 ~'~ I979 Taylor & Francis Ltd
Q
222
N . D . Carter et al.
Of the four populations studied, three were different ethnic groups living in London (Northern European, Negro, and Asian), from which blood samples were routinely obtained by the Metropolitan Police Forensic Laboratory. The fourth population was a genetically isolated group of about 1500 people in Newfoundland, being studied as part of an extensive health survey (Buehler, Firme, Fodor, Fraser, Marshall and Vase 1975). Primate bloods were supplied by the Wellcome Institute of the Zoological Society of London.
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3.
Results
Figure 1 shows diagrammatically the ten phenotypes of P G M 1 on isoelectric focusing and indicates how they are derived from the three phenotypes described for starch gel electrophoresis; the nomenclature is that introduced by Bark et al. (1976). Examples of all 10 phenotypes have been observed in the populations studied, with different frequencies for each of the four populations. Table 1 gives allele frequencies for the four populations; significant differences may be observed between these frequenAllele frequency Population N. European Newfoundland Negroes in London Asians in London
1-
1+
0.132 0.153 0-t26 0.096
0.627 0.537 0.672 0.537
2 0.058 0.076 0-027 0-121
2+
No.
0.182 0.233 0-173 0-246
2773 296 384 120
Table 1. Frequencies of the four human P G M ~ alleles determined by isoelectric focusing. 'Africans' include a random selection of people of African origin living in London; 'Asians' include a random selection of people of Indian origin who have immigrated to London from Asia and Africa.
i
2-I
d
m
c
a
b
m
2
m m
mm
m
mm
Starch gel electroph(~esis POM L bands
m
2+
IN mm
~-
mm m
m mmm
m
m |scelectric focusing PGM
m
l-ma ,-
m
m
m
n
m
I
bands
m
m
Figure 1. P G M 1 phenotypes on starch gel electrophoresis and isoelectric focusing. For clarity the isoelectric focusing diagram shows the separation of the 'a' and 'b' isozymes delineated on starch gel, ie. the primary P G M 1 gene products. The 'c' and 'd' bands on starch gel are thought to represent secondary post-translational components.
223
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lsoelectric Jocusing o f P G M 1
cies. Chi z tests comparing each of the populations show P < 0.001 for Europeans and Asians, Europeans and Negroes and Asians and Negroes; the Newfoundland population is included for comparison, although, as will be discussed below, it is not a randomly selected population. Kfihnl and Spielmann (1978) have quoted gene frequencies for the four P G M 1 alleles in a German population, and these agree closely with those given in table 1 for a Northern European population in London. Although allele frequencies are significantly different for each of the populations studied, all have a highest frequency of P G M I + and all show lower frequencies of the 1 - and 2 alleles. To compare the frequency data produced by isoelectric foc.using with information already published from starch gel electrophoresis (Spencer et al. 1964, Hopkinson and Harris 1966, Hopkinson and Harris 1968, Hopkinson and Harris 1969), the allele frequencies for P G M I + and P G M I - were combined (table 2). This gave a frequency for the P G M I allele very similar to frequencies ah'eady quoted for the populations under investigation. The isoelectric point of each P G M 1 isozyme band was measured by a surface pH electrode in 250 samples, the average result for each band was as follows: P G M 1 1 - pH 6.127, P G M 1 l + p H 6'075, PGM1 2 - p H 5"975 and PGM1 2 + p H 5-9.
Population
N. European Newfoundland Negroes in London Asians in London
PGMI~ gene t?equency detected by 1.E.F.
Published frequency(starch gel) 0'738 0"764
Spenceret al. (1964) Hopkinsonand Harris (1966)
0"74 0.767
Hopkinsonand Harris (1968) Hopkinsonand Harris (1969)
0.782 0.72
Hopkinsonand Harris (1966) Hopkinsonand Harris (1969)
0.759 0"69 0-798 0.633
Table 2. Summedhequenciesofthc PGM{ ~ and PGM{ allelescompared with published PGMI allele frequencies.
Family groups from the Newfoundland community were examined to confirm the Mendelian inheritance of the four alleles, and to provide linkage data with other gene loci on chromosome 1. Figure 2 gives one pedigree from the family showing autosomal codominant inheritance of the alleles: data from other family groups examined confirm this. Linkage has been reported (Robson, Cook, Corney, Hopkinson, Noades and Cleghorn 1973) between the loci for P G M 1 and the rhesus complex on Chromosome 1. Studies using isoelectric focusing to type individuals for P G M 1 show from L O D scores that linkage is present but weak in males; it could not however be confirmed in females. Table 3 gives LOD scores for all informative families, and recombination frequencies for phase known families. A male LOD peak of0-138 occurred at 0 = 0.3; in females the peak was 0.0 at 0 = 0.5. In four families the phase was known for female meioses, giving a recombination frequency of 53"8~o; in only one family was the phase known for male meioses, two of the three children showed recombination of the paternal haplotype. Q2
N.D. Carter et al.
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Figure 2.
1-1-
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1 6
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1+1-
2-1-
,, 2-1+
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L O D scores at recombination frequency 0
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Pedigree of a Newfoundland family.
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0.1
0-15
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0.25
0.3
0.35
0.4
-5,2 -2-35 -1-02 -0-33 0.01 0.14 0.13 0.06 -8.16 -3.96 -3.56 -2.13 -1.39 -0.86 -0.5 -0.24
0.45 -0,001 -0.08
Linkage data for P G M 1 and Rhesus alleles.
A range of primate species was screened for PGM 1 phenotypes, to determine whether isozyme patterns resembling one or more of the human allelic products occurred among them. Primate lysates were examined for PGM1 by both starch gel electrophoresis and isoelectric focusing, and isozyme patterns were compared with those from human lysates. All the Hominoidea studied (Pan(7), Gorilla(4) and Pongo(3)) showed an isozyme pattern identical to the human PGM ~pattern on Starch gel electrophoresis (figures 3(a) and 3(b)) and to human PGMI + on isoelectric focusing (figure 3(c)). Among old world monkeys, Presbytis(1), Cercocebus(1) and Macaca mulatta(8) all showed the same pattern, identical to human PGMI +, but Papio anubis(1) and Papio papio(1) gave different patterns with cathodal isozyme bands. Among new world monkeys Callithrix(4) showed an isozyme pattern identical to human PGMI +, but Saimiri(1) and Aotus(2) gave completely different patterns. The isozyme pattern in the lemur showed a band cathodal to human PGMI -. (Figures in brackets indicate the number of each species examined.) Where more than one sample from a species was available, PGM isozymes observed were identical; however intraspecific variation has been reported for Pan, Gorilla and Macaca iris (Schmitt, Lichte and Fuhrrnann 1970, Goodman and Tashian 1969, Barnicot and Cohen 1970); each was found to possess additional alleles, with different electrophoretic mobilities from common human phenotypes. 4.
Discussion The results from electrophoresis and isoelectric focusing of primate bloods suggest that the PGM~ + allele, or one identical to it in electrophoretic mobility and isoelectric point, is common to man and a number of higher primates.
Isoelectric Jocusing of PGM 1
225
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Figure 3 (a)
Origin
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+ m m
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lsoelectric focusing
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~'igure 3. Primate P G M phenotypes. (a) Starch gel electrophoresis, l, H u m a n 2 1; 2, Presbytis; 3, H u m a n 2 - 1; 4, Aotus; 5, Lemur; 6, H u m a n 2-1; 7, Pongo; 8, Pongo; 9, H u m a n 2 1; 10, Gorilla; 11, Gorilla,• 12, H u m a n :2 1. (a') Block d i a g r a m of figure 3(a). (b) Starch gel electrophoresis. 1, t t u m a n 2 -- 1, 2, Papio pupio: 3, CalIithrix, 4, H u m a n 2 - 1; 5, Saimiri sciureus: 6, Papio anubis; 7, H u m a n 2 1; 8, Pan; 9, Macaca mulat tel; 10, H u m a n 2 1. (h') Block diagram of figure 3(b). (c) isoelectric focusing• 1, H u m a n 2 + 1 .- ; 2, Papio anubis; 3, Saimiri seiureus: 4, H u m a n 2 - 1 + ; 5, Callithrix; 6, Papio papio; 7, H u m a n 2 + 1 - ; 8, Gorilla; 9, Gorilla; 10, H u m a n 2 ,- I + ; 11, Ponqo; 12, Pongo; 13, t l u m a n 2 + 1 - ; 14 Lemur; 15, Aotus;16, H m n a n 2 1 + . (c') Block d i a g r a m of m a i n bands in figure 3 (c), for ease of interpretation only b a n d s in the zone between h u m a n P G M 1z ~ and P G M ] and cathodal to this zone are indicated in all 'lanes' of this gel. K n o w n h u m a n P G M 1 allelic products are indicated with a white spot.
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N . D . Carter et al.
Despite the differences in gene frequencies between the four human populations in this study, all show similarity, having P G M ~+ as the most frequent allele, and all except the Asian population have PGM~z- as the least frequent allele. The high frequency of the P G M ~+ allele could relate to evidence from primate studies that this is an ancestral allele in man, from which the other three common alleles, as well as rarer alleles have evolved. I f P G M I + is taken as the ancestral P G M 1 gene there are several different methods by which the other alleles could have evolved; these may be considered in two categories, (a) three independent mutations, and (b) two independent mutations and a cross-over between variant allelic genes to form the fourth PGM1 allele. There is no direct evidence for either hypothesis, but it is interesting to speculate on the mechanism if (b) is correct. Starch gel electrophoresis of P G M 1 gives three phenotypes suggesting two alleles (see figure 1), but when the same phenotypes are subjected to isoelectric focusing, four distinct allelic products can be recognized giving a total of ten phenotypes (see figure 1). This latter observation leads to the hypothesis that an original PGM1 gene, for example PGMI + mutated by one charged amino acid substitution to form a second allele, for example PGM~ +, which is clearly seen as a faster moving band on starch gel electrophoresis, and differs in isoelectric point by about 0.13 pH units. At some other time another amino acid substitution occurred in the P G M I + cistron, forming a gene coding for a protein with a slightly more basic isoelectric point, differing from the original gene by about 0.05 pH units. At this point there are three PGM1 alleles, the original PGM~ + and the two mutated alleles, PGM~ + and PGM~ - ; if an intracistronic cross-over then occurred between these two mutated genes forming a new cistron containing both mutations, its enzyme product would be expected to differ in isoelectric point from the original PGM~ + by the large positive charge that formed the P G M 2 + isozyme plus the additional smaller negative charge that resulted in the P G M ~ - alMe (see figure 4), and would focus in approximately the same position as the PGM~ isozyme, which has an isoelectric point that is more basic than P G M 2 + by about 0.08 pH units. Point mutations as the source of new alleles have been well documented (c.f. Lehmann and Kynoch 1976), and intracistronic crossing over has also been implicated for example, in the production of new alleles at the haptoglobin locus (Smithies, Connell and Dixon 1962). P G M is a relatively large monomeric enzyme, molecular weight approximately 60000 (i.e. 180000 coding nucleotides) and there is therefore an increased likelihood for an intracistronic cross-over to take place at this locus. It would be very interesting to undertake a peptide analysis of the products of these four P G M 1 alleles to verify this mutational hypothesis, but since the amount of enzyme protein in red cells is extremely small, it may be necessary to investigate such tissues as muscle, which have markedly higher glycolytic activity. The increase in the number of alleles available for study through the technique of isoelectric focusing gives more variability and is particularly useful for linkage analysis. A greater number of informative families may now be recognized than was previously possible using starch gel electrophoresis. For example, if one parent was typed homozygous P G M 1 1-1 on starch, and the other homozygous P G M 1 2-2, no recombinants with the other locus being investigated would be recognized among the offspring; however, ifisoelectric focusing showed that one parent was heterozygous, e.g. PGM1 1 + 1 - , recombinants could be recognized among the offspring. The LOD scores for PGMa/rhesus linkage give a recombination fraction of 30~ between the loci in males and this is in close agreement with the data provided by
229
Isoelectric focusing o f P G M 1 Chromosome 1
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PGM 1 locus
1 + Allele
Mu,ad/ t,=
Mutation
2-P Allele
1 - Allele
2 - Allele
Figure 4. Hypothesis for the evolution of three PGM 1 alleles from an ancestral PGMI allele.
Robson et al. (1973), who also show that the recombination fraction in females is higher than in males. O u r results indicate no clear linkage association between these two loci in females.
Acknowledgements This work was supported by grants from the U.S. National Institutes of Health (1 ROI CA19821~)1) the Canadian Medical Research Council and the Canadian National Cancer Institute. We are also grateful for the continued support from the Newfoundland T.B. and R.D. Association and the Newfoundland Cancer Treatment and Research Foundation. The pedigrees were collected and organized by Sharon Buehler and Joyce Crumley and the rhesus typing was performed by Michael Newton using antisera that was a gift from the Canadian Red Cross Blood Transfusion Service, courtesy of Dr P. B. L. Moore. Primate bloods were kindly supplied by Dr Christine Hawkey of the Zoological Society of London.
230
Isoelectric f o c u s i n g o f P G M a
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References BARK, J. E., HARRIS, M. J., and FmTlq, M., 1976, Typing of the common PGM variants using isoelectric focusing--a new interpretation of the P G M system. Journal of the Forensic Science Society, 16, 115 120. BARNICOT,N. A., and COHEN,P., 1970, Red cell enzymes of primates (Anthropoidea). Biochemical Genetics, 4, 41:-57. BUEHLER, S. K., FraME, F., FOOOR, G., FRASER, G. R., MARSHALL, W. H., and VAZE, P., 1975, Common Variable Immunodeficiency, Hodgkin's Disease, and other malignancies in a Newfoundland family, The lancet, i, 195-197. GOODMAN, M., and TASHIAN, R. E., 1969, Geographic variation in the serum transferrin and red cell phosphoglucomutase polymorphisms of chimpanzees. Human Biology, 41, 237-249. HOPK1NSON,D. A., and HARRlS,H., 1966, Rare phosphoglucomutase phenotypes. Annals ofiluman Genetics, 30, 167 168. HOPKINSON, D. A., and HARRIS, H., 1968, A third phosphoglucomutase locus in man. Annals of Human Genetics, 31,359 367. HOPIONSON, D. A., and HARRIS, H., 1969, Red cell acid phosphatase, phosphoglucomutase and adenylate kinase. In Biochemical Methods in Red Cell Genetics (New York: Academic Press), 337-374. KOHNL, P., SCUMIDTMANN,U., and SPIELMANN,W., 1977, Evidence for two additional common alleles at the PGM~ locus (phosphoglucomutase--E.C.: 2.7.5.1.). Human Genetics, 35, 219-223. KOrrNL, P., and SPEILMANN,W., 1978, Investigations on the PGM] polymorphism (phosphoglucomutase EC 2.7.5.1) by isoelectric focusing. Human Genetics, 43, 57-67. LEHMANN, H., and K~q~OCH, P. A. M., 1976, Human Ilaemoglobin Variants and their Characteristics (Amsterdam, New York, Oxford: North Holland Publishing Company). MAYNARD-SMITH,S., PENROSE,L. S., and SMm~, C. A. B., 1961, Mathematical Tablesfor Research Workers in Human Genetics (Edinburgh: Churchill Livingstone). ROBSON,E. B., COOK,P. J. L., CORNEY,G., HOPKINSON,D. A., NOADES,J., and CLEGHORN,T. E., 1973, Linkage data on Rh., PGM, PGD, Peptidase C and Fy from family studies. Annals of Human Genetics, 36, 393 399. SCHMITT,J., LICHTE, K. H., and FUHRMANN,W., 1970, Red cell enzymes of the Pongidae. Humangenetik, 10, 138-144. SMI1:mES,O., CONNELL, G. E., and DIxoN, G. H., 1962, Chromosomal rearrangements and the evolution of haptoglobin genes. Nature (Lond.), 196, 232 236. SPENCER,N., HOPK1NSON,D. A., and HARRIS,H., 1964, Phosphoglucomutase polymorphism in man. Nature, 204, 742-745. SUTTON,J. G., and BURGESS,R., 1978, Genetic evidence for four common alleles at the phosphoglucomutase1 locus (PGM 0 detectable by isoelectric focusing. Vox Sanguinis, 34, 97 103. Address correspondence to: Dr. N. D. Carter, Department of Child Health, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE. Zusammenfassung. Jiingst wurden durch Isoelektrofokusierung vier Allele des PhosphoglukomutaseLokus des MensChen (PGM 1) dargestellt. In dieser Arbeit werden ffir vier Bev61kerungen Genfrequenzen gesch/itzt, die Ergebnisse zeigen signifikante Unterschiede. Die Stammbaumanalyse des PG M t- und RhesusLokus bestg.tigen eine schwache Koppelung bei Miinnern, keine jedoch bei Frauen. Eine Prtifung der PGM lPh~inotypen bei einer Reihe yon Primaten zeigt, dab ein Teil von ihnen ein Band besitzt, das beim selben isoelektrischen Punkt wie menschliches PGM~ + fokusiert. Wir schliegen daraus, dab alle Allele des Menschen und der iibrigen Primaten aus einem gemeinsam,' PGM~ +-Lokus evoluiert haben k6nnen. R6sum~. Quatre alleles du locus de la phosphoglucomutase ( P G M 0 chez l'homme ont 6t6 r~cemment mis en 6vidence par focalisation iso~lectrique; dans la pr6sente 6tude les fr6quences g6niques en ont ~t~ estim~es dans quatre populations, et des differences significatives sont apparues entre les r~sultats. L'analyse du p6digr~e des locus PGM1 et Rhesus confirment un faible linkage dans le sexe masculin, mais aucun dans le sexe f~minin. Un examen des ph6notypes P G M t dans une s~rie de primates montre qu'une certaine proportion poss~dent une bande focalis~e au m~me point iso~lectrique que le PGMI + humain. Nous proposons que tousles alleles de l'homme et des primates peuvent avoir d~riv~ d'un locus ancestral commun PGM ~+.
