Gm and Km Allotypes in Disputed Parentage ELIZABETH S. SEBRING, B.S., M.T.(ASCP), HERBERT F. POLESKY, M.D., AND MOSES S. SCHANFIELD, PH.D.
Sebring, Elizabeth S., Polesky, Herbert F., and Schanfield, Moses S.: Gm and Km allotypes in disputed parentage. Am J Clin Pathol 71: 281-285, 1979. Immunoglobulin allotyping in 925 cases of disputed paternity provided evidence of exclusion for 70 alleged fathers. Combining results from erythrocytic antigen, enzyme, and serum protein tests, 230 men were proven to be falsely accused; in 15 cases the immunoglobulin allotype provided the only evidence for exclusion, in 67 no exclusion would have been identified if testing had been limited to ABO, Rh, and MNSs. Various cases in this study illustrate the importance of using an extensive battery of immunoglobulin reagents in order maximally to exclude falsely accused men, and to identify infrequent haplotypes, which might erroneously be interpreted as indirect exclusions or may indicate a high likelihood of paternity. The problems of Gm typing of very young children are described. When the child is less than 6 months of age, direct exclusions may be missed by the Gm allotypes; indirect exclusions are valid only when the phenotype differs from that of the mother. Km allotypes are not age-dependent. (Key words: Paternity testing; Gm; Km; Immunoglobulin allotyping.)
of 2,680 individuals involved in 925 disputed parentage cases. Material and Methods Specimens Data from 2,680 samples collected by or submitted to the Minneapolis War Memorial Blood Bank for paternity testing during the years 1973 through May 1977 were used for analysis. Some of these specimens were referred to us after questionable exclusions were found in initial testing in other laboratories. These samples are grouped into 925 cases, a case consisting of any set of alleged father, mother, and child in which at least one person differs from those in any other set. Methods All individuals were typed for the following erythrocytic antigens: A, A,, B, O; M, N, S, s; D, C, C w , c, E, e; K, k; for the following erythrocytic enzymes: acid phosphatase, phosphoglucomutase (PGM,, PGM 2 ), adenosine deaminase, adenylate kinase, 6-phosphogluconate dehydrogenase; and for the following serum proteins: transferrin, group-specific component, haptoglobin, ceruloplasmin, Gm (see Table 1), and Km(l). Some specimens were also typed for Fy a , Fy'\ Jk a , Jk b , and esterase D. All blood grouping was done by standard methods by the use of reagents according to the manufacturers' directions. The methods used for erythrocytic enzyme and serum protein electrophoresis are described or referenced by Dykes and Polesky.1-2 Ig Allotyping
Received November 29, 1977; received revised manuscript and accepted for publication January 19, 1978. Presented in part at the 1976 Annual Meeting of the American Association of Blood Banks, San Francisco, California, October 30-November 5, 1976. Address reprint requests to Dr. Polesky: Minneapolis War Memorial Blood Bank, 2304 Park Avenue, Minneapolis, Minnesota 55404.
Gm and Km typing was done on microtiter plates using a passive hemagglutination-inhibition system. This method has been described by Schanfield and associates. 9 Liquid-nitrogen-frozen erythrocytes from a single donor (most probable genotype cDE/cDE) were thawed each day just prior to coating and use in the test system. All sera were typed at a 1:20 dilution in saline solution with appropriate controls. Many of the
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TRADITIONALLY, most laboratories involved in testing individuals in disputed parentage cases have used only the ABO, Rh, and MNSs systems. 7 The chances of identifying a falsely accused man with these tests is just 58%.' Several court decisions, which increase the legal status of children born out of wedlock, and the adoption of the Uniform Parentage Act, 13 which facilitates the utilization of medical evidence regarding parentage in the courts, make the testing of additional polymorphic systems worthwhile. In fact, justice is more completely served when experts involved in providing medical evidence in paternity cases use additional reliable tests to increase their chances of reaching as correct a determination as possible. The need for this testing has been forcefully pointed out in the most recent revision of the Joint AMA-ABA Guidelines: "Present Status of Serologic Testing in Problems of Disputed Parentage." 3 The costs of such tests are small compared with the cost of child support. This report is to summarize the results obtained when immunoglobulin (Ig) allotyping was included in the testing
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SEBR1NG, POLESKY, AND SCHANFIELD
Table I. Reagents Used for Immunoglobulin Allotyping
erythrocytic typing limited to ABO, Rh, and MNSs provided evidence for only 163 exclusions.
Specificity*
Age of the Child and Gm Results
Numeric
Agglut inator
Coat
1 2 3 17
Hel, Pan Max Sta Pon
Dwi Yap Dan Dwi
s t
5 10 6 24 21 15 16
Ble Arr Alt' Hod Leh Gai Ros
Hun Hun 1872 1872 Sul Puh Puh
1
1
Sim, Cla
Abr
lameric a X
f z bl b5 c3 c5
e
younger children's samples also were typed at 1:5 and 1:10 dilutions. Any serum with agglutinating activity in saline solution was inactivated at 65 C for 10 minutes and retyped. Sera were typed with the reagents and for all of the markers listed in Table 1. In the results, any person who was positive for G3m (bl, b5) has been designated G3m(b). Definitions Exclusions were classified as direct or indirect. 4 Direct exclusions are those in which a haplotype present in the child is absent in both the mother and alleged father, or by the absence in the child of either of two nonidentical haplotypes demonstrable in the alleged father. Indirect exclusions are those in which a child lacks a genetic marker while the alleged father is homozygous for it, or the child is homozygous for a marker not present in both parents. Results Analysis of the results of erythrocytic antigen, erythrocytic enzyme, and serum protein typing tests in 925 cases of disputed paternity revealed exclusions in 230 cases (Table 2). Immunoglobulin (Ig) allotyping provided evidence for exclusions in 70 of these 925 cases. Direct evidence for exclusion was found in 39 cases by Gm typing and in 13 cases by Km(l) typing. Indirect evidence in the Gm system in 21 cases was corroborated by exclusions in other systems. In 15 cases the Ig allotypes provided the only evidence for exclusion; of these, nine were direct exclusions by Gm, three by Km(l), and three by both Km(l) and Gm. Ig markers and erythrocytic antigens provided evidence for exclusion in 203 of the total 230 exclusions found; however,
Racial Classification and Gm Results A difference in exclusion rates was observed when those cases with children 6 months old or older were divided into three racial classifications: those cases involving Caucasians, those cases involving Afro-Americans, and those cases in which the persons involved were not of the same racial heritage (Table 4). The ten Gm exclusions in cases involving non-Caucasians were Table 2. Exclusions in 925 Cases of Disputed Paternity
Systems Erythrocytic antigens Non-lg serum proteins and erythrocytic enzymes Ig allotypes Gm Km Cumulative total
Number of Individuals Excluded
Percentage of Cases with Exclusions
180
19.5
105 70* 60 13 230
11.4 7.6 6.5 1.4 24.9
* Three individuals were excluded by both Gm and Km(l).
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* Nomenclature recommended by the WHO Workshop on Human Immunoglobulin Allotypes, July 16-19. 1974. Rouen, France.
In order to ascertain the effect of age on Gm phenotype and the rate of direct Gm exclusions found, we have classified this series of cases into four groups according to the age of the child when tested (Table 3). Before 3 months of age, 83% of children were allotype-identical to their mothers, compared with 49% of children 1 year old or older. A Gm haplotype absent in the mother was found in 11% of children 0 - 3 months of age and in 29% of those 1 year old or older. The percentage of direct Gm exclusions showed an age relationship. It was 1.0% in children less than 6 months of age and 6.0% in children 1 year old or older. This difference, which is statistically significant, also exists when cases involving only Caucasians are compared: in 199 cases with children 0 - 6 months of age, two direct Gm exclusions were found (1.0%); in 459 cases with children 1 year old or older, 21 direct Gm exclusions were found (4.6%). Although there were 27 cases in which indirect Gm exclusions were found, only the 21 in which this was corroborated have been included in Table 2. Of the six cases not included in Table 2, four were omitted because the child was less than 6 months old when tested. It is possible that the IgG detected in these four children was maternal. In two cases the only indication that the alleged father may have been falsely accused was the indirect evidence found in the Gm system.
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GM AND KM ALLOTYPES IN DISPUTED PARENTAGE
Tabic 3. Effects of Age of Child on Gm Phenotype and Rate of Direct Gm Exclusions Found Gm Phenotype of Child Partially Identical to Mother's Phenotype
Age of Child (Months)
Number of Children Tested*
0-3 3-6 6-12 312
94(101) 102(105) 220(223) 484 (496)
Identical lo Mother's Phenotype
Non-identical Haplotype Present
Homozygous for One of Maternal Haplotypes
(%)
(%)
83 63 55 49
II 25 28 29
Direct Gm Exclusions
(%)
Number of Cases
Percentage of Cases
6 12 17 22
1 1 7 30
1.0 i.q 3.1 6.0
Number of cases in piirentheses. Difference is due to instances in which two men were accused of falhering the same child.
Km Results Analysis of Km(l) typing of mother-child pairs showed that 7% of children 0-3 months of age and 6% of those over 12 months of age were Km(l) positive with Km(l) negative mothers. Comparison in cases involving children of four different age groups showed no significant difference in Km(l) exclusion rates. Analysis of Km(l) typings by racial classification showed that 18% of Caucasians and 52% of AfroAmericans were Km(l) positive. The observed Km(l) exclusion rates were 12/871 (1.4%) in Caucasians and 1/42 (2.4%) in Afro-Americans. This difference is not statistically significant. Uncommon Gm
Haplotypes
Three infrequent Gm haplotypes were found in these cases: (1) Gm-'-'-h—One mother was of the phenotype Gm(z,a;g,b) with a most probable genotype of Cw z a ; B / Gm'-aM. However, two of her children were Glm(z,a) negative. Family studies, including testing for G2m(n), showed her true genotype to be Gmz-a- • ;B /Gm- : - : ". Negative results obtained for Glm and G2m markers have been indicated by ••. (2) Gmf;B—In three cases (six individuals), the recombinant haplotype Gm'-* was found. (3) GmKM'—In one case a child was of the phenotype Gm(x,f; b). There are no frequency data for the haplotype Gmx,Ub; however, it has been found in many Caucasian populations. Although the most likely genotype for a Gm(z,a,x,f; g,b) parent is Gm z,a ' !i;e /Cm f; ', the genotype Gm'-a'-R/Gmx-''b is also possible. In three cases either a child inherited Gmz-a:g from a (z,a,x,f; g,b) parent or there was indirect evidence for exclusion of the
alleged father. In another case, a child of Gm phenotype (z,a,x,f; g,b) either inherited GmxM' from his mother or there was indirect evidence for nonpaternity of the alleged father whose Gm type was (z,a; g). These four cases were not interpreted to be Gm exclusions; however, each father was excluded by findings in other systems. In three of the 925 cases an increased likelihood of paternity was indicated by Gm typing. In two cases involving Caucasians the alleged father and child had the Oriental haplotype G m z a ; b 5 s l . In one case the alleged father and child both had the recombinant Gmm. Two Men Accused of Fathering the Same Child Ig allotyping was informative in 25 instances in which the woman accused two different men of fathering the same child. Combining all systems, 23 of the accused men were excluded. In each of the two instances in which neither man was excluded, the alleged fathers were brothers. Ten alleged fathers were excluded by Ig allotype. Seven of these men would not have been excluded if only erythrocytic typing had been done: the evidence for nonparentage was found for two in both the Gm and acid phosphatase systems, one in the group-specific component protein system, one in Table 4. Observed Gm Exclusion Rates by Race— Child 6 Months Old or Older
Caucasians AfroAmericans Mixed
Excluded All Systems Combined
Excluded All Systems Except Gm
Excluded Gm
Number of Cases
No.
%
No.
%
No.
672
172
25.6
165
24.6
46
6.8
37 10
18 4
48.6
17 3
45.9
7 3
18.9
7c
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fourld by G1 m (a, x, or f) typing in three, G3m (c or s) typing in six, and G3m (g) typing in one.
SEBRING, POLESKY, AND SCHANFIELD
284
Table 5. Exclusion Rates in Caucasian Cases with Children >6 Months Old Exclusion Rate Observed
Expected
Erythrocytic antigens ABO Rh MNSs K
124 39 70 56 8
.6425 .2021 .3627 .2902 .0415
.5935 .1537 .2767 .3112 .0400
Erythrocytic enzymes acP PGM ADA AK 6-PGD
50 29 15 8 5 2
.2591 .1503 .0777 .0415 .0260 .0104
.4121 .2231 .1541 .0516 .0429 .0146
Non-Ig serum proteins Hp Gc Cp Tf
26 20 7 0 0
.1347 .1036 .0363 .0000 .0000
.3260 .1845 .1618 .0123 .0018
Ig allotypes Gm Km(l)
54 46 10
.2798 .2383 .0518
.2881 .2394 .0640
Cumulati ive total
171
Fy* EsD* Cumulative total
14 4 172
.8865 .1647 .0471
.1840 .0740 .9167
* Subset of 290 of above cases (78 exclusions) also typed for Fy", Fy\ EsD.
the adenosine deaminase system, and three only by immunoglobulin allotype. Observed Exclusion Rates In Table 5, the number of observed exclusions, the expected exclusion probability (based on appropriate gene frequencies), and the observed exclusion rate (based on the number of proven exclusions divided by the theoretical number of falsely accused men) are shown by individual test system for those cases involving Caucasians and children 6 months old or older. The theoretical number of falsely accused (193) was obtained by dividing the number of proven falsely accused (171) by the cumulative exclusion probability (.8865). Discussion Methodology The selection of genetic systems for use in paternity testing should be predicated on the usefulness of the system, the ease of testing, the specificity and sensitivity of the test, and the stability of the markers to be tested. Ig allotyping is simple, sensitive, and reliable. The actual testing can usually be completed in two
hours. Unlike reagents for HLA testing, Ig allotyping reagents are normally monospecific, or can be rendered monospecific quite readily by absorption. The majority of Ig typing systems will detect 1-10 /u,g/ml of Gm- or Km-specific antigen; thus, normal adult sera can be diluted 1/20-1/40 and normal infant sera 1/5-1/10. Unlike many genetic systems that undergo rapid changes during storage, the antigens of human Ig are remarkably stable. Usefulness The Ig markers proved to be a useful system in our paternity test battery. Combined results for all test systems (cumulative exclusion probability .9167, excluding Jka, Jkb) provided evidence for exclusion of paternity in 230 of the 925 cases studied. Erythrocytic typing for ABO, Rh, and MNSs (cumulative exclusion probability .5784') provided evidence for 163 of these exclusions. Including Ig markers with the tests for erythrocytic antigens provided evidence for exclusion in 203 cases. In 15 of the 230 cases in which men were proven to be falsely accused, the Ig allotype provided the only evidence for exclusion. In 25 instances in which two men were accused of fathering the same child, Gm typing excluded ten of the alleged fathers. Five of these ten men would not have been excluded if only erythrocytic typing had been done. The observed exclusion rates for Gm and Km (Table 5) were close to the expected values, confirming in practice the usefulness of these markers in determining nonparentage. We have no explanation for the deviations in observed exclusions compared with expected exclusions in the other test groups. The usefulness of Gm allotyping in paternity testing is dependent on the number of factors tested and the haplotypes detectable. The incidence of Gm haplotypes varies with racial heritage. The probability of exclusion in Caucasians based on a representative Minnesota population11 and use of reagents for Gm (a, x, f, and bl) is 0.24. These reagents are capable of detecting the three common Caucasian haplotypes. With these same reagents, the exclusion probability for Afro-Americans based on gene frequencies obtained in a Northern United States Black population8 is 0.17. However, the exclusion probability for this same Afro-American population is 0.33 when reagents for G3m (z, g, b5; c3, c5; and s) are added to the test battery. Of ten exclusions in non-Caucasians, seven would not have been identified if only reagents for Gm (a, x, f and bl) had been used. Not only is the use of an extended battery of reagents important in order to maximally exclude falsely accused men, but in some instances the results may indicate a high likelihood of paternity. In three Caucasian cases, the alleged father and child were both
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No. of Individuals Excluded
A.J.C.P. . March 1979
Vol. 71 • No. 3
GM AND KM ALLOTYPES IN DISPUTED PARENTAGE z,a:b5,s t
11
Although the Gm exclusion rate was affected by the age of the child when tested, this was not true of Km(l), a marker found on kappa light chains of all immunoglobulins. The exclusion probability of Km(l) in Caucasians is .064 and for Afro-Americans .119. In this series, the observed exclusion rates, 1.4% and 2.4%, for these two groups were not significantly different. Pitfalls In interpreting evidence for exclusion in the Gm system, certain sources of error should be kept in mind. As described above, the presence of only maternal IgG or transient hypogammaglobulinemia in very young children may lead to a misinterpretation of results. It is also essential that the Ig test system include a screen for saline antibody activity in each serum against each coated erythrocyte. When interpreting indirect evidence of exclusion, as in other systems, deleted, recombinant, or mutant haplotypes and all possible genotypes must be kept in
mind. In the present study, the haplotypes Gm--'-*, Gmle, and Gm x,f;b were observed. In addition, the haplotypes Gm z ' a : - ; -, 5 Gmz'-'-R,e Gm-'-'-s,10 and Gm'-"'-14 have been found. Of these uncommon haplotypes, Gm f;g , Gm-'-'-z, and Gmz'-:s cause problems in interpretation of paternity test results when G3m(g) testing is not done. Unfortunately, dosage studies of serum in an individual with a deleted Gm haplotype may not show decreased levels of antigen due to compensatory increases in the allelic antigen. 5 The possible presence of Gmx-!'-b should be considered in all cases in which the alleged father or mother is Gm(z,a,x,f; g,b). It should be remembered that the majority of individuals with the phenotype Gm(z,a,x; g) are Gw z a x ; K /Gm z a ; B and not G/n z a x : g /G/n z a x ; B . Many of the totally deleted haplotypes originally reported in the Gm system have proven to be recombinants or only partially deleted haplotypes when tested with a more complete battery of reagents. The null haplotype found by Steinberg and co-workers 12 in a group of related individuals of a Hutterite community has not been observed in other populations. References 1. Dykes DD, Polesky HF: The usefulness of serum protein and erythrocyte enzyme polymorphisms in paternity testing. Am J Clin Pathol 65:982-986, 1976 2. Dykes DD, Polesky HF: Paternity testing by using erythrocyte enzyme esterase D. J Forensic Sci 22:173-177, 1977 3. Joint AMA-ABA Guidelines: Present status of serologic testing in problems of disputed parentage. Family Law Q 10:247— 285, 1976 4. Lee CL: Current status of paternity testing. Family Law Q 9: 615-633, 1975 5. Lefranc G, Rivat L, Rivat C, et al: Evidence for "deleted" or "silent" genes homozygous at the locus coding for the constant region of the gamma 3 chain. Am J Hum Genet 28:51-61, 1976 6. Natvig JB, Michaelsen TE, Gedde-Dahl T Jr, et al: IgG, subclass protein with the genetic Gm markers Gm (z+ non-a + ): Another example of intragenic hybridization among IgG subclass genes. J Immunogenet 1:33-41, 1974 7. Polesky HF, Krause HD: Blood typing in disputed paternity cases—Capabilities of American laboratories. Transfusion 17:521-524, 1977 8. Schanfield MS: Unpublished data 9. Schanfield MS, Polesky HF, Sebring ES: Gm and Inv typing, Paternity Testing. Edited by Polesky HF. American Society of Clinical Pathologists, Chicago, 1975, pp 45-53 10. Sebring ES: Unpublished data 11. Sebring ES, Polesky HF, Schanfield MS: The risk of immunization to IgG following Rh immune globulin therapy. Transfusion 14:220-225, 1974 12. Steinberg AG, Muir AW, Mclntire SA: Two unusual Gm alleles: their implications for the genetics of the Gm antigens. Am J Hum Genet 20:258-278, 1968 13. Uniform Parentage Act, adopted by the National Conference of Commissioners on Uniform State Laws (1973) 14. van Loghem E, Natvig JB: Uncommon Gm gene complexes. Vox Sang 18:421-434, 1970
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found to have either G/M - or Gm'' . These two haplotypes have an approximate frequency of 0.005 in Caucasians. Neither of these haplotypes would have been evident with the use of a limited number of reagents. Usefulness and interpretation of Gm results in paternity testing are partially dependent on the age of the child when tested. At birth IgG is almost totally maternal, in the ensuing months a transition occurs to IgG with an individual's own genetic determinants. Therefore, two problems may arise in the interpretation of Gm typing of very young children. One is that an indirect exclusion of paternity may be thought to exist when it does not. In this study, 83% of children 0 - 3 months old were phenotype-identical to their mothers, compared with only 49% of children who were 1 year old or older when tested. Because of this age effect, Gm exclusions of paternity in very young children are valid only when the allotype of the child differs from that of the mother. The second problem is that a child may be too young to express IgG of paternal type, and it is thus possible to miss a direct exclusion. The frequency of detectable paternal antigen in children of this study who were 3 months old or younger was significantly less (x 2 (ldf) = 26.7, P =
Sebring, Elizabeth S., Polesky, Herbert F., and Schanfield, Moses S.: Gm and Km allotypes in disputed parentage. Am J Clin Pathol 71: 281-285, 1979. Immunoglobulin allotyping in 925 cases of disputed paternity provided evidence of exclusion for 70 alleged fathers. Combining results from erythrocytic antigen, enzyme, and serum protein tests, 230 men were proven to be falsely accused; in 15 cases the immunoglobulin allotype provided the only evidence for exclusion, in 67 no exclusion would have been identified if testing had been limited to ABO, Rh, and MNSs. Various cases in this study illustrate the importance of using an extensive battery of immunoglobulin reagents in order maximally to exclude falsely accused men, and to identify infrequent haplotypes, which might erroneously be interpreted as indirect exclusions or may indicate a high likelihood of paternity. The problems of Gm typing of very young children are described. When the child is less than 6 months of age, direct exclusions may be missed by the Gm allotypes; indirect exclusions are valid only when the phenotype differs from that of the mother. Km allotypes are not age-dependent. (Key words: Paternity testing; Gm; Km; Immunoglobulin allotyping.)
of 2,680 individuals involved in 925 disputed parentage cases. Material and Methods Specimens Data from 2,680 samples collected by or submitted to the Minneapolis War Memorial Blood Bank for paternity testing during the years 1973 through May 1977 were used for analysis. Some of these specimens were referred to us after questionable exclusions were found in initial testing in other laboratories. These samples are grouped into 925 cases, a case consisting of any set of alleged father, mother, and child in which at least one person differs from those in any other set. Methods All individuals were typed for the following erythrocytic antigens: A, A,, B, O; M, N, S, s; D, C, C w , c, E, e; K, k; for the following erythrocytic enzymes: acid phosphatase, phosphoglucomutase (PGM,, PGM 2 ), adenosine deaminase, adenylate kinase, 6-phosphogluconate dehydrogenase; and for the following serum proteins: transferrin, group-specific component, haptoglobin, ceruloplasmin, Gm (see Table 1), and Km(l). Some specimens were also typed for Fy a , Fy'\ Jk a , Jk b , and esterase D. All blood grouping was done by standard methods by the use of reagents according to the manufacturers' directions. The methods used for erythrocytic enzyme and serum protein electrophoresis are described or referenced by Dykes and Polesky.1-2 Ig Allotyping
Received November 29, 1977; received revised manuscript and accepted for publication January 19, 1978. Presented in part at the 1976 Annual Meeting of the American Association of Blood Banks, San Francisco, California, October 30-November 5, 1976. Address reprint requests to Dr. Polesky: Minneapolis War Memorial Blood Bank, 2304 Park Avenue, Minneapolis, Minnesota 55404.
Gm and Km typing was done on microtiter plates using a passive hemagglutination-inhibition system. This method has been described by Schanfield and associates. 9 Liquid-nitrogen-frozen erythrocytes from a single donor (most probable genotype cDE/cDE) were thawed each day just prior to coating and use in the test system. All sera were typed at a 1:20 dilution in saline solution with appropriate controls. Many of the
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TRADITIONALLY, most laboratories involved in testing individuals in disputed parentage cases have used only the ABO, Rh, and MNSs systems. 7 The chances of identifying a falsely accused man with these tests is just 58%.' Several court decisions, which increase the legal status of children born out of wedlock, and the adoption of the Uniform Parentage Act, 13 which facilitates the utilization of medical evidence regarding parentage in the courts, make the testing of additional polymorphic systems worthwhile. In fact, justice is more completely served when experts involved in providing medical evidence in paternity cases use additional reliable tests to increase their chances of reaching as correct a determination as possible. The need for this testing has been forcefully pointed out in the most recent revision of the Joint AMA-ABA Guidelines: "Present Status of Serologic Testing in Problems of Disputed Parentage." 3 The costs of such tests are small compared with the cost of child support. This report is to summarize the results obtained when immunoglobulin (Ig) allotyping was included in the testing
Minneapolis War Memorial Blood Bank, Minneapolis, Minnesota
282
A.J.C.P. . March 1979
SEBR1NG, POLESKY, AND SCHANFIELD
Table I. Reagents Used for Immunoglobulin Allotyping
erythrocytic typing limited to ABO, Rh, and MNSs provided evidence for only 163 exclusions.
Specificity*
Age of the Child and Gm Results
Numeric
Agglut inator
Coat
1 2 3 17
Hel, Pan Max Sta Pon
Dwi Yap Dan Dwi
s t
5 10 6 24 21 15 16
Ble Arr Alt' Hod Leh Gai Ros
Hun Hun 1872 1872 Sul Puh Puh
1
1
Sim, Cla
Abr
lameric a X
f z bl b5 c3 c5
e
younger children's samples also were typed at 1:5 and 1:10 dilutions. Any serum with agglutinating activity in saline solution was inactivated at 65 C for 10 minutes and retyped. Sera were typed with the reagents and for all of the markers listed in Table 1. In the results, any person who was positive for G3m (bl, b5) has been designated G3m(b). Definitions Exclusions were classified as direct or indirect. 4 Direct exclusions are those in which a haplotype present in the child is absent in both the mother and alleged father, or by the absence in the child of either of two nonidentical haplotypes demonstrable in the alleged father. Indirect exclusions are those in which a child lacks a genetic marker while the alleged father is homozygous for it, or the child is homozygous for a marker not present in both parents. Results Analysis of the results of erythrocytic antigen, erythrocytic enzyme, and serum protein typing tests in 925 cases of disputed paternity revealed exclusions in 230 cases (Table 2). Immunoglobulin (Ig) allotyping provided evidence for exclusions in 70 of these 925 cases. Direct evidence for exclusion was found in 39 cases by Gm typing and in 13 cases by Km(l) typing. Indirect evidence in the Gm system in 21 cases was corroborated by exclusions in other systems. In 15 cases the Ig allotypes provided the only evidence for exclusion; of these, nine were direct exclusions by Gm, three by Km(l), and three by both Km(l) and Gm. Ig markers and erythrocytic antigens provided evidence for exclusion in 203 of the total 230 exclusions found; however,
Racial Classification and Gm Results A difference in exclusion rates was observed when those cases with children 6 months old or older were divided into three racial classifications: those cases involving Caucasians, those cases involving Afro-Americans, and those cases in which the persons involved were not of the same racial heritage (Table 4). The ten Gm exclusions in cases involving non-Caucasians were Table 2. Exclusions in 925 Cases of Disputed Paternity
Systems Erythrocytic antigens Non-lg serum proteins and erythrocytic enzymes Ig allotypes Gm Km Cumulative total
Number of Individuals Excluded
Percentage of Cases with Exclusions
180
19.5
105 70* 60 13 230
11.4 7.6 6.5 1.4 24.9
* Three individuals were excluded by both Gm and Km(l).
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* Nomenclature recommended by the WHO Workshop on Human Immunoglobulin Allotypes, July 16-19. 1974. Rouen, France.
In order to ascertain the effect of age on Gm phenotype and the rate of direct Gm exclusions found, we have classified this series of cases into four groups according to the age of the child when tested (Table 3). Before 3 months of age, 83% of children were allotype-identical to their mothers, compared with 49% of children 1 year old or older. A Gm haplotype absent in the mother was found in 11% of children 0 - 3 months of age and in 29% of those 1 year old or older. The percentage of direct Gm exclusions showed an age relationship. It was 1.0% in children less than 6 months of age and 6.0% in children 1 year old or older. This difference, which is statistically significant, also exists when cases involving only Caucasians are compared: in 199 cases with children 0 - 6 months of age, two direct Gm exclusions were found (1.0%); in 459 cases with children 1 year old or older, 21 direct Gm exclusions were found (4.6%). Although there were 27 cases in which indirect Gm exclusions were found, only the 21 in which this was corroborated have been included in Table 2. Of the six cases not included in Table 2, four were omitted because the child was less than 6 months old when tested. It is possible that the IgG detected in these four children was maternal. In two cases the only indication that the alleged father may have been falsely accused was the indirect evidence found in the Gm system.
vol. 7i . No. 3
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GM AND KM ALLOTYPES IN DISPUTED PARENTAGE
Tabic 3. Effects of Age of Child on Gm Phenotype and Rate of Direct Gm Exclusions Found Gm Phenotype of Child Partially Identical to Mother's Phenotype
Age of Child (Months)
Number of Children Tested*
0-3 3-6 6-12 312
94(101) 102(105) 220(223) 484 (496)
Identical lo Mother's Phenotype
Non-identical Haplotype Present
Homozygous for One of Maternal Haplotypes
(%)
(%)
83 63 55 49
II 25 28 29
Direct Gm Exclusions
(%)
Number of Cases
Percentage of Cases
6 12 17 22
1 1 7 30
1.0 i.q 3.1 6.0
Number of cases in piirentheses. Difference is due to instances in which two men were accused of falhering the same child.
Km Results Analysis of Km(l) typing of mother-child pairs showed that 7% of children 0-3 months of age and 6% of those over 12 months of age were Km(l) positive with Km(l) negative mothers. Comparison in cases involving children of four different age groups showed no significant difference in Km(l) exclusion rates. Analysis of Km(l) typings by racial classification showed that 18% of Caucasians and 52% of AfroAmericans were Km(l) positive. The observed Km(l) exclusion rates were 12/871 (1.4%) in Caucasians and 1/42 (2.4%) in Afro-Americans. This difference is not statistically significant. Uncommon Gm
Haplotypes
Three infrequent Gm haplotypes were found in these cases: (1) Gm-'-'-h—One mother was of the phenotype Gm(z,a;g,b) with a most probable genotype of Cw z a ; B / Gm'-aM. However, two of her children were Glm(z,a) negative. Family studies, including testing for G2m(n), showed her true genotype to be Gmz-a- • ;B /Gm- : - : ". Negative results obtained for Glm and G2m markers have been indicated by ••. (2) Gmf;B—In three cases (six individuals), the recombinant haplotype Gm'-* was found. (3) GmKM'—In one case a child was of the phenotype Gm(x,f; b). There are no frequency data for the haplotype Gmx,Ub; however, it has been found in many Caucasian populations. Although the most likely genotype for a Gm(z,a,x,f; g,b) parent is Gm z,a ' !i;e /Cm f; ', the genotype Gm'-a'-R/Gmx-''b is also possible. In three cases either a child inherited Gmz-a:g from a (z,a,x,f; g,b) parent or there was indirect evidence for exclusion of the
alleged father. In another case, a child of Gm phenotype (z,a,x,f; g,b) either inherited GmxM' from his mother or there was indirect evidence for nonpaternity of the alleged father whose Gm type was (z,a; g). These four cases were not interpreted to be Gm exclusions; however, each father was excluded by findings in other systems. In three of the 925 cases an increased likelihood of paternity was indicated by Gm typing. In two cases involving Caucasians the alleged father and child had the Oriental haplotype G m z a ; b 5 s l . In one case the alleged father and child both had the recombinant Gmm. Two Men Accused of Fathering the Same Child Ig allotyping was informative in 25 instances in which the woman accused two different men of fathering the same child. Combining all systems, 23 of the accused men were excluded. In each of the two instances in which neither man was excluded, the alleged fathers were brothers. Ten alleged fathers were excluded by Ig allotype. Seven of these men would not have been excluded if only erythrocytic typing had been done: the evidence for nonparentage was found for two in both the Gm and acid phosphatase systems, one in the group-specific component protein system, one in Table 4. Observed Gm Exclusion Rates by Race— Child 6 Months Old or Older
Caucasians AfroAmericans Mixed
Excluded All Systems Combined
Excluded All Systems Except Gm
Excluded Gm
Number of Cases
No.
%
No.
%
No.
672
172
25.6
165
24.6
46
6.8
37 10
18 4
48.6
17 3
45.9
7 3
18.9
7c
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fourld by G1 m (a, x, or f) typing in three, G3m (c or s) typing in six, and G3m (g) typing in one.
SEBRING, POLESKY, AND SCHANFIELD
284
Table 5. Exclusion Rates in Caucasian Cases with Children >6 Months Old Exclusion Rate Observed
Expected
Erythrocytic antigens ABO Rh MNSs K
124 39 70 56 8
.6425 .2021 .3627 .2902 .0415
.5935 .1537 .2767 .3112 .0400
Erythrocytic enzymes acP PGM ADA AK 6-PGD
50 29 15 8 5 2
.2591 .1503 .0777 .0415 .0260 .0104
.4121 .2231 .1541 .0516 .0429 .0146
Non-Ig serum proteins Hp Gc Cp Tf
26 20 7 0 0
.1347 .1036 .0363 .0000 .0000
.3260 .1845 .1618 .0123 .0018
Ig allotypes Gm Km(l)
54 46 10
.2798 .2383 .0518
.2881 .2394 .0640
Cumulati ive total
171
Fy* EsD* Cumulative total
14 4 172
.8865 .1647 .0471
.1840 .0740 .9167
* Subset of 290 of above cases (78 exclusions) also typed for Fy", Fy\ EsD.
the adenosine deaminase system, and three only by immunoglobulin allotype. Observed Exclusion Rates In Table 5, the number of observed exclusions, the expected exclusion probability (based on appropriate gene frequencies), and the observed exclusion rate (based on the number of proven exclusions divided by the theoretical number of falsely accused men) are shown by individual test system for those cases involving Caucasians and children 6 months old or older. The theoretical number of falsely accused (193) was obtained by dividing the number of proven falsely accused (171) by the cumulative exclusion probability (.8865). Discussion Methodology The selection of genetic systems for use in paternity testing should be predicated on the usefulness of the system, the ease of testing, the specificity and sensitivity of the test, and the stability of the markers to be tested. Ig allotyping is simple, sensitive, and reliable. The actual testing can usually be completed in two
hours. Unlike reagents for HLA testing, Ig allotyping reagents are normally monospecific, or can be rendered monospecific quite readily by absorption. The majority of Ig typing systems will detect 1-10 /u,g/ml of Gm- or Km-specific antigen; thus, normal adult sera can be diluted 1/20-1/40 and normal infant sera 1/5-1/10. Unlike many genetic systems that undergo rapid changes during storage, the antigens of human Ig are remarkably stable. Usefulness The Ig markers proved to be a useful system in our paternity test battery. Combined results for all test systems (cumulative exclusion probability .9167, excluding Jka, Jkb) provided evidence for exclusion of paternity in 230 of the 925 cases studied. Erythrocytic typing for ABO, Rh, and MNSs (cumulative exclusion probability .5784') provided evidence for 163 of these exclusions. Including Ig markers with the tests for erythrocytic antigens provided evidence for exclusion in 203 cases. In 15 of the 230 cases in which men were proven to be falsely accused, the Ig allotype provided the only evidence for exclusion. In 25 instances in which two men were accused of fathering the same child, Gm typing excluded ten of the alleged fathers. Five of these ten men would not have been excluded if only erythrocytic typing had been done. The observed exclusion rates for Gm and Km (Table 5) were close to the expected values, confirming in practice the usefulness of these markers in determining nonparentage. We have no explanation for the deviations in observed exclusions compared with expected exclusions in the other test groups. The usefulness of Gm allotyping in paternity testing is dependent on the number of factors tested and the haplotypes detectable. The incidence of Gm haplotypes varies with racial heritage. The probability of exclusion in Caucasians based on a representative Minnesota population11 and use of reagents for Gm (a, x, f, and bl) is 0.24. These reagents are capable of detecting the three common Caucasian haplotypes. With these same reagents, the exclusion probability for Afro-Americans based on gene frequencies obtained in a Northern United States Black population8 is 0.17. However, the exclusion probability for this same Afro-American population is 0.33 when reagents for G3m (z, g, b5; c3, c5; and s) are added to the test battery. Of ten exclusions in non-Caucasians, seven would not have been identified if only reagents for Gm (a, x, f and bl) had been used. Not only is the use of an extended battery of reagents important in order to maximally exclude falsely accused men, but in some instances the results may indicate a high likelihood of paternity. In three Caucasian cases, the alleged father and child were both
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No. of Individuals Excluded
A.J.C.P. . March 1979
Vol. 71 • No. 3
GM AND KM ALLOTYPES IN DISPUTED PARENTAGE z,a:b5,s t
11
Although the Gm exclusion rate was affected by the age of the child when tested, this was not true of Km(l), a marker found on kappa light chains of all immunoglobulins. The exclusion probability of Km(l) in Caucasians is .064 and for Afro-Americans .119. In this series, the observed exclusion rates, 1.4% and 2.4%, for these two groups were not significantly different. Pitfalls In interpreting evidence for exclusion in the Gm system, certain sources of error should be kept in mind. As described above, the presence of only maternal IgG or transient hypogammaglobulinemia in very young children may lead to a misinterpretation of results. It is also essential that the Ig test system include a screen for saline antibody activity in each serum against each coated erythrocyte. When interpreting indirect evidence of exclusion, as in other systems, deleted, recombinant, or mutant haplotypes and all possible genotypes must be kept in
mind. In the present study, the haplotypes Gm--'-*, Gmle, and Gm x,f;b were observed. In addition, the haplotypes Gm z ' a : - ; -, 5 Gmz'-'-R,e Gm-'-'-s,10 and Gm'-"'-14 have been found. Of these uncommon haplotypes, Gm f;g , Gm-'-'-z, and Gmz'-:s cause problems in interpretation of paternity test results when G3m(g) testing is not done. Unfortunately, dosage studies of serum in an individual with a deleted Gm haplotype may not show decreased levels of antigen due to compensatory increases in the allelic antigen. 5 The possible presence of Gmx-!'-b should be considered in all cases in which the alleged father or mother is Gm(z,a,x,f; g,b). It should be remembered that the majority of individuals with the phenotype Gm(z,a,x; g) are Gw z a x ; K /Gm z a ; B and not G/n z a x : g /G/n z a x ; B . Many of the totally deleted haplotypes originally reported in the Gm system have proven to be recombinants or only partially deleted haplotypes when tested with a more complete battery of reagents. The null haplotype found by Steinberg and co-workers 12 in a group of related individuals of a Hutterite community has not been observed in other populations. References 1. Dykes DD, Polesky HF: The usefulness of serum protein and erythrocyte enzyme polymorphisms in paternity testing. Am J Clin Pathol 65:982-986, 1976 2. Dykes DD, Polesky HF: Paternity testing by using erythrocyte enzyme esterase D. J Forensic Sci 22:173-177, 1977 3. Joint AMA-ABA Guidelines: Present status of serologic testing in problems of disputed parentage. Family Law Q 10:247— 285, 1976 4. Lee CL: Current status of paternity testing. Family Law Q 9: 615-633, 1975 5. Lefranc G, Rivat L, Rivat C, et al: Evidence for "deleted" or "silent" genes homozygous at the locus coding for the constant region of the gamma 3 chain. Am J Hum Genet 28:51-61, 1976 6. Natvig JB, Michaelsen TE, Gedde-Dahl T Jr, et al: IgG, subclass protein with the genetic Gm markers Gm (z+ non-a + ): Another example of intragenic hybridization among IgG subclass genes. J Immunogenet 1:33-41, 1974 7. Polesky HF, Krause HD: Blood typing in disputed paternity cases—Capabilities of American laboratories. Transfusion 17:521-524, 1977 8. Schanfield MS: Unpublished data 9. Schanfield MS, Polesky HF, Sebring ES: Gm and Inv typing, Paternity Testing. Edited by Polesky HF. American Society of Clinical Pathologists, Chicago, 1975, pp 45-53 10. Sebring ES: Unpublished data 11. Sebring ES, Polesky HF, Schanfield MS: The risk of immunization to IgG following Rh immune globulin therapy. Transfusion 14:220-225, 1974 12. Steinberg AG, Muir AW, Mclntire SA: Two unusual Gm alleles: their implications for the genetics of the Gm antigens. Am J Hum Genet 20:258-278, 1968 13. Uniform Parentage Act, adopted by the National Conference of Commissioners on Uniform State Laws (1973) 14. van Loghem E, Natvig JB: Uncommon Gm gene complexes. Vox Sang 18:421-434, 1970
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found to have either G/M - or Gm'' . These two haplotypes have an approximate frequency of 0.005 in Caucasians. Neither of these haplotypes would have been evident with the use of a limited number of reagents. Usefulness and interpretation of Gm results in paternity testing are partially dependent on the age of the child when tested. At birth IgG is almost totally maternal, in the ensuing months a transition occurs to IgG with an individual's own genetic determinants. Therefore, two problems may arise in the interpretation of Gm typing of very young children. One is that an indirect exclusion of paternity may be thought to exist when it does not. In this study, 83% of children 0 - 3 months old were phenotype-identical to their mothers, compared with only 49% of children who were 1 year old or older when tested. Because of this age effect, Gm exclusions of paternity in very young children are valid only when the allotype of the child differs from that of the mother. The second problem is that a child may be too young to express IgG of paternal type, and it is thus possible to miss a direct exclusion. The frequency of detectable paternal antigen in children of this study who were 3 months old or younger was significantly less (x 2 (ldf) = 26.7, P =
