UNIT VII. HEMOGLOBIN, SERUM GROUP SYSTEMS, HLA AND OTHER GENETIC MARKERS Hemoglobin

SECTION 38. HEMOGLOBIN 38.1 Introduction Hemoglobin (Hb)is the major protein of human red cells, forth. Hemoglobins exist which contain only one kind of comprising about 95% of their dry weight. Adult human chain: Hb H is B4, for example, and Hb Bart's is 7,. blood normally contains from about 4 million to 6.5 million Jones (1961) pointed out that hemoglobin structural red cells per mm3 blood, the average figure being slightly heterogeneity can be classified as follows: (1) Maturation higher for men. Hemoglobin itself is present in concentra- heterogeneity, which refers to the fact that different tions of about 14 to 16 g per 100 mP blood. It is the oxygen hemoglobins are normally synthesized during different transporting protein in higher animals; without a molecule stages of development. There are embryonic, fetal and adult having its properties, complex multicellular aerobic life as hemoglobins. (2) Minor hemoglobin heterogeneity, which we know it would not be possible. refers to the presence of small amounts of structurally dif- Hemoglobin is one of the most extensively studied of all ferent but normal hemoglobins along with the major proteins, and its literature fills many volumes. As noted in component characteristic of a particular stage of develop- section 5.1, it acquired its present name over 100 years ago ment; and (3) genetic heterogeneity, which refers to the (Hoppe-Seyler, 1864). In forensic serology, hemoglobiin is various "abnormal" hemoglobin variants. Many of these important in two principal contexts: (1) Blood is normally are thought to be the result of point mutations, and with a idenad in questioned samples by procedures designed to few exceptions, variant hemoglobins are very rare. The ex- demonstrate the presence of hemoglobin; and (2) hemo- cellent review of hemoglobins by Huisrnan (1%9) was globin exhibits a very large number of genetic variants, a organized according to this clasiication of heterogeneity. few of which are comparatively common and conveniently 38.2.1 Normal adult hemoglobins detectable by electrophoretic and/or isoelectrofocusing procedures. Many of the reactions of hemoglobin, which The structural studies on normal adult hemoglobins were form the basis of blood identification techniques, depend prompted in part by the recognition that the sickle cell con- upon the heme moiety of the molecule. These were discussed dition represented a diWete mol~~ularalteration in the in detail in unit 11, sections 4 through 7. Hemoglobin Hb molecule. Structural studies on nonnal adult and fetal, v-~s, which can serve as genetic markers, are discussed and variant hemoglobins were carried out simultaneously in in this section. ~bF, which discussed in section 8.3.1 as an effort to relate the detailed structures to the genetics. a marker for the bl&d of fetuses and young children, is also Most of these studies occurred in conjunction with the discussed here. Studies on hemoglobin variants and their development of presentday understandings of biochemical detailed structures have contributed sisnificantly to our cur- genetics (section 1.2.2), and have contributed importantly to rent understanding of molecular genetics. them. The major normal adult hemoglobin is called Hb A. It 38.2 Hemoglobin Structure has been called Hb A,, Ao and AII at different times (Holm- The structure of hemoglobin can be described at several quist and Schroeder, 1%6a), but these latter usages are now different levels. First, the molecule is a subunit protein, and discouraged (see in section 38.2.3.6). Hb A is a tetramer may be described in terms of the polypeptide chains that composed of two a and two /3 chains, and its structure is writ- make it up. Second, the detailed structure of each type of ten a2B2.Each polypeptide chain is associated with a heme subunit polypeptide chain can be described in terms of group (Figures 4.4 and 4.5). Intact Hb A thus has 4 heme amino acid sequence, and in terms of helical and nonhelical groups, and its MW is 64,450. The complete amino acid se- regions. Finally, the x-ray data have made possible a de- quences of the a and 6 chains were worked out in the early tailed description of the three-dimensional structure for 1%0's, and are shown in Figure 38.1 (Braunitzer et al., some of the hemoglobins. Detailed structures will be dis- 1%1; Konigsberg et al., 1%1). The sequencing studies have cussed briefly in the sections below. been well reviewed by Braunitzer et al. (1964). Part of the The hemoglobiin molecule is tetrameric, consisting of four stability of protein molecules arises from the a-helical associated polypeptide chains held together by noncovalent arrangement of the polypeptide chains (section 1.1.2.1). forces. One heme group is associated with each polypeptide This structw feature was predicted by Pauling and Corey, chain. Most hemoglobins consist of two a chains and two and has since been found to occur widely in nature (Pauling, none chains. The non-cr chainscan be 8, y, 6, e or J= Normal 1W). The helical structure of the polypeptide chains of Hb adult hemoglobm is designated Hb A, and consists of two a has been worked out using x-ray crystallographic and other aud two /3 chains. Its structure may thus be written: ~~282. techniques. The polypeptide chains consist of a series of Sily,Hb A2,a minor adult hemoglobin, has the struc- helical regions which are periodically interrupted. These tun ~26,.Normal fetal hemoglobin, Hb F, is azyl, and so helical regions are indicated by upper case letters, as shown Sourcebook in Forensic Serology, Immunologv, and Biochemistry

I Pha 40 (6 Pro - Thr - Thr . LI. - Thr .Tvr - Phe .Pm - Hi. - Phe - Asp - Lmu -S.r C - CO ICE1

m 66 80 55 Gly ,Ala -Val -Ah - Awn .Thr .L.u .AIa - Asp - Ah .Val - Lvr .Lvs - Glv -%- Gly - Lys -Val - Gln - AIa - Sn / "M /nh ' 0. I

80 90 AIa - Leu .Sar - AL. .Lau - Sm - Hh- L*. .LWU- Arp \ IFFG Val I 110 106 100 95 Asp Leu - Hb - Ah -Ah - Lou - Tkr .Val - Lou - Leu - Cvs - His - Sar .Leu .Lw - Lva .Phe - Ann -Val - Pro

115 A$ on Glu 120 126 130 135 140 - me- Tkr .Pm .Ah -Val - Hls .All. Ser - Leu - Asp - Lys - Phs - Leu - AID - Sar -Val - Sar .Thr - Val - Leu. Thr - Sar .I.*. - Tvr - Arp n HC

6 10 15 Val - His .Leu - Thr - Pm - Glu - Glu - Lvs - Sar - Ala - Val - Thr - Ala - Lau - Tq- Gly - Lvs - Val NA L 6. cnnw 25

36 Tvr 40 45 W 55 Pm -Try - Thr - Gln - Arg - Phe - Phe - Glu - S.r - Phs - Gly - Asp - Leu .Ser .Thr .Pro .Asp .AIa . Val - Met C- CB 0 -1 GlY 75 70 65 60 Asn ,Hh Ah bu Glv Asp S.r .Phe Ah Gly Lau Val Lvs Lvr Gly Ah Lvs Val Lvs Pro - - - - - . . . - . - - [email protected] - . - - I L*U 0, AS; 1 80 Am EF I Leu \ T*. \ Gq 15 90 % Thr- Phe - Ah .Thr - Lwu - Ser - Glu - Leu .%-Cys - Asp - Lvs 7 -F Leu I FG 115 110 106 100 ni. GI* .Phe .His . Hi. .Ah - L.u .Val. Cv8 - Val - Leu - Val - Asn .Glv - Leu - Lmu - Arr - Phe - Asn - Glu .Pro - Asp -Val ./ I 120 LVS I ..- I GH Glu 125 130 135 110 145 \ ph. .mr.pro . pro .Val - Gln .Ah - Ah - Tyr - Gln - Lvs - Val .Val .Ala - Gly - Val - Ah - Asn - Ala - Leu - Ala - His .Lys .Tyr - His ' I nc

Figure 38.1 a and 6 Chain Sequences in Hemoglobin. Helical regions are denoted by capital letters. The a 50 - 51 residues are denoted D6 - D7, Dl - D2. and CD8 - CD9 by different authors. There are no non-helical sequences BC and DE in a ; since there is no helical sequence D, CD is followed by E. There are no nonhelical sequences AB, BC and DE in 6. The proximal and distal histidines, which interact with heme, are boxed. Hemoglobin in Figure 38.1. The location of a particular amino acid genetically distinct from Hb A. In 1%1, Ingram and Strat- residue in the chain may thus be indicated in two ways: fust, ton discovered that Hb k2is not made up of the same poly- all the amino acid residues are numbered sequentially from peptide chains as Hb A, consisting instead of or chains and 6 the N-terminal end of the chain (as is the usual convention); chains. Hb A2has the subunit formula ~~262.The sequence of and second, the position of the amino acid in a particular the 6 chain has been determined, and is shown in Figure helical region (numbering from N-terminal to C-terminal) 38.2. Hb A2 represents about 2% of hemoglobins as a rule, may also be given. Transitional regions, which lie between but this proportion can be altered by pathological condi- two helical regions, are designated using two letters, the one tions. The other "minor" hemoglobins are not as well char- representing the immediately preceding (N-terminal side) acterized in general, but are thought to result from post- helical region, and the one representing the immediately synthetic alterations of the polypeptide chains, rather than following (C-terminal side) one. The N-terminal amino from distinct genetic loci. Hb AI,, for example, appears to acids which precede the first (A) helical region are denoted have hexose (probably glucose) condensed with the "NA", while the C-tennind amino acids which follow the N-terminal amino acid of the @chain (Holmquist and last (ZF) helical region are denoted "HC". A few examples Schroeder, 1%4, 1% and 1966b; Lehman and Huntsman, of this nomenclature (see in Figure 38.1): (1) the N-terminal 1974). Hb AI, comprises 5 to 7% of normal hemoglobins. amino acid of the a! chain, valine, can be called "I", or Another minor hemoglobin may come about through the "NA 1"; (2) the Asn residue of the a! chain is "68" or combination of Hb A with glutathione. It is now recom- "E17"; (3) the 6th residue of the i3 chain is Glu, and is mended that the minor hemoglobins be designated Hb Az designated @6(A3);and (4) the Phe residue of the 6chain at (ad2) and Hb AI (fast moving zone in electrophoresis at position 42 is @2.(CDl). The helical designations are ex- alkaline pH). Components of Hb AI are Hb Ah, Ab, etc., tremely useful in comparing different chains for structural and components of the subfractions are AI,,, Am, etc. homologies and differences. Present nomenclature recom- mendations call for the use of both the absolute position 38.2.2 Nonnal embryonic and fetal hemoglobins and the helical region position designations in specifying a In 1867, Korber first described the differentiation of particular residue (see in section 38.2.3.6). The exact loca- animal hemoglobins by the rate at which they denature in tion of the helical regions in the polypeptide, and the exact alkali. In addition, he found that fetal Hb was much more three-dimensional structure of the hemoglobin molecule resistant to alkali denaturation than adult Hb. This property have been determined for the most part by the x-ray may still be used to distinguish between fetal and adult crystallographic studies of Perutz, Kendrew and their col- hemoglobins (section 8.3.1). The principal hemoglobin of laborators in England. These studies actually began in the cord blood at birth is called Hb F, normal fetal hemoglobin. 194.03, but were interrupted by World War 11. They were Hb F is found in fetuses after about 10 weeks of gestation. continued after the war, and were directed at determining Its level increases for a time until, at bi, it is found the complete structures of human and animal hemoglobins together with Hb A. After about 6 months, Hb F all but and myoglobins. The solution of these structures represents disappears. Hb F comprises only about 1% of normal adult one of the brightest moments in protein chemistry. The blood hemoglobins (Chernoff, 1953b). Hb F can be distin- structure of horse Hb was obtained before that of the guished from Hb A by differential sensitivity to alkali dena- human molecule (Perutz, 1%2 and 1%5; Perutz et al., 1960, turation (Kiirber, 1867; Singer et al., 1951), chro- 1%4 and 1%5; Muirhead and Perutz, 1%3). In these papers matographic techniques (Allen et al., 1958; Huisman et d., as well as in many of the reviews cited in this section will be 1958), electrophoresis (Culliford, 1%; Huehns, 1968) or by found drawings of the threedimensional mnfonnation of immunological methods (Chernoff, 1953a and 1953b; hemoglobin and photographs of the molecular models that Goodman and Campbell, 1953; Diacono and Castay, 1955). were constructed based upon the measurements. Hb F is made up of two cr chains and two y chains (Schroeder It has been known for many years that Hb A is not the and Matsuda, 1958). Its tetrarneric formula is azy2.The a only hemoglobin in adult red cells. Hb A2 was observed by chains in Hb A and Hb F are identical (Schroeder et al., Kunkel and Wallenius in 1955. Chromatographic techniques 1%3a). The amino acid sequence of the y chain has been have revealed the presence of several "minor" hemoglobins worked out (Schroeder et al., 1%3b) and is shown in Figure in adult blood. Huisman et al. (1958) detected two minor 38.2. It is now known that at least two slightly different y fmctions in addition to A. Others detected a number of chains are normally synthesized, and occur in Hb F (see minor components, which were designated AI, AIII, A,, A2, below). There are minor components of Hb F, just as there A, and so forth. Subfractions related to AI were called AI, are of Hb A (Allen et al., 1958). Hb FI, which can account through AI, (Allen et al., 1958; Clegg and Schroeder, 1959; for 10% of the fetal Hb, differs fmm Hb F in that the Schnek and Schroeder, 1961; Jones and Schroeder, 1963; N-terminal residue of the y chain is acetyiated (Schroeder et Atassi, 1%; Holmquist and Schroeder, 1964, 1%6a and al., 1%2). It was first thought that Hb FIwas a2FyN,where 1966b). The nomenclature of the components is quite com- -yF was the usual Hb F y chain, and yN was the plex, because different workers have used different designa- N-acetyl-Gly. ... . y chain. It appeared that there was tions, and because the number of minor hemoglobins seen more to it, however (Huehns and Shooter, I!%), and with depends on the separation technique employed. Hb A2 is the finding of the different kinds of y chains, the detailed Sourcebook in Forensic Serology, Immunology, and Biochembtry

5 10 15 20 25 Val - - - Leu - Smr - Pro - Ala - Aap - Lys - Thr - Asn - Val - Lys - Ale - Ab - Try - Gly - Lya - Val - Gly - Ale - His - Ala - Gly - Glu - Tyr - Gly - Ala

5 10 15 20 25 Gly - His - Phe - Thr - Glu - Glu -Asp - Lys - Ala - Thr - Ile - Thr - Ser - Leu - Try - Gly - Lys - Val - Asn - Val - - - - - Glu -Asp - Ala - Gly - Gly - Val - His . Leu - Thr - Ro - Giu - Glu . LvS - Ser - Ale - Val .Thr - Ab - Leu - Try .Gly . Lys - Val - Asn - Val - - - - - Asp - Glu - Val - Gly . Gly . Val - His - Leu - Thr - Pro - Glu - Glu - Lya - Thr - Ah - Vel - Asn - Ab - Lau - Try - Gly - Lys - Val - Asn - Val - - - - - Asp - Ale - Val - Gly - Gly -

30 35 40 45 U) Glu - Ab - Leu . Glu - Arg - Met - Pha - Leu - Sar - Phe - Pro - Thr - Thr - Lvs - Thr - Tvr - Phe . Pro - His - Phe - - -Asp - Leu - Ser . His - Gly . Ser -

30 35 40 45 50 Glu - Thr - Leu - Gly - Arg - Leu - Leu - Val - Val - Tyr - Pro - Try - Thr - Gln - Arg - Phe - Phe -Asp. Ser - Pha - Gly - Aan . Leu - Ser - Ser - AIa - Ser . Glu . Ala - Leu .Gly - Arg .Lau - Leu - Val - Val - Tyr - Pm - Trv - Thr - Gln - Arg - Phe - Pha - Glu - Ssr - Phe . Gly - Aap - Leu - Ser - Thr - Pro .Asp - Glu - Ala - Lau - Gly - Arg - Leu - Lau - Val - Val . Tyr - Pru - Try - Thr - Gln - Arg - Phe - Phe . Glu . Ser - Phe - Gly - Aap . Leu - Ser - Ser - Pro -Asp -

% BO 65 70 AI~- - . ------Gln-Val -Lys-Gly -a-Gly. Lys- Lys-Val -Ale -Asp-Ale- Leu.Thr-Asn- Ala. Val - Ala- His-Val -Asp-

I 60 65 70 75 Ala - IIe - Met - Gly - Asn - Pro - Lys - Val - Lya - Ala - $484#& - Gly - Lys - Lya - Val - Leu - Thr . Ser . Leu - Glv - Asp - Ale - IIe - Lys - His - Leu - Asp . Ale - Val - Met- Gly - Asn - Pro - Lys - Val - Lys . Ab -$@;- Gly - Lys - Lys - Val . Lau - Glv . Ala - Phe - Ser - Asp - Gly . Leu . Ala - Hia - Lau .Asp. Ab - Val - Met - Glv - Aan - Pro - Lys - Val - Lys . Ala -a!.Gly - Lys - Lys - Val - Leu - Gly . AIa - Phe - Ser - Asp - Gly . Leu - Ala - His - Leu - Asp -

75 (10 85 90 95 100 Asp - Met - Pro - Aan - Ala - Lau - Ser - Ala - Leu - Ser - Asp - Leu .m-AIa - His - Lyr - Leu . Arg - Val - Asp - Pro - Val - Am - Phe - Lys - Leu - Leu -

a0 85 90 95 100 105 Asp - Lau - Lvs - Gly - Thr - Phe - Ab - Gln - Lau - Ser - Glu - Lmu - - Cya -Asp - Lys - Leu - HIS - Val .Asp - Pro - Glu - Asn . Phe - Lys - Leu . Leu Aen - Leu - Lys - Gly - Thr - lha - Ah - Thr - Leu - Ser - Glu - Leu -B- Cys -Asp - Lys - Leu . HIS - Val -Asp . Pro - Glu - Aan - Phe - Arg - Leu . Leu ,yx Asn - Leu - Lys - Gly .Thr - Pha - Ser - Glu - Leu - Ser - Glu - Leu -&- Cys -Asp. Lys - Leu - His - Val - Asp - Pro - Glu - Aan - Pha . Arg . Leu - Leu

106 110 115 120 125 Ser - Him - Cyr - Leu - Leu - Val - Thr - Leu - Ala .Ala - His - Leu - Pro - Ale - GIu - Phe - Thr - Pro - Ala - Val - His - Ala . Ser - Leu .Asp - Lys - Phe -

110 115 120 125 130 Gly -Am - Val - Lau - Val - Thr - Vel - Leu - Ala - Ile - His - Phe - Gly . Lys - Glu - Phe - Thr - Pro - Glu - Val - Gln . Ala - Ser - Try - Gln - Lys -Met. Gly - Aan - Val - Leu - Val - Cya - Val - Leu - Ala - His - His - Phe - Gly - Lys - Glu - Pha - Thr - Pro - Pro . Val - Gln - Ala - Ala - Tyr . Gln - Lys - Val . Gly -Am - Val - Lou - Val - Cya - Val - Leu - Ala .Arg - Asn - Phe - Gly - Lys - Glu - Phe - Thr - Pro - Gln - Met - Gln - Ala - Ala - Tyr - Gln - Lys - Val -

134 135 140 Leu - Ale - bar - Val - Ser - Thr - Val - Lau - Thr - Sar - Lys - Tyr - Arg.

136 140 145 Val - Thr - 'Iy - Val - Ah - Ser - Ah Leu - Ser - Ser - Arg - Tyr . Mia. A* - Vel - AIa - Qly - Val - Ala - Aan - Ale - Leu - Ab .HIS .Lys - Tyr - His. Val - AIa .Gly - Val - AIa - Asn - Ala - Leu - AIa . His - Lys - Tyr - His.

Figure 38.2 Sequences of the a, B , Y and b Chains of Hemoglobin. Sequences are arranged to show the maximum sequence homologies between chains. Large dashes represent 'Braunitzer gaps' (see text). The histidyl residues which interact with heme are half-toned. Not all authors place the gaps identically. Hemoglobin structure of Hb FIwill undoubtedly be found to be more nated Ty. Schroeder et al. (1979) believe that Ty is a product complex than originally thought. of a mutant Ay locus, and suggested that the chain be called The sequences of the a,B,y and 6 chains are shown to- T%. A review of fetal and embryonic hemoglobins was gether in Figure 38.2. One of the interesting features of the given by Lorki (1973). Recommendations have recently sequences, in view of what is now known about the genetics been made by an international body regarding standardized of Hb,is the sbdarity of sequence structure among the dif- Hb F preparations (International Committee, 1979). ferent chains. This similarity is called structural or chemical The tetrameric structural formulas of the normal adult, "homology". The structural homology can be maximized fetal and embryonic hemoglobins are summarized in Figure by introducing theoretical "gaps" into the chains at certain 38.3. points. These are called "Braunitzer gaps", and they do not Adult really exist, of course. figure 38.2 has the Braunitzer gaps in order to illustrate the maximum chain homologies. In 1954, Drescher and Kiinzer noticed that there seemed to be another hemoglobin in fetal blood that differed from Hb F. Its alkali denaturation curve was intermediate be- tween that of Hb A and Hb F. Halbrecht and Klibanski Fetal and Embryonic (1956) confirmed another hemoglobin in very early fetuses by electrophoretic and alkali denaturation differences. Hb F a2 YZ (aZGYAY) Allison (1955) referred to this early hemoglobin as "primitive". In 1%1, Huehns et al. found not one but two Hb Gower I ?, &2 hemoglobins in early fetuses (less than 10 weeks of gesta- Hb Gower II a2 &Z tional age), which they called Hb Gower 1 and Hb Gower 2. These names are still used. Additional studies on these "em- Hb Portland I y2 < bryonic" (as against "fetal") hemoglobins indicated that they contained a new polypeptide chain, designated E. Hb Gower 2 had the structure a2e2,while Gower 1was thought Figure 38.3 Tetrameric Structure of Adult, to consii entirely of the new chain, i.e. e (Huehns et al., Fetal and Embryonic Hemoglobins 1964 and 1%4b). In very young embryos, Hb Gower 1 is the dominant Hb (Hecht et al., 1%6), and as the embryo 38.2.3 Genetic v-ts of hemoglobin develops the proportion of Gower hemoglobins decreases as 38.2.3.1 Introduction. Obviously, the "normal" hemo- that of Hb F increases. In 1%7, Capp et 01. found a new globins just discussed are genetically different, in that dif- hemoglobin in a baby girl in Portland, Oregon, who had ferent genes are responsible for the synthesis of the different been born with multiple abnormalities. This hemoglobin chains comprising them. The "genetic variant" hemo- had two y chains and two chains that were not yet identified, globins, however, are those which exhibit structural differ- and was called Hb Portland 1. This Hb was soon found in ences from the "normal" hemoglobins. Hundreds of such normal infants as well (Hecht et al., 1%7), and is now variants are now known, and in most cases the exact struc- believed to be a normal embryonic hemoglobin. The second tural difference is known. Many of the variants are very rare kind of polypeptide chain in Hb Portland 1 was a new one, and can be explained by mutations. A few variant kinds of and was designated c (Capp et al., 1970). Hb Portland 1 is Hb reach polymorphic frequencies in certain populations, thus y2c2,and it is now thought that Hb Gower 1 is be2,in- presumably because there is some selective advantage to stead of e, as first thought. There are thus three embryonic their possessors. In some cases, the selective advantage can hemoglobins, distinct from Hb F. be explained, such as in the case of Hb S. As noted briefly above, there is some further complexity The fvst "abnormal" hemoglobin to be studied in detail in Hb F, which has the structure a2y2.Additional sequence was Hb S, or sickle cell hemoglobin. The recognition that studies on the y chains revealed a rather startling finding: sickle cell trait and sickle cell anemia represented the that Hb F normally contains two different kinds of y chains heterozygous and homozygous conditions of a variant gene, (Schroeder et al., 1968). These chains differ only at position and the fmding that Hb S differed from Hb A in a single 136, which may be occupied by Gly or by Ala. The resulting amino acid, ushered in an era in human biochemical y chains are designated Gy and %, respectively. All the Hb F genetics. Hemoglobin thus became a model system for re- samples studied had both kinds of chains, from which had lating genetic variation to protein structure at the molecular to be drawn the extraordinary genetic conclusion that there level. Developments in protein separation methodology were at least two y chain loci, and that they were nonallelic. along with the solutions to the three-dimensional stmcture There is now good evidence for the nonallelic y chain loci, of hemoglobin have made it possible to find many variant and the matter is discussed further in section 38.3. There is hemoglobins, and to diagnose the differences in structure another complexity in y chain structure. Ricco et al. (1976) between normal and variant polypeptide chains. Since Hb A found a Hb F with still a different y chain. The new chain and Hb F are constructed from a, 0 and different y chains, had Thr at position 75@19) instead of Ileu, and was desig- each coded for by different genetic loci, hemoglobin Sourcebook in Forensic Serology, Zmmunologv, and Biochemistry variants are often categorized according to the chain in serve as a means of differentiating them anthropologically which the variation occurs, i.e. a! chain variants, 8 chain (Beet, 1946). In addition, he recognized that "sickle" was variants and y chain variants. The simplest type of Hb inherited in a simple Mendelian fashion, based upon studies variants have a single amino acid substitution in one of the of a large pedigree (Beet, 1949). chains. There are variants which have two substitutions in In 1949, Neel proposed a fonnal genetic scheme for the the same chain. There are also more complex variants, such inheritance of sickle cell trait and disease. He regarded the as those with extended chains, frameshift mutations, deleted sickle gene as a dominant characteristic. Very shortly there- residue chains and the so-called fusion hemoglobins. after, Pauling and his collaborators showed that Hb A and 38.2.3.2 a! Chain variants. The a! chain consists of 141 Hb S could be separated electrophoretically, and further amino acids, and single amino acid substitution variants that sickle cell trait people had about half Hb A and half Hb have now been found at many of the positions. Huisrnan S, while sickle cell anemics had all Hb S. This finding in- (1%9) noted that 40 possible variants had been reported, 22 dicated a simple codominant manner of inheritance in which of which had been fully characterized. Lehmann and Hunts- both genes were expressed in heterozygotes. The presence of man (1974) could list about 65 a chain variants in which the an S gene could thus be shown to produce a separate mole- substitution had been characterized. In 1975, the Intema- cule, and Pauling et al. (1949) termed sickle cell anemia a tional Hemoglobin Reference Center was established at the "molecular disease' '. This new concept represented a differ- Medical College of Georgia in Augusta. Its purpose is to ent way of looking at numerous metabolic disease states, consolidate and organize all the information about Hb and hemoglobin served as the model system in its subse- variants that appears in the literature, and which may be quent development (Itano, 1953). Sickle cell Hb was clearly sent to the Center. A number of hemoglobin variants have different from normal adult Hb (Pauling et al., 1949; several names, because some variants discovered in different Allison, 1957). In 1956, Ingram and his collaborators began laboratories and differently named have later turned out to publishing their studies on the exact chemical difference be- be the same. The Center periodically communicates lists of tween Hb A and Hb S, the results of which opened up the variants in the new journal Hemoglobin. These lists are cited present period of molecular genetic inv-on. By a in this book as "IHIC Variant List, Year published", and technique called peptide mapping, Ingram (1956) showed the references are given under the same designation in the that Hb A and Hb S differed in a single peptide. Subsequent reference lists. By late 1976, the number of defined a! chain studies quickly demonstrated that Hb S differed from Hb A variants was 73 (IHIC Variant List, 1976a), and is probably in a single amino acid residue (Ingram, 1957, 1958 and 1959; greater now. A few u chain miants (not selected for any Hunt and Ingram, 1958a and 1958b). Hb S contained Val particular reason) are shown in Table 38.1. where Hb A had Glu. Hemoglobin C (Itano and Neel, 1950) 38.2.3.3 /3 Chain vmiants. The first variants of hemo- was soon found to have a Glu'Lys substitution at the same globin descriied were chain substitution vatiants, and a position as the one in Hb S (Hunt and Ingram, 1%0). description of the way the early variants were characterized Hemoglobin D was first described in Los Angeles, but is almost equivalent to a description of the early develop- reaches high frequencies in the Punjab. It is thus called Hb ment of hemoglobin biochemical genetics. Not surprisingly, D Los Angeles, Hb D Punjab, and a variety of other names; the first variants studied were those that are comparatively it is often simply called Hb D, although there are other common. Because of their comparatively more frequent hemoglobins called "D", which are different. The com- occurrence in populations, they are also the most important paratively common Hb D (Los Angeles or Punjab) has a genetic markers from a forensic point of view. Glu-~Glnsubstitution at position 121 (Baglioni, 1962a). Hb Sickle cell hemoglobin is the "premiere" variant of Hb A. D Ibadan, for example, is a 887 ThrLys variant, while Hb Many of the pioneering studies on Hb have been canied out D Iran is 822 Glu-Gln. Hb E was found by Itano et al. on sickle hemoglobin (Hb S). The sickling of red cells from (1954) using electrophoretic separations. It was shown to a patient was first noted by Dr. Herrick in Chicago, in 1910. represent a Glu-Lys substitution (Hunt and Ingram, 1959 Hahn and Gillespie (1927) studied the sickling behavior of and 1961). red cells from affected people, and showed that the red cells There are now known to be many 13 chain variants, and a could be induced to sickle in vitro under conditions of low sample of them is shown in Table 38.1. Huisman (1969) oxygen concentrations, and that they would regain their listed 45 variants. Lehmann and Huntsman (1974) showed shape if the O2level was brought back to normal. Detailed over 120. The IHIC Variant Lists (1976b, 1977a and 1978) studies on the medical and physiological consequences of showed 138, 164 and 173 variants, respectively. sickle cell trait and anemia have been dedout over the There are some hemoglobins that have a double substitu- years by Digs (Diggs and Ching, 1934, Dim, 1956 and tion in the 13 chain. Hb C Harlem is a good example (Book- 1%5), among others. The condition was recognized as being chin et al., 1%7). Its 6 chains are substituted 6Glu+Val and inherited, and largely restricted to Black populations, in 73Asp+Asn. Moo-Pem et al. (1975) found a quite remark- 1934. Before the genetic details of sickle cell had been able 35 year old Black man who was heterozygous for Hb S worked out, a government hospital worker in Rhodesia and Hb C Harlem. looked at the frequencies of "sickle" in two different popu- 38.2.3.4 y Chain variants. There are fewer y chain sub- lation groups, and concluded that this characteristic could stitution mutants characterized than there are a or /3 ones. Hemoglobin'

Table 38.1 Some Hemoglobin Variants

Common Nama Synonyms SclmntHic Dalgnnlon Refannca achaln

J Toronto a 6 1A3I AIa - Asp Cmokamn at -1.. IS66 Anamkanj a 11 IUI Llt - Glu Poomkul m al., 1976 IPhlhdalphb I:ITaras: IBurlington a 16 (A141 LV - GIU Baala and Lahmann. 1% Foe Worlh a 27 IEEI Glu - Glv Schnsidar at al.. 1971 Uml: Kokun: L Fanan Mlhlp.n I: Mlhban II: a 47 (CDS or CE~)bp - GW Bbnco m al.. 1- Yukuhashl II:L Gallni *We a 47 ICD6 or CSI Asp - Asn Rshbar at *I., 19% M~mpomrfv a 48 ICD6 01 CEO) L.u - Arg Brlmhlll at a!., 1976 J Maxlco: J Parb II: M~I~CO a 61 IE3) Gln - GIU Jonas at *I.. Ism: ksass m .I.. 119 upp.81a J RmCppan a M lFG21 Lw - Thr Hvda at el.. 19n Chhpam a 114 IGHZI Pro - Arg Jonas at *I.. 1%8

6 Cluln

S 6 6 IA3l Glu - Val Ingram. IS7and 1%9 C 6 6 (A31 GIu - Lvs Hunt and lnpnm. l9MI 8skI 6 14 (A111 Leu - Pro Bauasrd n al.. 1#76 J Trlnldad: J Ireland: J Balmore 6 16 (A131 Glv - Asp DaJong and Want. 1W J Gwrgb 0 Taip.1 6 P (B4l Glu - Glv Bbckwall at 11.. 1989 E 6 M lB6I GIu - Lvs Hunt and lngnrn. 1961 Abbmma 6 S) ICSI Gk - Lva Bwhall at .I.. 1975 AMln 6 40 (C6I Arg - %I Moo-Pmn at .I.. 1977 Ar)lans. Gaorgla Waso 6 40 IC6l Are - Lva Moo-Pann at at.. 1977 G Copanhagan 6 47 (C06l Asp - Asn Sick at 81.. 13~ J Kaohsiung J Honolulu 6 69 IE31 Lvs - Thr Bbckwmll at a!., 1971 J Cambrldga J bmhrn B 69 (El31 Gly - Asp Skk at a!.. 1967 Aanu 6 76 (El91 Lou - Pro Hubbard at at.. 1975 D Lo8 Angaln: D Punimb. D D Chlugo: D North hmllna: 6 121 lGH41 Glu - Gln Baplloni. l-: DaJonp and Went. 1968 D Potw0.1: Oak Ridge Bmognd B 121 IGH4l Glu - Val Efmmov at al.. 1973

Y Chaln

F Auskland 'Y 7 (A41 Asp - AM Canall .t .I.. 1974 F KUII. LUI~PU, ^Y nIWI ASP - GIY Lie-lnjo at al.. 1373 F Vlctori. Jubilaa '7 80 IEUI Asp - Tyr Aharn at at.. 1976

Chain

A, Rwsw-A b 20 1821 Val - Glu Rider at al.. 1976 A, Malbourne b 43 (CD2l GI" - Lvs Sharma st al.. 1974 A, Indonula 6 69 IE131 01.1 - Arp Lia.lnjo at at.. 1971 A, Coburp 8 116 lGl8l Arg - HI0 Shsrrna at at.. 1975

Dal.1.d Rnlduas

Laldan 6 6 or 7 IA3 or A41 Glu missing DaJong at .I.. 1966 Lyon 6 17-16 (Al4-A161 Lvs-Val mbslng Cohan.SoIal at el.. 1374 Fraiburg 6 23 1861 Val missing Jones at .I.. 1-

Extanded Chains

Con.um Spho a + 31C I142 Glnl Csgg st 81.. 1971

Fuslon Hmmogloblns

Lapon Holhndia 1 11-PI 6150-1461 Barnabas and Mullar. 1%2 Lapore Balmor* b 1140l ElaS-lwl Ostemp and Smkh. 1969 Sourcebook in Forensic Serology, Immunology, and Biochemistty

The finding that Hb F normally contains two different y they are abnormal. They are usually associated with ane- chains, Gy and % (section 38.2.21, means that the Presence mias and other hematological disorders. of either Gly or Ala at 7136 is nod. Variants at other There is a class of anemia syndromes known as tholas- positions thus have to be characterid at 7136 as well as at semias. The term is derived from the ancient Greek word for the "variant" position. Further, 775 can be occupied by "the Sea" (meaning the Mediterranean). It turns out to either Thr or by neu. It is now recommended (see in section mean "sea in the blood", rather than Mediterranean 38.2.3.6) that these chains be separately designated: anemia, which it was coined to denote (Lehmann and AYI = y75Ile; 136Ala; = y75Thr; 136Ala; Huntsman, 1974). Theusage has persisted in theliterature, GyI = y75Ile; 136Gly. There are now about 15 y chain even though the thalassernia syndromes are not restricted to variants (IHIC Variant Lists, l977b), and the subject has Mediterranean peoples. In effect, a thalassemia syndrome is been recently reviewed.by one of the pioneers in the field characterized by an imbalance of a or 8 polypeptide chain (Schroeder, 1977). A sample of y chain variants is shown in production. a-Thalassemias are those in which there is Table 38.1. underproduction of cu chains, while 8-thalassemias represent underproduction of 6 chains. 8-Thalassemia is the classical 38.2.3.5 Other vmiants and other hemoglobins. A kind of Mediterranean (aoley,s anemia), and is the number of variants of the 6 chain have been found in Hb Az. most impomt in terms of frequency of occurrence. It is The IHIC Variant Lists (Irn) lo, and a controlled by a gene which behaves like an "allele" of the P them is included in Table 38.1. A few other types of variants chain structurai gene. meterms "8 thalassemia major,, and are known. The majority of variants represent single amino thwmia minor- indicate ~omozygos~tyand he- acid substitutions; a few (like Hb C Harlem, mentioned in erozygosity, respectively, for the gene, although section 38.2.3.3) have a double substitution. Three other may these terms to indicate the severity of the types of variants will be mentioned here: deletions, extended -festations. In he a-thwmias, there is an under- chains and fusion hemoglobins. Deletion variants are those production of a and a coTTespondingexcess of P which lack one or more d o acids found in the usual chains in adults and of in fetuses and children. polypeptide hai in. A few them are shown in Table 38.1- Before any of this was clearly understood, two hemoglob'm~ Hb Freiburg is an example. It is missing the Val residue at without a were d-bed; ~bH is 8, and B ~is Y ~ 823, and is designated f123val-'0. The IHIC Variant y,. The latter was apparentlynamed after St. Bartholomew's (1977b) show lo variants. chain Hospital, where the baby in whom it was first seen was born hemoglobins have more amino acid residues on the ( ~ and H ~ ~ 1974).h ~is~ now known~~ that ~~b ~~ , C-tenllinal end of the chain than are normally found. Hb H ,d ~b b art'^ ariseby the same basic mechanism. There Constant Spring (Clegg et aL, 1971), for example, has 172 two models for the inheritance of a-thalasse-. In the residues in the a chain instead of 141. Some authors show it fust, there are two deliC a-thalaSSe- genes: the severe as "a141 31 additional residues" in a chain variant lists. a thb- gene and an a thalaSSemia2 gene. There are 7 extended chain hemoglobins in the IHIC The four possible com~inations give rise to a of in- Variant Lists (197%)- The fusion hemoglobins are very in- creasingly serious clinical conditions. The 2 teresting, and their existence has implications for the struc- heterOYgotes are least affected, while the atha[ 1 ture and arrangement of the 8, y and 6 chain genes at the homozygotes are stillborn in the last weeks of pregnancy. In level. In lgS8, and found a most the second model, one must accept that the a chain struc- peculiar in which they tural gene is duplicated (for which there is some evidence). "Lepore"* Characterization the @aglioni* Then, either one, two, three or all four a chain genes can be 1%2b) showed that the non-a chains appearedto be made Up affected by thalassemia, again giving rise to the series of of an N-terminal section of the 6 chain attached to a four conditions of increasing clinical severity ( K and ~ ~ ~ ~ C-terminal section of the 6 chain. The "break" Occurs Lehmann, 1970). The full explanation for all of the between what would be 687 and 8116, but because of the se- thal-- syndromes is somewhat more complex than has quence homology of the chains (Figure 38-21, the exact low- been indicated, and there is still more to be learned about tion cannot be determined. This 6-6 fusion chain was aPPar- them. Thal-fia syndromes have recently been reviewed ently made by a new gene having portions of 6 and portions by ~~~k (1978). of 8. There are other examples of these 6-8 fusion mutants Methemoglobine- simply means that met-Hb present now (Table 38.11, and they are sometimes called "Le~ore" i, the red cells at clinically sifl1-t levels. Met-Hb is hemoglobins. Individual examples are wmedby their hemoglobin in which the heme ironis in the ~ ~ 3 +and place of origin or discovery, such as Hb Le~oreBoston, Hb it -ot bind oxygen. Methemoglobinemia can come about Lepore Hollandia, etc. Huisman et 01. (1972) found a in a number of ways, only one of which fl be discussed hemoglobin, Hb Kenya, whose none chain is a 776 fusion here. As shown in Figure 38.1, the heme moiety of Hb is hybrid. The "break" is between 781 and 886. The -1MC slung between two histidyl residues (His58 and His87 in the a Variant Lists (1977b) show 7 fusion hemoglobins. chain, and His63 and His92 in the 8). The histidyl-nitrogen There are a few other hemoglobins which should prob- of the proximal histidine (a87 and 892) is coordinated to the ably not be considered "variants" strictly speaking, though Fez+of heme, while the distal hiiidyl-N (a58 and 863) per- Hemoglobin mits space for the binding of molecular oxygen. This struc- not followed because of the multiple polypeptide chain ture forces the iron to retain the ferrous state. A few structural loci controlling hemoglobin. By the time the letter hemoglobins have been found in which an amino acid sub- "Q" was reached, it was apparent that there were not going stitution occurs at a58 or 863. These disrupt the stability of to be enough letters in the alphabet. Hemoglobins began to the heme-a chain or heme-8 chain interaction, and allow be named after places, hospitals or people. Some exotic methemoglobin to form in quantity. As a result, they are names were proposed, such as 'Aida', 'Riverdale-Bronx' called the "M" hemoglobins. Substitutions at other posi- and 'Abraham Lincoln'. Some of the variants acquired tions in the chains can disrupt the heme-peptide chain many names, because they were discovered and rediscovered stability too. Hb M Milwaukee (898Val-Glu) causes the all over the world. It was not until the substitution or heme iron to take on the femc state. chemical alteration was clarified that the identities became A final condition that will be mentioned is called apparent. In these cases, the name given by the fust observer "hereditary persistence of fetal hemoglobin" or HPFH. usually prevails. There are a number of anemias and other conditions that A number of hemoglobin variants are distinguishable by can lead to the presence of Hb F after infancy. In HPFH, electrophoresis in various buffer systems. New variants have however, there is continued production of Hb F into the sometimes been designated according to their similar elec- post-infancy years because 8 and 6 chain synthesis has not trophoretic mobility, or their similarity in other properties, been "switched on". There are several possible genetic ex- to a known Hb,followed by the descriptive name. Thus the planations for this phenomen, but it has been attributed to original Hb D W121(GH4) Glu-Gln] is called Hb D Los the presence of a "high F" gene. While this explanation is a Angeles, D Punjab, D Chicago, D North Carolina, etc. But convenient way of thinking about HPF'H, it is probably too there are also Hb D Bushman (B16 GlpArg), Hb D simple. Indeed, different examples of HPFH may have dif- Iran U322 Glu-GIn) and Hb D Ibadan US7 WLys). ferent explanations. The "high F" heterozygotes have 10% Some of the variants have no capital letters in their designa- to 40% Hb F. Homozygotes who have been studied make tions: Hb Sawara (4Asp-Ala), Hb Winnipeg (a75 no Hb A nor any A2. hp-Thr), Hb Deer Lodge (82 Hi~Arg),Hb Alabama The material in section 38.2 has been reviewed on many (839 Gh-Lys), etc. occasions. The older literature is well reviewed by The latest recommendations for standardizing the Hb Braunitzer et al. (1%4), Schroeder and Jones (1965) and by variant nomenclature (Recommendations, 1979) cover a Huehns and Shooter (1%5). More recently, there is Giblett number of different aspects, some of which have been (1%9), Huisrnan (1%9), Lehmann and Huntsman (1974), pointed out in the foregoing sections. For the major nonnal Bunn et al. (1977a) and Bank et a[, (1980). The papers in the hemoglobins, Hb A (ad2)and Hb F (cu2y2)are used. Desig- major N. Y. Academy of Sciences conference (Kitchin and nations such as 4,Au, F,, and FU are discouraged. The Boyer, 1974) covered many aspects of hemoglobin. The so- embryonic hemoglobins are Gower-I (J;E~), Gower-I1 called hemoglobinopathies are well reviewed by Bunn et d. (0262) and Portland-I (S;yr). The only letter designations (197%) and by Huisrnan and Jonxis (1977). The latter book recommended for the abnormal hemoglobins are Hb C, Hb gives detailed procedures for the characterization of hemo- E, Hb S and Hb H. The remainder should have the descrip- globin variants. tive name following the letter, and many variants will have 38.2.3.6 Hemoglobin nomenclature. Just as in many only a descriptive name. Previously, it was the practice to other genetic marker systems, the nomenclature of Hb vari- show the alteration as a superscript. Thus, Hb G Georgia ants has developed somewhat unsystematically. More re- was cu295(G2)Pr0+~932,while Hb S was cr42qA3)Gh-va, and cently, there have been efforts to systematize it. Some old so forth. It is now recommended that only the variant chain, names are so entrenched in the literature that they have been residue number (sequential and helical) and the amino acid retained. With the IHIC now established (section 38.2.3.2), substitution be shown, without the use of superscripts. nomenclature standardization should prove to be consider- Thus, Hb G Georgia = &S(G2)Pro+Leu and Hb S = 86 ably easier than previously. (A3)Glu-Val. The designation "Hb M" is retained for ab- In the early years of Hb variant research, capital letters normal hemoglobins that have an increased tendency to were used to designate each new kind of hemoglobin that methemoglobin formation, e.g. Hb M Boston = a58(E7) was found. Hb A was nonnal adult hemoglobi. The letter WLys.The deletion mutants should signify which residues "B" was apparently skipped because sickle cell Hb had are "missing", e.g. Hb Freiburg = @23(B5) missing. Fusion sometimes been called B. Everyone agreed on Hb S for hemoglobi are designated so that the segments referring to sickle cell Hb, and soon after Hb S was characterized and the types of chains are identified, e.g. Hb Lepore- electrophoresis came into widespread use as a screening pro- Boston = 6(1-87)8(115-146). The elongated variants are de- cedure for hemoglobins, the commoner variants were noted by specifying the chain, the number of additional resi- quickly discovered. C, D and E were found not long after S dues found at the carboxy-terminus (C), and the residue im- had been characterized. Hb F has long stood for fetal hemo- mediately following the normal C-terminus, (i.e. position globin. The letter designation nomenclature was formalized 142 in CY and 147 in 8). Thus Hb Constant Spring = a by Chernoff et d. (1953). In 1955, AUison suggested that + 31C (142Gln). Hb H is retained for j3,, and Hb Bart's for

Wb be the gene locus name for hemoglobi, but this usage is 74. Sourcebook in Forensic Serology, Immunology, and Biochemistry

38.3 Biochemical Genetics of before the m-RNA is transported to the cytoplasm from the Hemoglobin nucleus. Introns have been identified in the non-cu chain coding regions of the human genome. The biochemical As has been noted, hemoglobin variants have provided a genetics of hemoglobin synthesis was recently reviewed by model system for human biochemical genetics. Now that the Forget (1979) and by Bank et al. (1980). genetic code is known (Table 1.4), it is possible to speculate In his remarks opening the major hemoglobin conference intelligently about the kinds of base sequence changes in at the New York Academy of Sciences in 1974 (Kitchin and DNA that could be responsible for the single amino acid Boyer, 1974), Prof. Motulsky summarized the importance. substitution variants. Indeed, this kind of analysis can help of hemoglobin research to modem biochemical genetics in the understanding of the evolution of mutations in the (Motulsky, 1974): human species (see, for example, Beale and Lehmann, 1965; Hemoglobin research plays a role in human biochem- Shaw et al., 1977; and Vogel, 1%9). ical genetics similar to that of drosophila research in In addition, studies of the Hb variants have yielded much formal genetics and analogous to work with microor- information about the molecular structure of the poly- ganisms in microbial genetics. Many fundamental con- peptide structural genes themselves. It has been clear for cepts have become clarified by investigations on human some time that the a and @ genes were on different hemoglobins. chromosomes (Diesseroth et al., 1976). It is now clear that the a gene is on chromosome 16, while the @ and y gene loci 38.4 Methods of Separating and are on chromosome 11 (Diesseroth et al., 1977 and 1978). Characterizing Hemoglobins There is growing evidence that the a chain locus is dupli- The most important method of separating normal and cated in most people (Nute, 1974; Forget, 1979). It is also common variant hemoglobins is electrophoresis. Dozens of quite clear now that there are two y gene loci per haploid set different electrophoretic procedures have been devised over of chromosomes, one locus coding for the y136Gly (Gy) the years for this purpose. Because some of the variant chain, and the other coding for the y136Aia (k/)chain hemoglobins have clinically sigdicant consequences, there (Schroeder et al., 1972; Schroeder and Huisman, 1974). have been a number of mass screening efforts in various This point was discussed in section 38.2.2. The chains can be populations. Apart from their medical value in identifying present in different proportions in Hb F, suggesting further people who may require treatment, or who may be advised complexity at the genetic level (Huisman et al., 1972). The to seek genetic counseling, many variants have been identi- so-called Ty chain, reported by Ricco et al. (1976) and fied through these studies. As has been noted, these variants discussed in section 38.2.2, has y75Thr instead of y75Ile. have been characterized and are of considerable biochemical Schroeder et al. (1979) regard Ty as the product of a genetic significance. Population distributions of the more modified ky locus and said that the chain should be called widely occurring variants are of anthropological and foren- Thus, it appears that most individuals have a duplicate sic interest as well. Although most hemoglobin variants are set of a genes on chromosome 16, and a series of closely link- rare, it has been estimated that 1 in every 2,000 persons car- ed y, 6, and @ genes on chromosome 11 (Schroeder and ries a detectable Hb variant (Motulsky, 1974). Huisman, 1974; Deisseroth et al., 1977 and 1978). The The oldest electrophoretic procedures employed paper or organization of the chromosome 11 genes is thought to be starch blocks as support media. Procedures using agarose, G4Lu6@ (Little et al., 1979). This arrangement helps in under- CAM, and polyacrylamide gels have since been devised. standing how control of the fetal-to-adult "switch" may Some workers still prefer paper electrophoresis (Lehmann come about at the level of DNA (Kabat, 1974), although the and Huntsman, 1974). Most forensic serologiscs probably exact control mechanisms are not yet known (Forget, 1979). utilize cellulose acetate membranes or starch or agarose gels In addition, the Lepore type (fusion) hemoglobins and Hb for Hb separation. Isoelectric focusing techniques have also Kenya (see in section 38.2.3.5) can be understood in tern been used to separate Hb variants. of non-homologous crossovers between 66 regions (Lepore) Depending upon which hemoglobins are to be separated, or between y@regions (Kenya) because of misalignment of different buffer systems are used. Hemoglobins which co- sister chromatids at meiosis. electrophorese in one system may separate in another. It is Until recently, it was assumed that the information base sometimes necessary, therefore, to run selected samples in sequence in DNA was colinear with that of the m-RNA, and more than one system to distinguish those types that have that the m-RNA sequence was, in turn, colinear with the the same mobility in the first system. Three "basic" buffer amino acid sequence of the protein (section 1.2.2). Recent systems are used for paper (and most other kinds of) electro- finc structure studies have revealed the extraordinary fact, phoresis, although dozens of minor variations have been however, that the coding sequences of DNA are commonly introduced. These are a barbital buffer, pH 8.6-8.9, a interrupted by intervening sequences of DNA of variable Tris-EDTA-borate buffer, pH 8.6-8.9 and a phosphate length (introns) which are not represented in mature buffer, pH 6.5. The alkaline buffers give good separations m-RNA. The sequences are transcribed initially into pre- of Hb A, S and C. The Tris system resolves Hb A2 much cursor m-RNA, and the m-RNA molecules then have these better than barbiial. Hb A and Hb F are not well resolved in sequences removed enzymatically by a splicing process these buffers; neither are C and E, nor S and D. At pH 6.5, Hemoglobin all the hemoglobins migrate cathodically except Hb H. terized. The antisera, however, are specific for the amino Paper electrophoresis is discussed in detail by Weiss (1%8) acid substitution in the chain, and are, therefore, extremely and by Lehmann and Huntsman (1974). specific if a mixture of hemoglobins is being investigated. Starch gel electrophoresis is somewhat more cumbersome 38.5 Medicolegal Applications and timeconsuming than CAM electrophoresis. For many applications, barbital or Tris-EDTA-borate buffers in the 38.5.1 Disputed parentage pH 8.68.9 range are used. The relative electrophoretic No references to the application of Hb variants as such to mobilities of a number of hemoglobins in pH 8.6 buffers are parentage problems were found, and it does not appear that indicated in Figure 38.4. Starch gel electrophoresis of hemo- hemoglobin is widely used for this purpose. According to a globins has been reviewed by Huehns (1968), and cellulose recent survey of laboratories in this country (Polesky and acetate membrane techniques are given by Chin (1970). In Krause, 1977), about 25% of the 30 AABB Reference Labo- 1957, Robinson et al. proposed using agarose gel electro- ratories could do Hb typing in the cases, and somewhat phoresis in acidic (sodium citrate, pH 6)buffer for Hb sepa- fewer did it routinely. About 17% of the other laboratories ration. The system gave a good resolution of Hb A and Hb said that Hb typing was available, but less than 2% used it F, and additionally differentiates Hb S from D and Hb C routinely. The main reason for using Hb typing would be from E. Relative electrophoretic mobilities of some hemo- detection of Hb S in cases involving Black subjects. The globins in acid citrate buffered agarose are shown in Fie probability of excluding a falsely accused Black father on 38.5. Agarose gels have been used at alkaline pH as well the basis of Hb S typing is about 4.5% (Chakraborty et al., (Lepp and Bluestein, 1978). Breen et al. (1%8) reported im- 1974). The value would be less for Hb C and Hb D, since proved resolution of hemoglobins on cellulose acetate with they are less common. Hb E would be a useful parentage the Beckman Microzone system using Tris-glycine, pH 9.3, marker in certain Asian populations. buffers. Jacobson and Vaughan (1977) reported a rapid starch gel procedure for mouse hemoglobins. Schneider and 38.5.2 Hb Typing in bloodstains Hightower (1977) have studied the behavior of dozens of For practical purposes, the hemoglobins that might be different hemoglobins on agarose gels using ciVic acid buf- diiosed in forensic cases are A, F, S and C. Hb D and E fers at acid pH. can be separated from Hb S and C under certain electro- In 1977, Burdett and Whitehead used polyacrylamide gel phoretic conditions. Investigations involving young children isoelectrofocusing to separate hemoglobins in the pH 5-8.5 may call for Hb F differentiation. This may be achieved in a range. Hb A, A2,S, F, D, C and E could be resolved in this number of ways, including immunological, electrophoretic, system. In 1978, Bassett et al. applied PAGIF in the pH and by alkali denaturation characteristics. This subject was range 6 to 9 to the study of about 70 different Hb variants. discussed in section 8.3.1. It would, of course, be infoma- They obtained good resolution of many of them, and said tive to find significant amounts of Hb F in an adult's blood, that the technique provided a good screening procedure if since this condition is comparatively rare. Hb F can be dis- very thin gels were used in order to conserve the costly tinguished from Hb A in three ways: (1) differential alkali arnpholines. denaturation behavior; (2) immunologically; and (3) electro- Electrophoresis is the method of choice for Hb screening phoretically. The fact that Hb A is considerably more alkali- in most laboratories. CAM methods are popular because labile than Hb F was noted by Kijrber (1867), and studied in they are so fast and require very small amounts of material detail by Singer et al. (1951). This method of differentiation if microtechniques are used. Identification of Hb A, A2, S, was applied to bloodstains by Culliford (1964). He followed F, D and E can be accomplished fairly readily by electro- the denaturation in the visible region. Watanabe (1%9) said phoresis. It is necessary to use different electrophoretic tech- that the reaction could be followed in the W. A description niques for certain of these. Samples running like Hb S at pH of the procedure is given in Culliford (1971), where he says 8.6, for example, can be run in citrated agarose to see that caution should be exercised in trying to differentiate Hb whether Hb D is present. Identification and characteimion F by this method in bloodstains more than about a week of other hemoglobins normally requires more sophisticated old. The technique is also described in the most recent techniques. PAGIF is a good approach to Hb separation in methods list from the London laboratory (MPFSL, 1978). laboratories equipped for it. If it is necessary to diagnose the Apparently, it is primarily a back-up for electrophoretic or amino acid replacement in a variant Hb, peptide "finger- immunological techniques. printing" and sequencing techniques must be used. Peptide Immunological procedures for the differentiation of Hb fingerprinting is discussed very clearly by Lehm and A and Hb F depend upon having comparatively qxcXic Huntsman (1974), as is the use of amino acid analyzers. anti-human Hb for the purpose. Efforts to prepare anti-Hb Cohen-Solal et al. (1974) discussed a specialized sequencing F have been going on for many years @arrow et al., lW, procedure. Recently, Garver et al. (1977) have employed Ikin et al., 1953), and antisera to human Hb was first RIA techniques to the identification and quantitation of prepared by Klein (1904, 1905a and 1905b), following the several variant hemoglobins. It is clearly necessary to have work of Leblanc (1901) and Ide (1902) on animal hemo- the monospecific antiserum in order to use this procedure, globins. This subject was discussed in sections 7.1 and 8.3.1. so it is useful for variants which have already been charac- Anti-Hb F procedures have been developed for bloodstains Soumebook in Forensic Serologv, Immunology, and Biochemistry

Hb A (Hb Seattle; Hb MI

Hb S (Hb D; Hb Zurich)

I Hb Bart's

Hb Gower I

Hb Gower II

t t t

ORIGIN A 2 A

Figure 38.4 Relative Electrophoretic Mobilities of Some Hemoglobins at pH 8.6 Hemoglobin

I HbA (Hb A,; HbII Hb F (Hb Victoria Jubilee)

I Hb S (Hb C Harlem)

Hb D

Hb Montgomery (Hb Mahidol)

Hb G Georgia (Hb Athens)

Hb G San Jose

Hb British Columbia

Hb 0 Arab

Hb H

t t t S A F ORIGIN

Figure 38.5 Relative Electrophoretic Mobilities of Some Hemoglobins of pH 6 in Agarose Sourcebook in Forensic Serology, Immunology, and Biochemistry using immunoelectrophoresis (Depieds et d, 1%0), Ouch- disulfide formation between glutathione and globin chains. terlony double diffusion, and crossed over electrophoresis These observations may be related to those of Barnard and (Baxter and Rees, 1974a; MPESL, 1978; Wiggins and Wrax- Grunbaum (1979). all, 1979). Most forensic science laboratories are not speciality Electrophoresis is the favored procedure for separating, hemoglobin laboratories. Differentiation of Hb A, F, S, C, and thereby identifying, not only Hb A and Hb F, but also D and E can be accomplished fairly readily. Beyond that, C, S, D and E. Pollack m al. (1958) fm suwedthe appli- though, known standards are unlikely to be available. There cation of Hb typing to bloodstain cases. They used paper are now hundreds of variants of Hb, and most of them are electrophoresis, and wuld differentiate Hb A and Hb S in not readily distinguishable by electrophoresis. More com- bloodstains on a robbery victim's clothing in a case in which plicated characterization techniques, such as peptide finger- the suspected assailant was Hb AS. They suggested that A printing and sequence analysis, are beyond the scope of and F might be separable as well. Hb A and F do not most forensic laboratories. In addition, fairly large samples separate very well in the classical alkaline (pH 8.6-8.9) buf- are required which may not be available, and it is not com- fer systems. In 1972, Wraxall reported that good A and F pletely clear that Hb isolated from dried blood is amenable separation could be obtained using barbital pH 8.6 cathodic to such analysis. Thus, while one might diagnose a sample tank and Tris-EDTA-borate pH 9.1 anodic tank buffers. as AC or AS or AD according to its electrophoretic mobil- This system was also applicable to A-S and A-C sepamtions ity, there is a possibility that the sample is really a rare and is described in Culliford (1971). Willdns and Oepen variant with a comparable mobiity. While the majority of (1977) confmed Wraxall's (1972) findings. In 1962, Hunts- variants are indeed rare, it would not be surprising for a man and Lehmann applied paper electrophoresis to the fairly busy casework laboratory that types Hb routinely to separation of Hb A, S, C, D and E in bloodstains a few days encounter one of them on occasion. According to Motulsky old. Hb D and F could be differentiated by alkali denatura- (1974) about 1 in 2,000 people has a detectable variant tion, they said. Culliford (1%4) used the discontinuous buf- hemoglobin. If a rare variant were encountered, and could fer system of Poulik (1957) to separate Hb A from S and C be identified (perhaps with the help of spechhed Hb labo- on Oxoid CAM. The procedure devised by Wraxall (1972) ratory), it would be highly individualizing and informative. was adopted later (Word, 1971), and Sartorius CAM's were preferred. Recently, Wiggins (1978) applied the acid 38.6 Distribution of Common citrate agarose electrophoretic method to the differentiation Hemoglobin Variants in U.S. Populations of Hb S from D and Hb C from E in dried blood. Using this The data are shown in Table 38.2. The most common procedure, Hb can be run in the alkaline system (Wraxall, variant hemoglobin is probably Hb S. The population 1972) to distinguish A, F, S and C, and a series of rare genetics of Hb S, and by analogy to what is known of other variants (MPFSL, 1978). The Hb AS or S, and AC or C Hb variants, suggest that the mutation causing S has oc- samples are then checked in the citric acid agarose system to curred more than once. There is not much doubt that West distinguish S from D and C from E, and thus giving far bet- Africa represents at least one point of origin. Hb S has ap- ter population discrimiaation when the rarer types are en- parently reached polymorphic proportions in areas of the countered. The way this technique works may be better ap- world in which malaria was (or is) endemic. Heterozygotes preciated in a general way by comparing Flgures 38.4 and (Hb AS) have an advantage against malaria, and this situa- 38.5. Baxter and Rees (1974b) described Hb typing in con- tion has been called "balanced polymorphism". The gene junction with their routine Hp typing procedure (section exists in other parts of Africa, in India and other parts of 39), and said that A, S, C and D could be distinguished. Mi,and in parts of Europe. The gene may now be seen in Hb F could be presumptively identified as well. any population into which there have been any significant Hb typing by electrophoresis is usually quite straight- migrations from high S regions. In this country, Hb S is forward, provided the Mons of the techniques are largely, but not exclusively, restricted to Blacks of African recognized and suitable Hb standards are available. In 1979, origin. Hb S may be seen in Mi,and it is occasionally Barnard and Grunbaum looked at a series of Hb A, AS and seen in Whites (e.g. Crane et al., 1977). It is not clear from AC stains up to 28 days old, however, and noted that most of the reports of Hb S in Whites whether the hemo- aqueous extracts of the bloodstains could exhibit altered globin has been rigorously identified as the 66Glu+Val mole- electrophoretic mobility relative to the standard hemoly- cule, or whether a different variant which looks like S elec- sates. The effect was reversible by extracting the bloodstain trophoretically could be present. It is, of course, possible with Cleland's reagent (Dm).Reversal of the obsewed ef- that I33 S that is 86Glu+Val arises periodically in a family by fects by DTT strongly suggests that these alterations were spontaneous mutation. Hb D was fm described in Los based on sulfhydryl effects of some kind. Some years ago, Angeles, but occurs in significant frequencies in certain Huisman et al. (1966) noted that Hb A, S and F showed Asian Indians. It is also seen in Pakistan, Iran and similar effects upon 4' storage of hemolysates. Dialysis Afghanistan. It is seen in Europeans, particularly those tended to prevent the effects, while reduced glutathione from countries which had close ties with India, and it has tended to enhance them. These workers thought that the been noted that Hb D was most common in this country in storage effects could be understood in terms of mixed people who were partly of English origin. Hb C was Hemoglobin detected in American Blacks. It is present in West Africa, site on DNA, adjacent to the 6 chain structuralgene. Hb E is but almost absent in East Africa. It is also seen in Euro- principally an Asian type, being found at appreciable fre- peans upon occasion. There is recent evidence (Kan and quencies in Burma and Indochina. It is not common in India Dozy, 1980) that the S and C mutations arose independ- nor in modem Chinese. Lehmann and Huntsman (1974) ently, and that Hb AS arose by several independent muta- provide a thorough discussion of the world population tional events m different places. This conclusion has been dimibutions of various hemoglobins. The best and almost reached by studying the relationship of Hb S and C to a only comprehensive reference source to population frequen- polymorphism for a restriction endonuclease recognition cies is Livingstone (1%7). Table 38.2 Distribution of Common Hb Phenotypes in U.S. Populations

Phenotypes - Number IPercentl Number Reference

CAUCASIAN

Beltlmore. MD Smlth end Conley, 1963

Ann Arbor. MI Noel. 1964

Houaton. TX Heynle el el.. 1967

Durham. NC 1"non-Black") ChernoH and Welchaelbeum, 1968

Southern Loulslmne lchlldrenl MoHltt and MoDowell. 1969

Memphla. TN erne Iautopayl McCormlch. 1960

St. Loulr. MO Ilnfentsl Mlnnlch et d., 1982

Baltlmora, MD (Infants) Weetherall. 1W3

M1dsslppl Thompson et rl., 1804

Callfornle Qrunbmum et el.. 1978

Detroit, MI Stolorow et el., 1979

Cellfornle Crunbeum et el.. 1960

NEGRO

Beltlmore. MD Smlth end Conky. 1863

Ann Arbor. MI Noel. 1964

St. Loula. MO ChernoH. 1%8

Oalveston. TX Ipatlentsl Schnefder, 1966

Houston. TX Havnle el el.. 1967

Durham. NC ChemoH end Welchaelbeum, 1SW

Southern Loulalena lchlldrenl Moffln end McDowell. 1969

Puerto Rlco Suaret et el.. 1969

Phlladelphie, PA lpetlents) Myeraon at el., 1968

Beltlmore. MD Marder and Conlsy 1969

Memphia. TN area leutopsvl McCormlch. 1980 Table 38.2 (Cont'd.)

Phenotypes - Number IParcentl Populetlon Number -A -AS S -A0 -D -AC -C Retarence Weahlngton. D.C. Ituberculousl 310 282 I90 28 I91 Ryan et el.. 1980 St. Louls. MO llntantsl 4490 369 179.9) 47 HO.61 2 10.41 9 I21 Mlnnlch st 01.. 1962 Wash. D.C. lpregnent womanl Study group 624 490 193.61 26 14.81 8 (1.6) Jenkins end Clsrk. 1962 Control group 304 283 193.11 11 13.61 10 (3.3)

Baltimore, MD Ilntentsl 900 784 187.1) 67 (7.41 28 12.9) Weatherell. 1983

Maryland 881 826 191.81 44 (6.61 12 11.81 Boyer et el., 1963

Sourheaat Qeorgla 237 214 (90.31 19 18) 4 H.71 Cooper et 01.. 1983 Galnesvllle, FL (pregnant woman1 944 869 192.1) 66 16.91 1 10.1) 9 10.961 Cotter end Prystowsky. 1963 Mlsslsslppl 1.310 1.100 I841 114 18.71 37 12.81 14 Ill 38 12.91 7 (0.61 Thornpaon at el.. 1964 Southern Louislene1tuberculousJ 220 211 196.9) 9 (4.11 Coulter. 1966 Alebeme 249.089. 220.405 188.61 21.423 18.61 674 10.21 6.074 12.41 102 10.04) Schnelder at el.. 1978

Callfornle 1.026 O 189.31 l8.6l (1.8) Orunbaum at el.. 1978

Detroit. MI 604 A 462 189.71 37 (7.3) 14 (2.81 Stolorow st al., 1979

Calltornla 792 A 718 190.4) , 64 (6.81 18 (2.31 Grunbaum el al., 1980 HISPANIC

Puerto Rlco I"Wh1te"l 1.487 1.486 199.91 1 10.06l Suerez st al.. 1969 Celltornla, IChicenolAmerlndlanl 1.696 * 199.6) (0.11 10.2) Orunbaum et el.. 1978 Celltornle IMexicenl 1.669 o 1.561 (99.61 3 (0.21 3 10.2) Qrunbeum st 01.. 1980 ASIAN CalltornlalHewail 3.053 - 199.9) Grunbaum at el.. 1978 Cellfornla/Hawali 1.451o 1.448 199.61 1 10.061 Grunbaum at al.. 1980

* one SC n those classitled four SC and three other 149 of the "S" were SIB+ thal. 3 0.4% ware rare 0.1% rare as AS by 73 were Slhlgh F: 329 were SIC. i( ten SC electrophoretic 0 32 other 11 were Slother and 7 were Clother: A one rare o two rare screening were 164 were rare varlanrs 0 two SC tested further 23 other A four rare

4 0' Serum Groups-Introduction

SECTION 39. INTRODUCTION TO SERUM (PLASMA) PROTEINS

Various polymorphic serum protein (serum group) this has been partially successful. This matter is considered systems will be considered in subsequent sections (40 in subsequent sections in connection with particular through 45) of this unit, and a brief introduction to serum systems. proteins is appropriately included here. Some of the en- The question of what actually constitutes a senun zymatic activities considered in Unit VI occur in serum, and (plasrna) protein is not that easy to answer. Some criteria they can be considered "serum proteins" as well. Any can be established, but there are always proteins that are dif- polymorphic system which exhibits enzymatic activity has ficult to classify, in part because they are poorly character- been included in the previous unit of this book, however. ized and/or have no known function. Putnam (1975a) dis- The serum proteins and serum goup systems included in tinguished between the "true" serum proteins, and what Unit VII do not possess known in vivo enzymatic activities, were called "passenger proteins". The latter may be found but are defined by a number of other properties (often im- in the serum at certain times, or even all the time, but they munological, serological or electrophoretic). Some of these traverse the serum in transit from one site to another as the proteins are not yet understood in tern of physiological result of a particular physiological condition (e.g. preg- function. The tenns "serum protein" and "plasma protein" nancy), as the result of disease, or because they are directly are used interchangeably in what follows. involved in transport processes. No clawification system can The development of various, reproducible protein separa- easily accommodate the number and variety of plasma pro- tion techniques which complement one another in terms of teins now known. There are some protein systems in plasma resolution and specificity, has enabled investigators to whose members act together in a concerted manner, such as separate, classify and characterize many of the proteins of the complement system, the lipoproteins, the coagulation human plasma (as well as thousands of other proteins). proteins, the protease inhibitors and the immunoglobulins. Human plasma possesses a large number and variety of pro- There are about 35 to 50 proteins generally recognized as teins, some of which are known to be under the control of true plasma proteins; but there are probably 100 or more polymorphic genetic loci. A number of the polymorphic other proteins which can be found in plasma, but have not senun protein systems may be utilized in forensic and been well characterized. medicolegal investigations. Putnam (1975a) presented several criteria for classifying a The nomenclature of plasma proteins is rather complex protein as a "plasma protein": (1) The protein must be pres- and cumbersome, because it incorporates a number of his- ent in plasma after the neonatal period; (2) Synthesis must torical classification designations along with the refinements take place in the liver or the reticuloendothelial system; that have been introduced as separation techniques im- (3) The primary function (if known) must be mediated in proved and the proteins have been better characterized. the vascular system, rather than in the target system; (4) The There has never been a coordinated international effort to protein should be actively secreted into the bloodstream, arrive at a truly systematic nomenclature, such as was done and not be there as a result of tissue damage or capillary for the enzymes by the Enzyme Commission (see section permeability; (5) The concentration should be higher in 1.1.3.2). Plasma proteins were first classified according to blood than in other fluids, except in the specialized cells their solubility in water and salt solutions, then grouped into which synthesize the protein; (6) It should have an appre- classes according to electrophoretic mobility at a restricted ciable half-life in plasma, and not be transitory; (7) Genetic pH, defined in terms of cold ethanol solubility, and even- polymorphism (if exhibited) should not be traceable to tually resolved by various kinds of electrophoresis, immuno- tissues of origin, as is the case with a number of the enzyme electrophoresis and electro-focusing techniques into various systems (see in Unit VI); and (8) A true plasma protein is not components, not all of which have yet been identified with a derived by proteolytic cleavage or catabolism of other particular function. An older review of serum proteins, plasma proteins, as for example the Fab and Fc fragments primarily of historical interest, may be found in Howe of immunoglobulins; however, true precursors, and their (1925). As happens'repeatedly, some of the proteins have active forms, e.g. plasminogen and plasmin, should both be been identified in a number of different laboratories, and considered plasma proteins. have been given a variety of names. In most cases, one of It is difficult to arrive at a criterion based upon minimal the names gains widespread acceptance. The nomenclature concentration in plasma, because the plasma proteins ex- of certain classes of plasma proteins (such as the immuno- hibit a very wide range of concentrations. Albumin is pres- globulins) has been standardized by international agree- ent in concentrations of about 5 g/lOO mP, while the ment. Some effort has been made to arrive at a uniform irnmunoglobuIin IgE occurs to the extent of about nomenclature for the genetic variants of serum proteins, and 5 x lo-' g/lOOmt. On the basis of concentration in normal Sourcebook in Forensic Serology, Immunology, and Biochemistry plasma, one can arbitrarily establish four classes of serum protein is altered. But the substitution of one uncharged, proteins (Putnam, 195a): Predominant proteins, such as straight chain amino acid for another, for example, could albumin and IgG (1-5 g/100 ml); Other major proteins easily escape electrophoretic detection. A one base change (1W1000 mg/100 me); Minor proteins (10-100 mg/100 mP); in a gene leading to a GUU codon instead of a GCU codon and Trace proteins (c10 mg/100 me). The lower the concen- would lead to a AladVal substitution in the polypeptide tration of a protein, the more sensitive must be a technique chain for example. used to measure its presence, and the less likely that normal The development of many of the methods used for the concentration ranges have been established. Some of the study of serum proteins was discussed in section 2. In many proteins occurring at very low normal concentrations have ways, the nomenclature of the serum proteins has grown up been studied because their levels are greatly elevated for and developed along with the methods used to study them. various reasons in certain individuals. A list of some of the In the last century, the terms "albumin" and "globulin" better characterized serum proteins, and some of their prop- were coined to represent serum protein fractions that were erties, is given in Table 39.1. The properties of many of soluble and insoluble, respectively, in water. With the these proteins have been reviewed by Putnam (1975b) and development of salt fractionation techniques, different by Cooper (1978). "globW7 were distinguished (euglobulins; pseudo- The serum proteins which exhibit genetic variation at globulins). An account of the many studies conducted, and polymorphic levels constitute an important class of genetic the terms that were introduced, may be read in Pedersen markers. They are discussed in more detail in the sections (1945). Development of moving boundary electrophoresis which follow. A review of the properties of a number of (Tiilius, 1930) by Tiselius and his collaborators was an im- polymorphic serum proteins was given by Gitlin and Gitlin portant step. Using the technique, four distinct fractions (1975). Genetic variation in a protein may be reflected in the could be distinguished in horse serum (Tiilius, 1937), and protein's structure itself, or in the amount of the protein these were named a, 6 and y globulin components. The that is present. Both structural and quantitative variation fourth fraction was albumin. Development of analytical may have a number of different genetic bases. The prin- ultracentrifugation, and its application to the study of ciples of biochemical genetics that were outlined in section serum proteins (see Pedersen, 1945), was also an important 1.2.2, that were seen to operate in the case of isoenzyme step in the effort to characterize the various components variation (Unit VI), and about which a great deal has been more completely. Subsequent electrohoretic invest&atiom learned from the study of Hb variants (section 38), are fully led to subdivision of the original serum protein fractions: crl, applicable to plasma proteins. Alterations, additions or a2,8,, &, 71, The subscript notation is traditional, but it deletions of bases in the coding sequence for a protein may is equally acceptable today to write al, ar2, 81, etc., or a-1, lead to an altered structure for that protein which is detect- or-2, 6-1, etc. (Putnam, 1975a). These electrophoretic char- able and recognizable. The alteration can be a single amino acterizations of serum proteins are still used to place many acid substitution, or a much more drastic one. Deletions or of them into generic classes, even though it is recognized additions of bases in the coding sequence can cause a shift in that any number of structurally and functionally diverse the "reading frame", and alter the structure of the protein proteins may be under a single electrophoretic peak. Devel- beyond the point of occurrence. The amino acid sequence opment of zone electrophoretic, irnmunoelectrophoreticand may be different, or the polypeptide chain can be shortened isoe1ectrofocusing procedures, as wen as immunofixation if a nonsense or termjnation codon was introduced. Un- detection techniques and their variations, have accelerated equal crossovers between genes can result in polypeptide the flow of new information about various serum proteins. chains much shorter or longer than is usual. Quantitative Zone electrophoresis was first performed on paper as a sup- changes are often genetically controlled as well, although porting medium (herand Tiselius, 1950; Kunkel and the mechanisms may be indirect. Alterations in genes con- Tiselius, 1951), and later on starch gels (Kunkel and Slater, trolling products responsible for conversion of one protein 1952). Starch gel electrophoresis was sigdicantly refined by to another, alterations in operator or regulator genes which the inves@tions of Smithies and collaborators (Smithies, control structural genes, or alterations in the production of 1955% 1955b, 1959% 1959b; Poulik and Smithies, 1958), m-RNA can all give rise to various quantitative variations. with the characterization of several serum proteins occur- The number and variety of genetic variants known for a par- ring along the way (see in subsequent sections). Resolution ticular system are dependent upon the likelihood with which could be acedby carrying out electrophoresis in two they will come to someone's attention, and upon the ability dimensions (Smithies and Poulik, 1956). Electrophoresis of the various techniques to discrjminaie them. Subtle, or may be carried out on cellulose acetate foils (Kohn, 1957 even not so subtle, variations which have no clinical rnani- and 1%8), agarose gels (Gordon et al., 1949), and festations or implications are less likely to be noticed than polyacrylamide gels (Raymond and Weintraub, 1959) as those which do have. Similarly, even in cases where various well. The sensitive and high-resolution polyacylamide disc populations are screened for variants or abnodties of a gel electrophoresis technique was introduced by Omstein certain protein, the technique being used is unlikely to be (1964) and Davis (1%4). Imrnunoelectrophoresis is likewise able to detect every kind of variant. Electrophoresis, for ex- an important and sensitive technique for the separation and ample, readily detects variants in which the net charge of the characterization of serum proteins (see in Grabar and Serum Groups-Introduction

Table 39.1 Properties Of Some Serum (Plasma) Proteins

PCE 3a.m ChE El 0.w R6.m Sourcebook in Forensic Serology, Immunologv, and Biochem&tv

Burtin, 1%4). The quantitative versions of irnmunoelectro- it "direct electrophoresis''. Immunofixation electrophoresis phoresis (such as rocket electrophoresis and crossed im- is used for the electrophoretic typing of several polymorphic munoelectrophoresis) yield additional information about a serum protein systems of interest in forensic science. The mixture of serum proteins (see Axelsen et al., 1973). One of relationship between various serum proteins as they appear the problems with many serum proteins is specSc detection. after separation by various forms of electrophoresis and im- Methods making use of specific antibodies are among the munoelectrophoresis is shown in Figure 39.1. most useful for this purpose, and electrophoretic separation Individual polymorphic serum protein systems are dis- may be combined with an overlay detection system that cussed in subsequent sections of Unit VII. Attempts to incorporates a specific antibody. The technique is usually diagnose individual differences in "serum protein profiles" called inimunofixation electrophoresis (Alper and Johnson, are discussed in Unit VIII. 1%9); it was orighlly devised by Win(1%4), who called Serum Groups-Introduction

- PA a 1s ORIGIN

Figure 39.1 Plasma (Serum) Protein Profiles by Various Techniques A. Moving Boundary Electrophoresis (pH 8.6) 6. Paper Electrophoresis C. Two-dimensional Starch Gel Electrophoresis D. lmmunoelectrophoresis Serum Groups-Hp

SECTION 40. HAPTOGLOBIN 40.1 Recognition of Haptoglobin (Hp) Free hemoglobin in plasma can pass across the renal glomerulus, whereas Hb complexed with Hp cannot In 1938, Polonovski and Jayle found a protein in serum (Allison and ap Rees, 1957). As a result, hemoglobin is which bound hemoglobin. They had noticed that the addi- cleared from piasma (at least in rabbits) significantly faster tion of serum enhanced the peroxidase activity of Hb (sec- than is Hb-Hp complex (Murray et al., 1%1). However, Hb tion 6 introduction). The Hb biding protein was fiuther is found to be cleared more quickly even in nephrectomized characterized, found to be an cr, glycoprotein, and given the animals. In 1958, AUison suggested that a principal function name "haptoglobin", from a Greek root meaning "to of Hp was to prevent the loss of Hb (and, hence, the loss of bind" (Polonovski and Jayle, 1940). They had considered iron) across the renal glomerulus. Both free Hb and Hb-Hp calling the protein "prosaptoglobii", but had settled on the complex are catabolized primarily in the liver (Keene and now familiar name. "Haptoglobin" is not a single molecule, Jandl, 1965; Murray et al., 1961). If free Hb is injected into but a group of closely dated ones, and it is proper to speak the bloodstream, it is rapidly complexed by haptoglobin, of "the haptoglobins". The multiplicity of molecular forms and the complex is cleared from circulation, thus temp is now known to be based on the control of Hp synthesis by rdy depleting the plasma of (the complexed) Hp (Noyes a polymorphic genetic locus, as a result of which one may and Garby, 1967; Laurell and Nyman, 1957). In order to ob- refer to the "haptoglobin system". Determination of serum serve hemoglobinuria, the level of free Hb in circulation haptoglobin levels was found to be a clinically useful and must exceed the level of Hp available for complex formation signif~cant measurement, and standardized procedures (Laurell and Nyman, 1957). Hemoglobinuric patients are based on Hb binding and peroxidase activity (Jayle, 1951; found to have little or no plasma haptoglobin (Allison and Co~dand Smithies, 1959) and using quantitative im- ap Rees, 1957). Free Hb may get into the circulation in munochemical techniques (rauthe et al., 1965) were devel- signihmt amounts as a resuit of hemolytic episodes. If an oped. It is now clear that the quantitative immunochemiml amount of free Hb sufficient to bid all the Hp is injected methods are not as straightforward as was first thought into circulation, Hp levels fall to undetectable levels within (Valetteet al., 1979), in that the results obtained depend- are about 24 hrs (Laurell and Nyman, 1957). Normal Hp levels ent on the Hp phenotype. This behavior can probably be ex- will return within a few days following total depletion. Hap- plained by irnrnunodetenninant differences in the Hp sub- toglobin is synthesized to the extent of about 3040% of the units (see below). The extensive studies of the Paris group intravascular pool each day (Noyes and Garby, 1967). There on haptoglobins were well summarked by Jayle and Moretti is no evidence that haptoglobin depletion causes an increase (1%2). in the rate of Hp synthesis by a "feedback" mechanism. The effectiveness of haptoglobi in consening hemoglobin 40.2 Haptoglobin Physiology (and, thus, iron) has been questioned on several accounts Many studies have been conducted on haptoglobin physi- (Giblett, 1%9), and the physiological role of haptoglobin is ology and function (Jayle, 1956, Kirk, 1% Giblett, 1%8 undoubtedly more complicated (Pintera, 1971). and 1969, Javid, 1%7b; Pintera, 1971), and not every Recently, Prof. Dr. Prokop and his collaborators have can recorded observation has been satisfactorily accounted for observed that human haptoglobins behave as antibodies as yet. The liver is probably the site of haptoglobii syn- against streptococci possessing the T4 antigen. This effect thesis, and the amount of Hp produced is subject to many varies according to the Hp phenotype, type 2-1 and 2-2 high influences. It can vary considerably in different physiolog- Hp's behaving as titered complete antibodies, while 1-1 ical and pathological states. There is some variation in the derived Hp acts like a "blocking" antibody (Kohler el al., normal level of Hp, values being of the order of 40 to 1978; Prokop and Kohler, 1979; Prokop, 1979). Homo- 180 mg/100 d,expressed in terms of Hb bi~ldingcapacity. logous animal haptoglobins exhibit the effect as well. These The quantity of Hb bound by Hp is related to the Hp type observations are particularly interesting in view of the struc- (see below). Haptoglobi may be catabolized as free Hp, or tural homology between the short polypeptide chains of Hp in compiex,with Hb. More is known about the latter route. and the light chains of the immunoglobulins (see below m Free hemoglobin released into circulation, is immediately 40.3.6). complexed with Hp, and the complex is removed by the 40.3 Genetics and ~iochemlstr~of reticuloendothelial system. The half-life of the complex in Haptoglobins circulation is a function of its concentration (Noyes and Garby, 1%7), the clearance rate tending to be more ex- 403.1 Genetic variation in haptoglobla ponential at lower concentrations of complex, and more Haptoglobin was well studied elemophoretically, both as linear at the bigher ones. free Hp and complexed with Hb, before the polymorphism Sourcebook in Foensic Serology, Immunology, and Biochemistry was fully recognized (Wieme, 1953; Jayie et af., 1952; to the presence of two Hpl alleles, HplF and Hpls. With Tuttle, 1955a and 1955b). In 197, Jayle and Gidnoticed three alleles, HpIF, HpIS and HP, six phenotypes could be that there appeared to be more than one kind of hapto- observed (provided appropriate electrophoretic conditions globin present in plasma on the basis of ammonium sulfate were employed): Hp 2-2, Hp 2-IF, Hp 2-IS, Hp IF-IF, fractionation, which was part of the Hp purification pro- Hp IF-1s and Hp 1s-IS. Family studies by Smithies et d. cedure. In 1955, Smithies found the genetically controlled (1x24 indicated that this genetic model was correct, and a variation in haptoglobin by starch gel electrophoresis. These survey of a small number of Europeans (from Toronto and studies were the same ones in which the now familiar zone Madison, WI) indicated that the frequencies of HpIF and electrophoresis technique on starch gels was elaborated (sec- HplS were about 0.16 and 0.24, respectively, assuming Hp2 tion 2.3.4). Three different starch gel electrophoretic pat- to be about 0.6. This genetic hypothesis has been confumed terns of serum proteins could be observed, the differences (Shim and Beam, 1964a; Ehnholm, 1%9; Fagerhol and lying in those proteins between the so-called "fast" and Jacobsen, 1%9). Fagerhol and Jacobsen (1%9) used a dif- "slow" a2gbbulias. Complex changes ocmmd in the band- ferent discontinuous buffer system for the resolution of the ing patterns depending upon the amount of Hb present. The Hp 1 subtypes, and originally called them "E,"K" and proteins all bound hemoglobin, and were easier to type in its "R", but said that they were equivalent to Hp IF-IF, presence (Smithies, 1955a). The different "groups" were 1F-1S and IS-IS, respectively. Electrophoretic patterns of originally called I, IIA and IIB, and were well distributed the three common Hp phenotyhpes as seen on starch gels at even in a small sample of sera from different individuals. It pH 8.6, and those of the a polypeptides in the subtypes was suggested (Smithies, 1955b) that these differences had a under denaturing conditions, are shown in Figure 40.1. The genetic basis. Studies on eighteen fdawere consistent biochemical genetics of haptoglobin types and subtypes is with a straightforward genetic model in which "group I" discussed further below. and "group IIB" were homozygotes for each of a pair of codominant alleles, and "group IIA" was the heterozygote 40.3.3 Other Hp vatiants and Hp 0 (Smithies and Walker, 1955). It was quickly realized that the A number of variants of haptoglobin have been found, proteins exhibiting this variation were identical to the and the genetic explanation for some of them is not as clear hemoglobin-binding proteins of Polonovski and Jayie as it is for others. The variants are described in this section. (1938), Wieme (1953), Tuttle (1955a), and others, namely The genetic basis for their occurrence is considered later in haptoglobin (Smithies and Walker, 1956). Accordingly, it connection with the structure and biosynthesis of the hapto- was suggested that this family of proteins be designated the globins. Giblett (1%9) usefully classified the Hp variant "haptoglobii system", that the genetic locus be called Hp, phenotypes as "quantitative" or "qualitative". and the two alleles Hp' and Hp2."Group I" was Hp 1-1 40.3.3.1 Quantitative variants. The majority of quanti- (Hp'Hpl), "IIA" Was Hp 2-1 (mpl)and "uB" WaS Hp .tative variants are modifications of the 2-1 phenotype. The 2-2 (Hp2Hp3. These features of this serum group system Hp 2-l(mod) (Giblett, 1959), or Hp 2-1M (Connell and were quickly confmed (Moretti et al., 1957; Sutton et al., Smithies, 1959) phenotype shows a spectrum of patterns, 1956), and the proposed mode of inheritance has been wide- ranging from a high concentration of the slower moving ly confmed by family and population studies (e.g. polymers (see below) with no visible fast Hp 1 band, to a Galatius-Jensen, 1956, 195th and 1960; Prokop et al., 1%1; high concentration of the fast band with only one of the Kirk, 1%8a and 1971). It was aiso clear that there were members of the polymeric series visible (Giblett, 1%9). significant differences between the Hp allele frequencies in Hp 2-1M (where "M" and "mod" indicate "modified") ~thnicallydistinct populations (Sutton et d., 1956; Allison occurs at appreciable frequencies in the Black population ?t QI., 1958; Giblett, 1959), and that some Black African (some 10% of American Blacks), but only occasionally in populations presented certain complexities in their Hp, not Europeans. Harris et al. (1960) described Hp 2-1M in a seen up to that time in Europeans (AUison et al., 1958; White family, and said that the pedigree indicated either a 3iblett, 1959). HpZMgene at the Hp locus, or mmer gene at another locus. If the HdMgene was present, it gave a Hp 2-1M pat- W.3.2 Additional genetic variation at the H'locus- tern in Hp'HPMpeople, but a 2-2 pattern indistinguishable FIaptoglobii "subtypes" from the usual one in H#Hp'Mpeople. Giblett and Stein- In 1%2, Connell et al. subjected partially purified hapto- berg (1960) examined about 500 sera from American Blacks gobins, representing the three common (1-1, 2-1 and 2-2) in 92 families, and found about 15% of a sample of unre- >henotypes, to electrophoresis after reductive cleavage in lated individuals to be Hp 2-1M. They thought that the he presence of mercaptoethanol. Two classes of products phenotype was a manifestation of an HdMallele. Sutton were obtained, one of which appeared to be common to all and Ihp (1%4) thought that Hp 2-1M could be divided 3p molecules, and which did not migrate in starch gels at into four classes, called b, c, d and e, on the basis of the shift Lcidic pH unless 8M urea was present. However, the other toward the faster moving bands. They believed that there xoducts migrated differently according to the phenotype. were two alleles, Hp" and HP, controlling the 2-1M Phis latter, called the "hp 1" product, showed two different phenotypes. Parker and Beam (1%3) offered an alternative Wterns, fast IF) and slow (S). This behavior was attributed explanation based on variation at a regulator gene locus, Serum Group-Hp

1s-IS IS-IF IF-lF 2-1s 2-1F 2M-IS 2M-1F 2-2 2J-1s ORIGIN or 1-1 2-1 2-2 2-1M I 2-2M

Conventional pH 8.6 Typing Chain Patterns on Reductive Acid Urea Starch Gels

Figure 40.1 Electrophoretic Patterns of Hp Types and Subtypes close to but not identical with the Hp locus. The genetic One of the more puzzling kinds of quantitative variation basis for Hp 2-1M is apparently still not completely is represented by the phenotype "Hp O",which denotes an- understood. haptoglobinemia or hypohaptoglobinemia. Failure to detect Galatius-Jensen (1958b) found a phenotype which repre- Hp by most of the electrophoretic methods used does not sented quantitative variability in the Hpl product. It was necessarily mean that it is completely absent; it usuaQy called 'Hp Cadberg', usually menHp Ca. It has been ob- means that the amount of Hp present is less than 15-20 mg/ served in other popWons as well (Giblett, 1%4; Nance lo0 mP expressed as Hb-binding capacity (Giblett, 1%9). and Smithies, 1%4; Harris et ol., 1959). The electrophoretic In 1958, AUison et d tested 99 Nigerian sera for Hp and pattern resembles that of a mixture of Hp 2-2 and 2-1 in found that a little over 32% of them had no detectable activ- variable proportions. Subtyping shows reduced Hpl product ity. The fmding was further discussed by AIlison in 1958. in relation to Hp2 product, although the ratio can vary even Sutton et d. (1956) saw no Hp in a small sample of bloods within the same family. Sutton (1%5) has suggested that Hp from the Ivory Coast. Giblett (1959) found that about 4% Ca may be the result of genetic mosaicism, different cell of about 400 Black people from Seattle were Hp 0. Hp 0 is populations in the same person producing Hp 2-2 and Hp much more common as the result of matings involving an 2-1. Hp Ca is inherited, however, and if mosaicism is the ex- Hp 2-1M or Hp 0 parent, and it is rate if the parez~tsare planation, then an inherited tendency toward cellular Hp 1-1 (Giblett and Steinberg, 1%0, Sutton and Karp, somatic mosaicism would have to be postulated (Giblett, 1%4). The HpMH# genotype, when expressed, cannot be 1%9). distinguished from HpZH#. The offspring of Hp 2-1M Two other variants, descrii by Giblett (1964), are x 2-1M matins are found to be Hp 1-1, Hp 2-1M or Hp 2-l(trans) and Hp 2-l(Haw). The Zl(trans) exhib'i a Hp 0. It appears that HPMHpMpeople are usually (or pattern that shows an increase in the faster moving compo- always) Hp 0. Black people who are phenotypically Hp 0 are nents and a proportionate decrease in the slower ones. This often HpMHplor HpMH# (Giblett and Steinberg, I=), shift is not as pronounced as in Hp 2-lM, and gives the ap- although they can have apparently normal genotypes pearance of a 'transitional' phase in going from an Hp 2-1 (H#Hpa or H#Hpl) as well. These genotypical interpreta- to an Hp 2-1M pattern (hence, the name 'trans'). Hp 2-1 tions were given on the assumption, of course, that there (Haw) was so named because it was found in the serum of really is an Hp2Mallele. Parker and Beam (1963) proposed an Hawaiian subject. The pattern is similar to 2-1M in hav- that Black populaiions canied a mutant in a regulator gene, ing a heavy concentration of the fastest bands, but differs in and that Hp 0 could represent homozygosity for this mutant that there is no associated increase in the intensity of the ale. There is evidence that such a gene may occur in second band nor a relative decrease in the intensities of the Caucasian populations as well, albeit at very low frequencies third and fourth bands. (Murray et al., 1966). In spite of the difficulties assodated Sourcebook in Forensii Serology, Zmmunologv, and Biochemistry with proving regulator gene hypotheses, they do appear (1%9) suggested that it should perhaps be ded'd' or 'd' more attractive than the original one based solely on HpM. (the latter, since it may be the result of a partial gene It is quite likely that the factors involved in determining Hp triplication-see in 40.3.6). When Hd is heterozygous with 2-1M and Hp Ca are applicable to Hp 0. There is some evi- Hp2, the haptoglobin synthesis is greatly reduced, and it is dence for the existence of a very rare HpOallele (Harris et only possible in the occasional specimen to determine the al., 1958a; Matsunaga, 1%2; Schwerd and Sander, 1967). In electrophoretic pattern (Giblett, 1%9). Isolated examples of these families, a homozygous Hp 1-1 or 2-2 parent had one Hp 1-J have been seen in such widely populations or more children who appeared to be homozygous for the as American Blacks, Kurdish Jewish, Australian aborigine other allele. In some cases, HpOHp'and HpoHp2 combina- and European (Smithies et d., 1%2b). Mukhejee and Das tions are expressed like Hp'Hp' or HpHp2, mpectively, (1970) described a 2-15 in a Bengali Hindu, and Hiiglund et but in other cases the phenotype is Hp 0. d. (1970) found 7 Hp 1-J types among 15,601 Swedish Giblett (1%9) noted that, in view of the fact that an Hp 1 adults. Minor differences in the patterns are consistent with phenotype could be the result of HplHp' or Hp'HpO independent origin of the genes according to the unequal genotypes, and similarly for the Hp 2 phenotype, the pheno- crossing over hypothesis (see below), although the samples typic nomenclature should reflect this fact. For some were not freshly drawn when compared. In 1966, Giblett et reason, homozygous haptoglobii types are more often ex- al. described two more structural variants, which were the pressed as "1-1" or "2-2", rather than just "1" or "2". result of a new allele H~B. Both 1-B and 2-B phenotypes For most other systems, phenotypic nomenclature denotes were observed. The a polypeptide conditioned by HP what is observed rather than implying a genotype. Giblett migrates between hp d and hp aIS. Hp 1-B and 2-B are said that this latter practice should be followed with hap referred to as the "Ba" types by Giblett (1%9). It is possible toglobin as well, and the suggestion is a good one. In sum- too, she said, that these represent B chain variations. Ren- mary, it appears that there is more than one genetic explana- wick and Marshall (1%6) debed the Hp 2-1D tion for Hp 0, and that the one based on an operator or phenotype, thought to be the result of an allele HpIDpaired regulator gene mutation is not completely developed as yet. with Hp2. In the presence of saturating amounts of Hb It is important to note, before leaving the subject of (which is the way Hp is usually typed), Hp 2-1D and Hp anhaptoglobinemia, that haptoglobin genotypes are not 2-1 are not distinguishable. But under "subtyping" condi- fully expressed in a majority of fetuses and newborns. Many tions, a band which runs faster than 1F is revealed are, therefore, "anhaptoglobiiemic", but this condition is (representing the aID peptide). The "D" stood for transitory and reflects the rate at which the Hp genes "dashing" in the name. become active. Detectable haptoglobin is usually found in The socalled 'Marburg' phenotypes were originally seen about 10-15% of cord blood and newborn sera (Ram et in a German family (Aly et al., 1%2), and have been exten- al., 1%1), but this percentage increases rapidly until it sively studied by Cieve and Deicher (1%5) and Weerts et d. reaches nearly adult levels by about 4 months age (Hauge et (1%5). AU the electrophoretic components of Hp 2-1Mb al., 1970). Few infants have less than 30 rng/100 mt hapto- demonstrated atypical immunological reactions with certain globin at 6 months age (Bergstrand et d., 1%1). The Hp anti-Hp sera. The antigenic detexminant called "B" (see type expressed in cord blood and newborn serum reflects the below), which is on the B chain and is normally blocked in genotype of the infant, and not that of the mother (Hirsch- the Hp-Hb complex, still reacted even when saturating feld and Lunell, 1%; Siniscalco et d., 1963). Hirschfeld amounts of hemoglobin were present. Subtyping revealed and Lunell (1%2) found an Hp 2-1 mother with a pair of no unusual a polypeptide chains, and it was concluded that twins (who had been stillborn), and the twins had Hp 1 and this variant represented a 6 chain mutation. Bowman and Hp 2 phenotypes. The data of Siniscalco et al. (1%3) sug- Cleve (1%7) have shown that the 'i%gerprinty of the 6 chain gested that the onset of Hp synthesis in infants might de- of Hp Mb is different from the usual one. Another pheno- pend on the maternal genotype, while Hauge et k (1970) type, ded Hp 2-1 Bellewe, was described by Javid noted that the Hp 2 phenotype tended to develop more (1%7a). It had immunological properties resembling those slowly than the others. of the 'Marburg' haptoglobii, but the electrophoretic pat- 40.3.3.2 Qualitative vmimtts. These variants contain tern differed. Electrophoresis under subtyping conditions components which are not present in the common pheno- suggested that the phenotype represented heterozygosity for types, and they are all very rare. They may be Werclassi- a chain variant. The 35 year old Black propositus had thee fied as a chain variants or B chain variants, since it is now sons who had apparently inherited the variant gene. clear that the two kinds of polypeptide chains are coded for Other vdtshave been described which may repnsent B by different and independent genetic loci (see below). The chain mutations. In 1%4, Robson et al. described five new best known rare variant is Hp 'Johnson', originally ob- phenotypes called Hp 1-P, 2-P, 1-H, 2-H and 2-L. The served in a Black woman and her daughter in Weby Dr. electrophoretic behavior of the components of these hapto- Giblett. It reveals one or the other of the 1S or 1F a peptides, globins differed in the presence and absence of hemoglob'm, and a much slower, heavily-staining a chain (Smithies et al., suggesting that something was unusual about their ability to 1%2a; Giblett and Brooks, 1%3). The polypeptide made by complex with Hb.The alleles thought to be responsible for the 'Johnson' allele was originally called hp 2Ja, but Giblett these types were called H~P,H~H and H+. A similar Serum Groups-Hp variant was reported by Giblett (1964). Hp Ab, as it was and his collaborators as summarized by Jayle and Moretti called, was found in a woman in Boston by Dr. Irving (1%2). One of the first purification schemes was devised by Umansky. The a polypeptides in these phenotypes migrated Jayle and Boussier (1954). Refinements were developed by like IF, 1S and 2 a chains usually do. The low pH of the gels Guinand e! al. (1956), Jayle et al. (1956), Moretti et al. used for subtyping might prevent separation of the variant (1958) and Herman-Boussier et al. (1%0). Haptoglobin has chain (if it were there) by protonating carboxyl groups been isolated from serum, from ascitic fluid in some pa- (Nance and Smithies, 1%4). Amino acid residues which tients, and from the urine of certain patients who excrete it were neutral or equally charged could also be involved. if it is of the 1-1 type. Purification methods for haptoglobin Shim et al. (1%5) said that the P and L variants might be are discussed in some detail in Pintera (1971) and in Putnam additional mutants at the P chain locus. (197%). The structure of haptoglobin is discussed below, but the The monomeric unit of human haptoglobin (represented molecule is known to consist of CY and P chains, coded for by by the molecule found in type 1-1 people) is composed of different loci. The so-called Hp locus is the a chain locus, four polypeptide chains, two cr chains and two chains, and most of the variants, including the three common types connected by disulfide bridges (Shim and Bearn, 1Wb; representing the polymorphism, are a chain variations. Malachy and Dion, 1973a and 1973b; Malachy et al., Some B chain vhtsare now known, however, and it 1973). The general structure of the molecule (Figure 40.3) is would be useful to distinguish between the structural loci in reminiscent of the structure of the IgG molecule Figure the nomenclature. Javid (1%7a) showed that the a and P 1.41). The chain can be dissociated by reductive cleavage loci are not closely linked, and suggested Bp as a symbol for with mercaptoethanol in the presence of 8M urea (Smithies the p chain locus. The common allele would be B+ and et al., 1966; Connell et a!., 1966). The Hp 1-1 molecule is B~Bwould represent the allele seen in Hp 2-1 Bellewe. Gib- the only one, from among the three common types, that ap lett (1%9) suggested, however, that it would be preferable to pears to be homogeneous. It migrates as a single band in call the a chain locus Hp, and the /3 chain locus Hpg, and starch gel electrophoresis, whether complexed with Hb or this seems like the best idea. The electrophoretic patterns ex- not, and exhibits a single ultracentrifuge peak. The 2-1 and hibited by some of the Hp variants are shown in Figure 40.2. 2-2 types contain more than one protein species, which can A useful table of many of the variants was given by Kirk be observed in the ultracentrifuge, or by starch gel electro- (1968a), and is reproduced in Pintera (1971) and in Putnam phoresis (Bearn and Franklin, 1958 and 1959), and which (1975~). differ to some extent immunologically (Korngold, 1%3; Eichrnann et al., 1966). The multiplicity of bands seen upon 40.3.4 Structure of the haptoglobins starch gels with 2-1 and 2-2 types is familiar to anyone who Efforts to purify and characterize haptoglobin have been has typed haptoglobin. These bands are now known to rep- undertaken by many workers, since the frrst work by Jayle resent a series of polymers, which are, however, very stable,

Figure 40.2 Electrophoretic Patterns of Some Haptoglobin Variants 573 Sourcebook in Forensic Serologv, Zmmunologv. and Biochemistry

Figure 40.3 Diagrammatic Structure of Monomeric Haptoglobin and not artifacts (Connell and Smithies, 1959). The charac- considered inwersible. The heme moiety of Hb is unimpor- teristic 2-1 pattern cmnot be produced by the mixing of 1-1 tant in the linkage, though Hp combines with oxyhemo- and 2-2 sera. The nature of the individual components in globin, and not with deoxyhemoglobin. The oxygen equilib- the 2-2 polymer series turned out to be rather complex, and rium of the complexed Hb is greatly altered (Nagel and was finally worked out by Fuller et a/. in 193. Until that Gibson, 1971). The combination is not species-specific, and time, there was disagreement as to the number of cz and B human Hp can combine with animal hemoglobins. Hp can chains in the polymers, and whether each successively larger likewise combine with Hb F and with some of the abnormal member of the series represented the addition of a B chain, a hemoglobins (see in section 38). Beam and Franklin (1958) half molecule (essentially 4)or an entire monomer. Fuller complexed Hp with Hb C in order to cause the complex to et al. (1973) isolated several discrete polymers, and could have a slower electrohoretic mobility than it would have had show that each consisted of a? (the cr chain coded for by the with Hb A. The cr chains of Hb are essential for binding to Hp allele) and chains in a 1: 1 ratio. Members of the series haptoglobin, though they bind more weakly than intact Hb, differed from one another by an average MW increment of but much more strongly than isolated Hb B chains. Human 54,500 daltons, approximately equivalent to one d chain Hb cy chains wiU bid animal haptoglobins, and Terpstra and (17,300) and one B chain (40,000). Reductive cleavage ex- Smith (1976) have, for example, studied Hb cz chain binding periments established that the "d/3"units were joined by to porcine Hp. There is recent evidence (Kazim and Atassi, disulfide bridges to form the polymers, and the polymers 1980), however, that Hp may in fact bid Hb chains quite corresponded to a series a$,, n = 3 to 8. Javid (1%4) car- strongly under certain conditions, but that special assay ried out experiments on the differences between the 2-1 and techniques are required to demonstrate the interaction. 2-2 patterns, confirming the thinking of Allison (1959) and Laurel1 (1960) suggested that the binding of Hp to Hb oc- Parker and Beam (1%3) that a' chains (the a chain product curs through the 4 dimer of hemoglobin. The fully of the Hpl allele) are incorporated into the polymers formed saturated Hp 1-1 (the simplest haptoglobin) is, in this view, by the a chain products of Hp2 (a2is the higher MW chain, bound to two halves of an Hb molecule rather than to one and is responsible for the polymerization phenomenon) in intact molecule, and there is considerable experimental the 2-1 type. The Hp 1-1 molecule is thus while the evidence that this view is correct. The data of Nagel and 2-2 haptoglobins consist of a series of molecules (dB),, Gibson (1971) suggested that Hp possesses four biding n = 1,2,3...... Type 2-1 haptoglobins are expected to sites, two for each @3 Hb dimer. One pair of sites in Hp bids be a series of polymers of the kind (a18)m-(a?B)n. a Hb aj3 dimer, and thereby induces an allosteric change in The nature of the Hp-Hb complex has been widely Hp creating a second site for an Hb cr8 dimer. The exact studied using a variety of methods, and these are reviewed nature of the biding regions in the two molecules, and of by Putnam (1975~).The exact nature of the biding has not the amino acids actually involved, is not completely known. been completely worked out, and this is still an active area Most of the evidence is indirect, and based upon various of investigation. Haptoglobin combines stoichiometrically molecular probe studies, e.g. Russo and Chen (1976), Osada with hemoglobin A to form a very stable complex, the bind- et al. (1978), Katnik and Dobryszycka (1978) and Hwang ing being so tight that the complex formation reaction is and Greer (1979). Confoxmational changes in Hp are clearly Serum Groups-Hp involved, and Hevdr (1977) has shown that the reduction of One of the repeated sequences is from alSand the other is Hb binding capacity by the haptoglobin in heated serum from a'' (which can be discerned because of the Glu/Lys varies with Hp type and subtype. difference mentioned above). The hp ar2 polypeptide chain represents the first example of a partial gene duplication 40.3.5 Subunit and polypeptide chain structure fully documented by amino acid sequence analysis. The Haptoglobin resembles the immunoglobulins in subunit point is discussed more fully in the following section. The structure, as noted above. There are two a (light) chains and hp a chains contain Cys at dland form an dl41inter-or two B (heavy) chains (Fig. 40.3). There are a number of dif- chain disulfide bridge. There is an a354intra-a chain ferent a chains, because it is the a chain structural locus disulfide linkage, and the Cys forming the a-chain half of which exhibits most of the genetic variation except for a few the interchain diiuKde bridge to the B chain is at a73. The rare variants of the 0 chain. The three major Hp types can be sequence of the B chain has been almost completely worked divided into six subtypes on the basis of the electrophoretic out (Barnett et al., 1972; Kurosky et al., 1976), and the a behavior of their polypeptide chains following reductive chain is attached to it through 0103 Cys. All the carbo- cleavage in urea (Connell et al., 1%2). Electrophoresis hydrate in haptoglobin appears to be assodated with the 0 under these conditions reveals that there are only two poly- chain, and is probably attached (at least in part) through peptide chains in Hp, a and 8, in spite of the multiplicity of 1323 Asn (Xurosky et al., 1976). bands seen in starch gels at pH 8.6 (explained above). Only the a chain patterns are of interest in electrophoretic subryp- ing (Fig.40.1), since all six types have common B chains 40.3.6 Biochemical genetics (Cleve et al., 1%7) which migrate very slowly in acid-urea Soon after the observation of the haptoglobin polymor- starch gels. It has become more or less conventional to phism, it became clear that the monomeric molecule was of designate haptoglobins with the symbol "Hp", the poly- the a2b2structure, and that the a chain locus was responsi- peptide chains as "hp", and the genes as Hp. The a chain ble for the genetic variation. Early peptide analyses of the structural locus should really be designated Hp,, as sug- alS,ulF and d chains indicated considerable similarity be- gested by Giblett (1%9), to distinguish it from HpS, now tween ds and alF,but a great difference in size between that B chain variants have been found. The a chains have them and the at. The peptide analysis suggested to Smithies been designated in a number of different ways by various et al. (1962b) that the c? chain was almost a duplicate of the workers, and these usages can be quite confusing to non- a1chains, and that at had arisen because of a partial gene haptoglobii specialists. There are, essentially, three kinds of duplication of the a' genes caused by a homologous but un- a chains, produced by the Hp,ls, Hp,IF and Hpa2genes. The equal crossover at the Hp, locus. This went gives rise to an Hpals locus produces hp alS,which has also been called "hp a chain which contains most of aiFand most of als in the 1Sor"; similarly, Hp,lF produces alF,and Hpa2produces d, same polypeptide chain, namely a2, and for this reason the which have also been designated in the alternative ways d chain can be designated aZFS.The way in which the par- shown for alS. tial gene duplication may have occurred is shown in Figure The classical Hp 1-1 phenotype can have haptoglobin 40.4A. During meiosis in an Hp 2-1 individual, when homo- with hp alFchains, with alschains, or with equal quantities logous chromosomes pair with one another, the aigene on of both. The subtype phenotypes (and genotypes) are then, the one chromosome (whether it is alSor a'? will find itself respectively, Hp IF-IF (HplFHp'3, Hp IS-IS (HplSWp1S) unable to pair with the almost twice as long a2 gene; and Hp 1F-1s (HplFHplS).The classical 2-1 phenotype may however, because of the structural homology, the a1can be subtyped into Hp 2-1F or Hp 2-IS, which contain hp a2, readily pair with one or the other half of a2 (the duplicated and either hp alFor hp alSchains, but not both. Hp 2-2 con- half). Therefore, as Smithies (1%4) discussed in detail, once tains hp d chains (although there may well be molecular the partial gene duplication has occurred, the possibility for variants of a2chains-see further below). further genetic variation is increased. Because perfect synap- The primary structure of the Hp constituent polypeptide sis is imposs%le between either a1gene and the dFSgene in a chains has been determined, and the a chain sequences have 2-1 individual, the alSor alFgenes will pair with a segment been most informative from a genetic point of view. The se- of the dFSgene, as shown in Fig. 40.4B. Crossovers in such quence work on the a chains may be found in the papers of heterozygous synapses can then lead to different a2chains, Black and Dixon (1968 and 1970), Black et al. (1970), namely atw, dSSand dSF.In a similar way, because of the Malachy and Dixon (1973a and 1973b) and Malachy et al. duplicative chain homology, displaced synapsing could oc- (1973). The als and alF chains contain 84 amino acid cur in homozygous 2-2 individuals @g. 40.4C). Here, part residues, and are identical except that alshas Glu at position of one CPgene paits with an (almost) homologous part of 54 where alFhas Lys. The a2chain is almost twice as long as the other dFSgene (the "F" and "S" pares differ in a single the a' chains, containing 143 amino acid residues. But what amino acid). A crossover under these circu-ces could is most extraordinary about the hp cr' chain sequence is that lead to two varieties of triplicated genes, giving flor @ it consists of the frst 71 and the last 72 amino acid residues chains. It is believed that Hp Johnson contains just such a of hp al, joined together to form a 143 residue chain. The chain, and these are thought to have arisen independently in Asp13-Ala71 of a' is repeated in hp cu2 as Asp72-Ala130. the various different populations in which they have been Sourcebook in Forensic Serology,Immunology, and Biochemimy

2 A. Possible Mechanism for Formation of the Hpa Gene

B. Unequal Crossing Over in Hp 2-1 Heterozygotes

C. Mechanism of Partial Triplication by Displaced Synapse in Hp 2-2 Homozygotes

Figure 40.4 Diagrammatic Representation of Possible Unequal Crossovers in Hp a Chains (modified from those in Giblett, 1968 and 1969). Serum Groups-Hp observed. Such genetic events are not expected to be com- is also more frequent in people of Black African origin, ap- mon, particuIarly in the Hp 2-2 case, where completely pears to be related in some way to the anhaptoglobinernia homologous pairing is possible, and it is not particularly phenomenon, and could, if encountered, lead to difficulty surprising, therefore, that 'Johnson' phenotypes are quite in the interpretation of the inheritance patterns. Most rare. Giblett (1968 and 1969) has suggested that if the laboratories utilize conventional Hp typing in these cases, triplication hypothesis is confiied for Hp 'Johnson', then under which circumstances the three common phenotypes the resulting a chain should be called d. Nance and can be detected. The discriminating power of the system Smithies (1%3) subtyped many sera and found a chains in would be increased by the application of Hp' subtyping. some with migration rates that could well correspond to dkF 40.4.2 Haptoglobin typing in dried bloodstains or P. These genes are apparently infrequent, however (Shim and Beam, 1944a). While the nature of many of these A number of different methods have been used for the "probable" Hp a chains will eventually be determined by phenotyping of Hp in bloodstains. Early studies on the use sequence analysis, there are considerable technical dif- of starch gel techniques similar to those used for serum typ- Mesassociated with telling the difference between chains ing were only partially successful (Diirwald, 1%1 and 1%3; like $ and GF,for example. Haptoglobin biochemical Falk and Bundschuh, 1%3), although some stains could be genetics is discussed in Giblett (1%9), Harris (1975) and typed. Prokop and Bundschuh (1%3) pointed out that the Putnam (197%). results to be expected from stains depended on the sub- Another point of some interest is the structuralhomology stratum upon which the blood is deposited because of the between the Hp polypeptide chains and those of other pro- differences in extractability of the serum. Culliford (1%3) teins. The a chains bear structural homology to the light (K obtained fair, but not completely satisfactory results with or A) chains of IgG. On this basis, it was expected that the bloodstains using starch gels and the discontinuous buffer Hp @ chain would show structural homology with the IgG system of Poulik (1957). heavy chains, but this expectation has not been fulfilled. In- In 1%6, Culliford and Wraxall descrii an immunoelec- stead, an unexpected homology was found between Hp @ trophoretic technique for Hp phenotyping in bloodstains and the corresponding regions of the so-called chymotrypsin which was found to be more reliable than starch gel pro- family of serine proteases. This matter is futher discussed cedures. Immunoelectrophoretic methods had been applied by Putnam (197%) and by Kurosky et al. (1976). to Hp typing in serum by Hirschfeld (1959~)and by Fme and Battistini (1%0). Hirschfeld (I%&) ' ed the 40.4 Medicolegal Applications studies on the irnmunoelectrophoretic method. In this pro- cedure, serum or bloodstain em is electrophoresed in 40.4.1 Disputed parentage agar gel at about pH 8.6, after which a trough is cut in the The Hp system is applied in cases of disputed paternity in gel and filled with an anti-Hp serum. After a suitable diffu- many laboratories. The use of the system is discussed exten- sion period, precipitin arcs develop, and those representing sively by Galatius-Jensen (1956, 1958% 1960 and 1%2), Hp 1, 2-1 and 2 phenotypes can be distinguished by their Baitsch (1%1), Giblett (1%3) and Prokop and Bundschuh somewhat different positions (electrophoretic mobilities). (1%3). The probability of excluding a falsely accused father Bargagna and Cave Bondi (1%8) conhed the usefulness in Western European populations is about 18070, placing it of Culliford and Wraxall's (1966) procedure in bloodstain among the better systems for this purpose. In the U.S. pop typing. They typed stains up to 69 days old. Whitehead and ulation, the chances of excluding a falsely accused father are Morris (1969) presented a modification of the method which about 18% for both White and Black people according to they said gave precipitin arcs that were easier to interpret in Chakraborty et al. (1974), and about 18% for Whites and terms of phenotype. Katnik and Dobryszycka (1977) have 15% for Blacks according to Dykes and Polesky (1978). The canied out immunochemical studies on Hp in an effort to system is not widely employed in U.S. laboratories, however find out which amino acid residues in the molecule are in- (Polesky and Krause, 1977), but the percentage of labora- volved in the antigenic determinants. The results indicated tories using Hp routinely is higher among AABB Reference that tyrosyl residues are not essential. The immunochemical Laboratories than among other laboratories. studies of Shim et al. (1%5), in which anti-@and anti-a? The most serious problems that could arise in the applica- chain sera were used, showed that the former reacts only tion of Hp to disputed paternity cases would involve the ap- with free Hp but not with HpHb complex, while the latter parently very rare silent allele HpO.Many newborns and reacted with either. The reason for the difference is that the infants are temporarily anhaptoglobinemic, and their sera B chain contains the Hb binding sites. The immunoelectro- cannot be typed until they are a little bit older. In addition, phoretic technique for bloodstain Hp typing is described in there are occasional anhaptoglobiiemic adults (section Culliford (1971) as well. 40.3.3.1). These people would simply not give a typing Electrophoresis is the preferred technique for Hp group- result. There may be more than one genetic basis for anhap- ing. For whatever reasons, the starch gel methods that are toglobinemia in older children and adults, and the condition usually used for serum typing are not very satisfactory for is comparatively more common in people of Black Africau bloodstain typing (Diirwaid, 1x1and 1%3; Falk and Bund- origin than in Europeans. The Hp 2-1M phenotype, which schuh, 1%3; Culliford, 1%3 and 1971; Turowska, 1969, Sourcebook in Forensic Serology, Immunology, and Biochemistry

Hilgermann, 1972b). As a result, other electrophoretic sup- tion products are extracted from the stain along with the port media have been employed for blood stains. Poly- Hp. Various methods have been proposed to get around this acrylamide gels appear to have proved most satisfactory. In difficulty. A related issue is the exmaability of Hp itself in 1971, Culliford descnied a reliable procedure for Hp typing older bloodstains. It is common knowledge that older in stains based on the use of polyamyhmide gel gradients. bloodstains do not yield up their constituent proteins to These were prepared in slab form using a gradient former, aqueous extraction media as readily as do fresher ones, and and the polyacrylamide concentration varied from about 5 there is no reason for supposing that haptoglobii is an ex- to about 30%. More recently, gels of this kind have become ception in this respect. Thus, one might fail to obtain commercially available. Earlier applications of horizontal readable typing results from a bloodstain for a variety of nongradient polyaaylamide gel electrophoresis to Hp stain different reasons, such as (1) the sample could have been an typing were not particularly successful (Gervais and Hp 0, or some other weakly expressed phenotype to begin Viescou, 1%5), but Felix et al. (1977) reported satisfactory with; (2) Sufficient Hp was not extracted to be detectable by results using such a technique. Polyacrylamide disc gel elec- the methods being used; (3) Degradation of the haptoglobii trophoresis has also been successfully used for bloodstain protein may have occurred; or (4) Hemoglobin degmhtion typing (Castilla et al., 1972; Hilgermaun, 1972a and 1972b). products may interfere with the electrophoretic typing. The Hilgermann (197%) could type stains up to 4 weeks old by first three of these problems could apply to any plasma pro- polyacrylamide disc gel electrophoresis, and said that the tein or isoenzyme genetic marker. Studies by Nikolenko technique was much better than starch gel or immunoelec- (1972 and 1975) in the U.S.S.R. and by Shaler et al. (1977) trophoresis. The gradient gel procedure is fully described in in this country indicate that procedures designed to better MPFSL (1978). It has been used in connection with the extract the Hp from bloodstains greatly improve the results Microzone electrophoresis system (Grunbaum, 1975), and with older specimens. Nikolenko's (1972 and 1975) results Baxter and Rees (1974b) reported that Hp phenotypes and indicate that the addition of urea to the extraction buffers several Hb variants could be determined simultaneously in allows typing of somewhat older stains, presumably because this way. Wraxall and Stolorow (1978) described a pro- haptoglobin is more efficiently extracted. The details of his cedure using horizontal nongradient polyacrylamide gels work must be left to those with a better reading knowledge which is fully satisfactory for typing bloodstains. Hp can be of Russian. Shaler et al. (1977) extracted bloodstains with rapidly typed in serum samples on agarose gels (Rafowin buffer alone, and with buffers containing the detergents and Lavergne, 1974). sodium dodecyl sulfate (SDS), Triton-X-100 and Tween- Haptoglobin is normally typed by electrophoretic separa- 20,at pH's of 4.6 and 8.4. The quantity of Hp extracted was tion of Hp-Hb complexes, and the separated proteins are determined by "rocket electrophoresis" (section 2.4.3.1). detected through their peroxidase activity (which the Hb still At low pH, Triton and buffer extractions were about equally possesses). Many of the reagents used for presumptive (cata- effective with stains of comparable age, while at higher pH, lytic) blood identification tests, based on the peroxidase ac- Triton and Tween were more effective. More important tivity of hemoglobin (section 6),have been employed for the than the detergent effect, however, was the observation that detection of Hp-Hb complexes in gels following electropho- the amount of immunologically detectable Hp extracted in- resis as well. Benzidine was often preferred until its use was creased significantly (as much as 133%) with 24 hrs extrac- discontinued because of its carcinogenicity. Owen et al. tion as compared with 6 hrs. Extraction with detergent at (1958) tested a series of oxidizable catalytic substrates, in- low pH, and with buffer at high pH, was essentially com- cluding guaiacol, leucomalachite green, amidopyridine, plete at 24 hrs. Stains from 44 to 202 days old were tested p-anisidine, benzidine, o-tolidine and o-dianisidine. They with and without Triton in the extraction buffer, and the preferred odianisidine, which was also recommended by differences were not significant. Extraction at pH 8.4 was Compton et al. (1976). Queen and Peacock (1%6) liked far superior to that at pH 4.6 with older stains, however. guaiacol; Burdett (1977) found leucomalachite green to The common practice of soaking stains for a matter of be preferable in comparison with several noncarcinogenic minutes in electrophoretic gel buffer prior to electrophoresis compounds including phenolphthalin, diphenylamine, is not, therefore, the best approach if one wants to type Hp 2,Michlorophenol indophenol, N,N1-diethyl-p-phenyl- in an older stain. enediarnine sulfate and 2,2'+iiazo-di-(3+thyl~ Improvements in bloodstain typing results have also been lined-sulfonic acid). Singh (1%7) recommended initial observed if the Hb and its apparent degradation products benzidine-peroxide staining, followed by an Amido Black are separated from the stain extract prior to electrophoresis. 10B staining step to give clearer, more permanent bands. Fresh Hb A must, of course, be added back to the sample Many investigators have noticed that older bloodstains before insertion into the typing gel. Gazaway (1976) become difficult to type for Hp. Until fairly recently, it was separated the Hb matafrom the Hp by an agar gel elec- seldom possible to type bloodstains more than a few weeks trophoresis procedure in cylindrical tubes. Conditions were to a few months old. Older stains tended to give streaky, employed in which the unwanted hemoglobin material overstained, and frequently unmdable patterns. One of the migrated into the gel while Hp did not. A simpler procedure reasons apparently has to do with the fact that hemoglobin has recently been described by Stolorow and Wraxall(1979). undergoes degradation in bloodstains, and the Hb degrada- The stain extract is subjected to a simple CHCl, extraction Serum Groups-Hp in a test tube, and the aqueous layer removed for electro- Reviews of many aspects of the haptoglobi system may phoretic typing. Chloroform extraction is an old procedure be found in Harris et al. (1959), Lauren (1960), Galatius- for this purpose, dating back to the early studies of For- Jensen (1960 and 1%2), Jayle and Moretti (1%2), Giblett manek (1900) and of Kriiger (1901). This procedure is appli- (1%3, 1%8 and 1%9), Prokop and Bundschuh (1%3), cable to the preparation of samples for electrophoresis by Javid (1%7b), Kirk (1968a), Prokop and Uhlenbruck any desired typing method, and it is possile to type blood- (1%9), Pintera (1971), Braun (1972), Harris (1975) and stains some months older in this way than has routinely been Putnam (197%). possible. A rather different approach involving wit immuno- precipitation of Hp from bloodstain extracts was reported by Blake and Sensabaugh (1978b). Subsequent typing in 40.5 Distribution of Hp Phenotypes in SDScontaining polyacylamide gels was possible in blood- U.S. Populations stains up to 18 months old. The data are shown in Table 40.1. The worldwide Haptoglobin cannot be detected in saliva or in semen distniution of Hp phenotypes was discussed by Walter and (Blake, 1976; Blake and Sensabaugh, 1976 and 1978a; SteegmiiUer (1%9), and extensive tables may be found in Schwerd and Fehrer, 1979). If the protein is present, the Mourant et al. (1976). The highest Hp' frequency in major concentrations are extremely low, and the system is not ethnic groups is found in Australian aborigines, and the applicable as a genetic marker in these fluids. In seminal lowest is found in Lapps. Walter and Steegmiiller (1%9) had plasma, Blake and Sensabaugh (1978a) could have detected data on almost 145,000 Caucasians. Hpl in European Hp at concentrations of 2-5.6 pg/mP, and they were unable Caucasians was 0.386, and somewhat lower in those of non- to do so. European derivation (0.254). Hp 2-1M was seen at frequen- Chun and Sensabaugh (1979) reported preliminary but cies greater than 1% only in Negroes, but non-European promising results on the possibility of subtyping Hp in Caucasians had a frequency of 0.5%. Hp 0 occurred in blood stains. Immunoprecipitation and absorption pro- 4.5070 of Negroes, and in 1-2% of non-European Cauca- cedures were employed for the isolation of the Hp from sians, Mongoloids, Australian aborigines and Lapps. Euro- stain extracts. The discriminaton index for the Hp system pean Caucasians showed 0.5% Hp 0. Hpls seems to be more would be improved signiricantly if the Hp 1 subtypes could common than HplFin most populations. be discriminated. Sourcebook in Forensic Serology, Irnrnunologv, and Biochemistry

Table 40.1 Distribution of Hp Phenotypes in U.S. Populations

-- - Frequency. Number IPersantl Popubtion -2-1 -2-1M -2 CAUCASIAN Ann Arbor. MI Sunon at at.. lSE9 Michigan and Illinois Bmyani-Siosln at 01.. 1%2

Saanla. WA Giblen and Brooks. 1963

Southaastarn GA Cooper at 01.. 1963 Mawland Quaen and Paacock. 1966 Chicago. IL Shih and Hsia, 1%9

Tscumreh. MI SchrafRar at at.. 1971 Orange Counw. CA Fitrp8trick at 81.. 1976

Baxar Countv. TX Ganawav and Lux. 1976

Pinsburgh. PA Hagins at 01.. 1978 California Grunbaum at el.. 1978

Detroit. MI S~OIO~OWat 01.. ism Miami. FL Stuvar. 1979 Lot Angalas. CA Slglar. 1979 California Grunbaum at al.. 1980

NEGRO smanla, WA Giblan. 1959 Ann Arbor. MI Sunon at el.. 1959

Seattle. WA and Cleveland OH Giblatl and Steinberg. 1960 New York. NY Parker and Beam. 1961

Seanla, WA Giblen and Brooks. 1963 Southaastarn GA Cooper at a!.. 1963 Chicago. IL Shih and Hair, 1969

Baxar Countv. TX Ganaway and Lux. 1978 Pinsburgh. PA Haglns at 01.. 1978 Cmlifornia Grunbaum st at.. 1978

Detroit. MI Stolorow at al.. 1979 Miami. FL Stuvsr. 1979

Los Angelas. CA Siglar. 1979 California Grunbaum st al.. 1980 HISPANIC Barar Countv. TX Ganawav and Lux. 1978 California Grunbaum st al.. 1978

Miami. FL Stuver. 1979 Lo5 Angalas. CA Siglar. 1979 California Grunbaum at 01.. 1980

OTHER U.S. Japanese Harria at a!.. 1959 New York. NY Chineas Parker end 8earn. 1961 Saanle. WA Orwntal Giblen and Brooks. 1963

NOWYork. NY Chinese Shim and Beam. 19Wa Calitornia and Hawaii Asian Grunbaum at al.. 1978 California and Hawaii Asian Grunbaum at 01.. 1980

1. 66 Hp 1 and 2.1 wars subtvpad: 6. 3 ware "rare" 416.111S.l: 517.611s-IF: 36154.512.1s: 21131.612-1F 9. "Chiuno/Ammrind'an" population 2. 4 were HpCa and 2 wars 'Johnson' 10. "Maxisen" population: 1 war "rara" 3 Approximately 15% 01 the nample ware Hispmnic Ibv surnamal and 2-1 subtvpad: 4. And see Shaler 119781 14117.5)lS-1s: lIl.3llS.lF: 64l8012.lS: lll.312.1F 5. 222 Hpl and 2-1 ware rubtvpad: 12. Subtyplng: 25l11.711S.1S; 2019.OllF.lF: 491P.lIlS.lF: 61127.512-15; a11 16 1.1 warn 1s-1s: all 45 2-1 were 2-1s

55124.812-IF; 612.712M.lS: 512.312M.lF 13. 1.1% wars "rare" 6. 3.2% ware "rare" 7. 410.791 were "rare" Serum Groups-Gc

SECTION 41. GROUP SPECIFIC COMPONENT

41.1 Recognition-Genetic Variation trophoresis and by starch gel electrophoresis (Arfors and Group specific component (Gc) was first recognized Beckman, 1%3). Gc phenotypes have been determined because of its polymorphism. Hirschfeld (1959b) found that under a number of different conditions on starch gels normal human sera subjected to imrnunoelectrophoretic (Parker et al., 1%3; Bearn et al., 1%4a) and on poly- analysis showed qualitative differences in proteins in the a2 acrylamide gels (Kitchi and Bearn, 1966). Under some elec- globulin region. Sera could be classified into three groups, trophoretic conditions, heterogeneity in the homozygous orighally called "l", "2" and "3". These differences were types was noticed. It was thought that this might indicate not attributable to the Hp polymorphism, and those seen in something about the subunit structure of the molecule. The human sera were analogous to qualitative variation seen in matter is discussed further in a subsequent section. Azen et rabbit sera when the latter were tested by immunoelectro- al. (1%9) separated the Gc proteins on starch at high volt- phoresis with a horse anti-rabbit serum (Hirschfeld, 19%). ages, and said that better resolution was obtained, but that A preliminary report on the variation in human sera was an additional component appeared in the 1 and 2-1 types. given at a meeting late in 1958 (Hirschfeld, 1W). The type Gc phenotypes can be determined by a number of variations "1" and "3" human sera showed single immunoprecipitin and combinations of electrophoresis and immunoelectro- arcs, while type "2" showed both. The type "2" pattern phoresis, as is discussed further below. was indistinguishable from that given by an equal mixture of type "1" and type "3" sera, and the patterns were in- 41.2 Further Gc Phenotypes variant in a given person over the course of time. Family, Hirschfeld (1%2) found two Gc types that did not corre- and mother-child pair studies (Hirschfeld et al., 1W) in- spond to the common phenotypes. One, from the serum of dicated that this was an inherited variation in which types 1 an African Negro, appeared to represent Gc 1 and a faster and 3 were homozygous, and type 2 was the heterozygote. moving component called "Y"; thus Gc 1-Y. The other, AU the observatons could be explained by a pair of alleles, from a Caucasian serum, had a component that was faster and it was suggested (Hirschfeld and Beckman, 1960) that than 2, but slower than 1, and it was called "Gc X". Parker the system be called the 'group smc component', and the et al. (1963) also reported a fast variant in a Black subject, genetic locus controlling its expression be called Gc. The and one of intermediate mobility in a Caucasian. Family alleles were designated Gc' and Gct, and the system was in- studies could not be carried out in either of these cases to dependent of Hp, Gm, Tf and pseudocholinesterase. Types prove that the vdtswere reflections of other alleles at the "1" and "3" thus became Gc 1-1 and Gc 2-2, respectively, Gc locus, however. The 'Caucasian' variant of Parker et al. and type "2" was Gc 2-1, by analogy to the nomenclature (1%3) is sometimes called 'Gc Caucasian' or 'Gc Cau', and that had been developed for haptoglobin. Family and popu- it is probably the same as 'Gc X' but the two have never lation studies soon established that the two allele hypothesis been directly compared (Johnson et al., 1975). Cleve et al. of inheritance was correct (Hirschfeld et al., 1%0, (1%3a) found several additional phenotypes that could be NerstrBm, 1%3a and 1%3b; Reinskou and Mohr, 1%2; accounted for by two additional variant alleles. The first Mama et al., 1%3; Biitler et al., 1%3; Reinskou, 1%5a; was seen in Chippewa Indians, and was called 'Gc Chip- Seppda et al., 1%7; Suyama and Uchida, 1%9), and that pewa' or 'Gc Chip'. Gc Chip1 was hard to distinguish by the gene frequencies differed in racially and ethnically immunoelectrophoresis, but was distinctive by starch gel distinct populations (Hirschfeld and Beckman, 1%1; Cleve electrophoresis. It was due to an allele designated G~P. and Beam, l%la and l%lb; Baitsch et al., 1%3; Blumberg Evidence of the second allele was detected in Australian et al., 1%; Hurnmel et al., 1970). The earlier studies on aborigine sera, which was called GcAb. All three the Gc polymorphism were well reviewed by Hirschfeld phenotypes, Gc Ab-1, Gc Ab-2 and Gc Ab-Ab, were (1%2). observed. The Gc Ab band was faster than Gc 1 or Gc Chip, The Gc types are detectable electrophoretically as well. In and was best detected in polyacrylamide gels (Kitchin and 1959, Smithies had noticed that there were differences in the Beam, 1966). No differences could be detected by Kitchin migration patterns of proteins in the "post albumin" and Beam (1966) between Gc Ab, Gc Y and another variant region, and thought that these might have a genetic basis originally described in Greenland Eskimos by Persson and (Smithies, 19% and 1959b). In 1%2, Schultze et al. (1%2a) Ti~lgsgaard (1%5), either by immunoelectrophoresis or showed clearly that the Gc types were determinable by polyacrylamide gel electrophoresis, and these were thought starch gel electrophoresis. The following year, it became to be the same. In a direct comparison on thin layer agarose clear that the Gc types and the "post albumin" variations gels, followed by immunofixation detection, however, were identical, as sera were examined both by immunoelec- Johnson et al. (1975) found that Gc AbN was different Sourcebook in Forensic Serologv, Immunology, and Biochemistv from the Eskimo variant, which they called 'Gc Esk'. In ad- tions of heterogeneous bands of Gc 1-1; (3) This group con- dition, 'Gc Esk' could not be distinguished from 'Gc D' (see sisted of 'subgroups' of the Gcl allele, as disclosed by a par- below), and so 'Eskimo' and 'Darmstadt' may be identical. ticular antiserum, raised in sheep against Gc 2-2 (Ruoslahti, Reinskou (196%) found another pair of variant types in 1%5). Type 1 sera could be clasdied as 'reacting' or Norwegian sera, which could be designated 'Now-1' and 'nonreacting', and the 'reactor' characteristic was in- 'Now-2'. He did not care for the descriptive nomenclature herited in the manner of a Mendelian dominant. (4) The that had developed, though, and suggested that the gene fourth group comprised the silent alleles of Gc. These are responsible for the 'Norw' phenotypes be called GcIC, and apparently very rare. Henningsen (1W reported the possi- the phenotypes 1-1C ('Norw-1') and 2-1C ('Now-2'). The biity of GI?' in a family with peculiar Gc inheritance. 'Nonv' and 'Chip' phenotypes appear to be characterized Prokop and Uhlenbruck (1%9) reported a family in which a by altered proportions of the heterogeneous Gc 1-1 pro- Gc 2 father had a Gc 1 daughter, who, in turn, had a Gc 2 teins. Gc Chip has an increased anodal portion, and Gc son. There was no question about the maternal relationship Norw an increased cathodal one. The "C"in the GciCdesig- of the daughter to her son, and the easiest explanation was nation stands for "cathodal", and Reinskou (I%%) said that the father was G?W, his daughter GciGP, and her that G ~ shouldP be designated GciA,where "A" stands son GcZGc". Patscheider and Dirnhofer (1979) described an for "anodal". The Norw variants are apprently quite rare. extraordinary sibship in which GP and ESP were appar- A second example was reported by Rittner and Dahr (1969). ently segregating together. A child in the third generation Other variants that have been reported are: GG,observed was believed to be GciG&,ESDIEW,and the family study as 1-Z and 2-2 (Hennig and Hoppe, 1%5; Cleve et d., indicated that the Gc" had been inherited from the maternal 1%6); Gc Bangkok or GC~~~,seen as Gc 1-Bangkok grandfather, and the ESP from the maternal grandmother. (Rucknagel et al., 1%8); Gc Darmstadt, or GcD, seen as The child's mother was also, thus, a double heterozygote for D-1 and D-2 (Cleve et al., 1970); Gc Wien, or GcW,seen as the silent alleles. Gc W-1 and W-2 in 8 members of a family in Vienna This cW1cation of the Gc phenotypes by Clwe (1973) (Wien) (Speiser et d., 1972); Gc Japanese or G$, seen as would have to be revised somewhat in view of the hding Gc 5-2 (Omoto et al., 1972); Gc Opava, or GcO~,seen as that there are two Gc' alleles, whose products are revealed Gc Op-1 and Op-2 (Cleve, 1973; Vavrusa and Cleve, 1974); by isoelectric focusing (section 41.3). In addition, the best Gc Boston, or G@ (if genetic transmission can be proven), technique for typing most of the Gc variants seems to be seen as B-2 (Johnson et al., 1975); and Gc Toulouse, or thin layer agarose electrophoresis, followed by immunofixa- GCT, seen as Gc T-2 (Constans et al., 1978). tion detection (Johnson et al., 1975). Their studies clarified Cleve (1973) reviewed all the known whtsto date, and some of the relationships Meenthe rare variants. Patterns could classify them into four major categories: (1) Molecu- of some of the Gc types as seen using this technique are lar variants, with electrophoretic mobility different from shown in Fie41.1. Gc 1and Gc 2. The faster ones are Gc Ab, Gc D, Gc Wien The majority of the Gc variant phenotypes are rare. Gc and Gc Japanese; Gc Opava migrates in between Gc 1 and Chip reaches polymorphic frequencies in the Indians after Gc 2; and the slower ones are Gc Bangkok and Gc Z. (2) This whom it was named. (3cAb, giving rise to the Gc Ab group included Gc Chippewa and Gc Norway variants, phenotypes (likely equivalent to 'Gc Y' in Black popula- which, as noted above, seem to represent altered propor- tions), occurs in appreciable frequencies in the aborigines,

ORIGIN

1 2-1 2 Ab-1 Ab-2 Ab-Ab J-2 D-2 1-C~U W-2 2-IC 8-2 Op-2 2-2 2-IA

Figure 41.1 Electrophoretic Patterns of Some Gc Phenotypes (after immunofixation agarose gel electrophoresis. pH 8.61. Identities and probable identities: Gc Ab = GcY; Gc X = GcCau: Gc 2-1A = Gc 2-Now; GC 2-1A = Gc 2-Chip. Serum Groups-Gc and is occasionally seen in Black people. There is little 41.4 Methods of Phenotyping Gc population data, but GCAb had a frequency of 0.015 in 492 A number of procedures have been employed to Black Americans from Pennsylvania and Georgia (Kueppers distinguish the Gc phenotypes in serum. The three wmmon and Harpel, 1979). Gc Ab phenotypes are seen in various phenotypes, 1, 2-1 and 2, can be diagnosed with any of populations in Oceania as well (Kirk et al., 1%3). them in experienced hands. Some of the rarer variant types are more difficult to discriminate by certain methods, and 41.3 Additional Genetic Variation at the easier using other ones, and this has been one of the dif- Gcl Locus-The Oc 66Subtypes99 ficulties in the unequivocal classiiication of the variants ob- served in different laboratories using different methods. The In 1977, Constans and Viau found that "subtypes" of Gc original procedure, immunoelectrophoresis, serves well in 1 wuld be distinguished by polyacrylamide gel isoe!lecrric many laboratories (see in Hhchfeld, I%&). Interpretation focusing (PAGIF), and detection of the Gc bands by im- of the patterns is made considerablyeasier if specific anti-Gc munofixation. The "fast" and "slow" bands of Gcl were serum is used rather than anti-human serum. Both starch designated "F" and "S", and accounted for by the alleles and polyacrylamide gel electrophoresis have been used for GclFand GcIS.These Gc alleles, along with Gcf give rise to typing Gc, as discussed above. A polyacrylamide disc gel the phenotypes lF, IF-IS, IS, 2-IF, 2-1s and 2. The GclF procedure was devised by Raunio et al. (1%6). Polyacryla- frequency was comparatively low in a Pyrenean valley pop mide gel electrophoresis can be employed on a preparative ulation, but about 0.58 in a central African pygmy group. These observations, and the proposed genetic explanation for them, have been amply confirmed (Constans et al., 1978; Kiihnl et al., 1978; Ishimoto et a!., 1979; Hoste, 1979; Kueppers and Harpel, 1979). The GcIFand Gc'S frequencies in Hessen were 0.125 and 0.603, respectively (KW et al., Table 41.1 Gc Variant Nomenclature Equivalents 1978), while in a Belgian population, they were 0.167 and - 0.543 (Hoste, 1979). Constans et al. (1978) extended their New Qc Nommshn Old Dnl#n.tlorn original studies, and found two additional vhtsof Gc. 1s 1s One, found in an African pygmy population, exhibits a 1F IF band in about the same position as the second band of a 1-1 2 2 on polyacrylamide gel electrophoresis. It was a variant of Gc 1A1 Ab:Vi* 2, and was called '2a' (for 'anodal'). It was inherited, and 1A2 0r:O J: OMS %:@ 4.0 the responsible allele was designated GP. Both 2a-1 and 1A.3 OMS 6:O OMS P:O N: J~O 2a-2 phenotyes were observed, and the fonner can be fur- lA4 Igloo: Y : Eskho~ ?As YO ther classified as 2a-1F or 2a-1s by isoelectrofocusing. The lM 1co other variant was found in a tribe of South American In- 1A7 OP, dians from Bolivia. It was called 'Gc Am-1' and was indis- lM Am1:O TK2.OOMS 10:O JdO tinguishable from Gc Ab-1 by electrophoresis, but it was as- 1A.9 Amt: TKl:@OMS 14:o Js 0 sumed that Gc Am types reflect a new allele, G@. Ishi- 1Cl T. mot0 et al. (1979) subtyped Gc in Japanese populations, and IC2 OMS 1:O 0kO found that the Gc J phenotypes could be further subdivided tw 10: YO into a series of phenotypes representing wmbiinations of 1C4 OMS 2I:o hllao 1CS Bognk.0 G$~,GCT~, G$C and ~c'~with GclS, GcSFor W.They 1M ?Dl0 found some further variants as well, which were called Gc 1C1 ID20 Or. Ja, Jb, Jc, Jd, Ok and Mie. In a note artachedto the reprint, 1- 1E Dr. Ishimoto noted that these variants were equivalent to 1C9 SI the Gc 1A2, 1A3, 1A9, 1A8, 1C2 and 1C4, mpectively, of lC10 1Q0 Dr. Constans. This latter nomenclature resulted from an in- zA1 2co ternational conference on Gc variants and nomenclature, 242 or. held in Paris in 1978 (Constans and Cleve, 1979). Cone- zA3 2A zA4 2Y spondence between old and new nomenclature for about 30 zA5 v?o tB known variants was given, along with representationsof the ?A6 Wh electrofocusing patterns. Briefly, the "A" and "C" 2C1 ZD@ designations mean "anodal" and "cathodal", and variants 2CZ 7. are then classified according to mobility. For example, Gc t Accordhg to Conmne and Cbw 1197s): only one pm. produn b sham: only rerlano Ab is now Gc 1A1, and the phenotype that would previously *chYIv comp.nd bv hooMric focusing anshan have been called Gc 'Ab-IF' is now Gc 'lA1-IF'. The t Tho '7"mnk Black populationsla thought to be eguln*nt to "Ab paper must be read by those interested in Gc typing. %en h Jam-. 0 Accodh~to lmhimoto n -1.. (197'91 Equivalences are shown in Table 41.1. 0 hmlU.1 w.nenb.lon not clum nublhhod Sourcebook in Forensic Serology, Immunologv, and Biochemistry scale (Simons and Bearn, 1%7). Prokop (1963) dmia Vitamin D, binding were detected among the variant types. kind of two-dimensional immunoelectrophoretic procedure The MW of the purer preparations of Gc is in the neighbor- suitable for Gc. Electrophoresis was performed as usual, but hood of 52,000 (Irnawari et al., 1976) to 58,000 (Bouillon et then two antibody troughs were cut in parallel on either side al., 1976). of the sample position, and electrophoresis performed at There have been various reports of the Gc level in nonnal 90" to the original direction. This procedure was much serum. Published values range from about 0.28 to about faster than classical immunoelectrophoresis, in which the 0.75 mg per me of serum. The higher values are those of Kit- antibodies are allowed to diffuse into the gel. The antigen- chin and Beam (l%S) using quantitative immunoprecipita- antibody crossed electrophoresis procedure of LaureU tion, and the lowest values were obtained by radial immuno- (1%5), which is described in detail by Weeke (1973), has diffusion measurements (Kueppers and Harpel, 1979), who also been used for Gc typing (see in section 2.4.3.2). used a commercial standard serum for calibration. There Sutcliffe and Brock (1973) used this technique, and it has have been suggestions, too, that the normal Gc content of also been found useful in comparing Gc variants. In 1%9, serum differs according to phenotype, but this variation was Alper and Johnson descrii a techhique called immunofix- not seen by Kitchin and Bearn (1%5) nor by Kueppm and ation electrophoresis (section 2.4.2); the technique is further Harpel (1979). discussed by Ritchie and Smith (1976). Here, the separated components are allowed to react with specific antibody in 41.5.2 Biochemical studies the gel, after which the gel is washed free of excess protein, The amino acid composition of Gc has been studied and the specific complexes stained. This technique has been by Cleve et al. (1%3b), Bowman and Bearn (1%5) and used extensively for the detection of Gc types following elec- Bowman (1%7 and 1%9) among others. The fast and slow trophoresis (Johnson et al., 1975; Martin and Kopietz, 1976; bands of 1-1 and 2-2 are all very similar, and the Asp and Grunbaum and Zajac, 1977; Wraxall and Stolorow, 1978). Glu content is quite high. The molecule contains carbo- Irnmunofkation may also be used to detect Gc following hydrate but little is known about how it is arranged. isoelectric focusing. Most of the techniques that have been Schultze et al. (1%2b) found the equivalent of 7 hexose, 5 found suitable for bloodstain typing utilize some form of acetylhexosamine and 1 fucose per 51,000 MW,but no sialic immunofvtation detection scheme (see below). Hoste (1979) acid. The data of van Baelen et al. (1978), however, strongly detected the Gc bands on polyacrylamide gels after isoelec- suggest that the Gc 1 molecule must contain sialic acid. trofocusing by a simple non-immune sulfosalicylic acid Bowman (1%9) proposed a molecular model for Gc, staining procedure. based on end group analysis, peptide maps and other chem- ical data. The model is further discussed in the review by 41.5 Physiological and Biochemical Putnam (1977) as well. The molecule is a dimer, according Studies on Gc to her proposal, which can be made up of three different kinds of chains controlled by three separate genetic loci. The 41.5.1 The function and properties of Gc protein chain called 6 is common to fast bands of both 1-1 and 2-2. For years, Gc proteins were a genetic curiosity, the system In 1-1, 6 is combined with crl to give crlS which is character- being well studied as a genetic marker, but the protein con- istic of 1-1. In 2-2, 6 is paired with an & chain to give the stituents of it having no known physiological function. It d6 characteristic of 2-2. A B chain combines with or1 to give has been known for many years that serum contains some the cr1/3 slow band of 1-1. Van Baelen et al. (1978) canied sort of Vitamin D binding protein, and that this protein is out studies on the Gc proteins as separated by isoelectro- an or-globulin (Thomas et al., 1959). Belsey et al. (1974) focusing. Gc 2-2 contained a single protein, where Gc 1-1 found that rat and chick, in addition to human, sera all have had two bands with lower PI. The 2-1 contained all three. this protein, and that it binds 25-hydroxy-Vitamin D An anodal shift was observed after incubation of the preferentially. The Vitamin D binding protein was isolated apo-Gc with excess 25-hydroxy-Vitamin D. Sialidase treat- from human serum and partially purified by Imawari et al. ment had no effect on Gc 2-2, but the faster Gc 1-1 band (1976). Daiger et al. (1974 and 1975a) then observed focuses in the position of the slower band after treatment, polymorphism in the human serum Vitamin D binding pro- strongly indicating N-acetyl neuraminic acid in the Gc 1-1 tein, and quickly realized that they had 'rediscovered' the fast band. Gc polymorphism. Their studies indicated that Gc and Vita- Reviews of the Gc polymorphism may be found in Cleve min D binding proteins are identical. Bouillon et al. (1976) and Bearn (1%2), Beam et al. (I%&), Reinskou (I%&), rapidly confmed the identity using an extensively purified Giblett (1%9), Prokop and Uhlenbruck (1x9) and Putnam preparation. They said that the protein should be renamed (1977). "tmwalciferin". Haddad and Walgate (1976) purified 41.6 Medicolegal Applications the serum Vitamin D binding protein, and likewise showed that it was immunologically identical to Gc. Cieve and 41.6.1 Disputed parentage Patutschnick (1977) used autoradiography to show that the The Gc polymorphism has been used in disputed paternity components of the common Gc phenotypes, as well as those cases in a number of laboratories for quite some time of many of the variants, bind Vitamin D,.No differences in (Hirschfeld and Heiken, 1963; Nerstrem, 1%k, Ritter, Serum Groups-Gc

1%3; Marek et al., 1%3; Biitler et al., 1963; Holzhausen et stains on linen. Similar observations were made by Brzecka al., 1%4; Reinskou, I%&). The probability of excluding a and Mikulewicz (1966). They said that a major difficulty falsely accused father of Western European origin is about was in concentrating the protein in bloodstain extracts to a 15-16% and the system is considered reliable (Reinskou, sufficient extent that the Gc types were detectable. Gc could 1%6a). In U.S. populations, the exclusion probabilities are be typed in fairly fresh post mortem blood, but older about 16% for falsely accused White fathers, and about hemolyzed samples were unsatisfactory. Tumanov and 74% for falsely accused Black fathers (Chakraborty et al., Il'ina (1974) utilized an immunoe1ectrophoretic technique. 1974; Dykes and Polesky, 1978). The system is apparently but with a monospecific anti-Gc serum. Stains on a variety employed only rarely in disputed parentage cases in this of substrata could be typed for up to 18 months time, a con- country (Polesky and Krause, 1977). The exclusion prob- siderable improvement compared with the older procedures. abiities given above are based on the detection of the three In 1975, Wraxall reported briefly on efforts to apply the common phenotypes. Serum phenotyping is generally antigen antibody crossover electrophoresis technique to straightforward. The possibility of rare or silent alleles, bloodstains. Apparently, many of the problems encoun- though they are not often encountered, must be kept in tered in immunoelectrophoretic techniques are alleviated by mind. If isoelectrofocusing equipment is available and the the use of electrophoretic separations followed by immuno- Gcl subtypes can be determined, the exclusion probability is fixation detection. Johnson et al. (1975) said that they had increased significantly. Kiihnl et al. (1978) indicated that the no difficulty typing aged, contaminated or jaundiced sera in figure would be 24Vo in the Frankfurt population; Hoste this way, in contrast to the difficulties reported previously (1979) said it would be almost 32Vo in Belgium. when immunoelectrophoretic techniques were being used. They preferred agarose gels for immunofvraton electropho- resis over polyacrylamide or starch gel media. Martin and 41.6.2 Gc phenotypiug m bloodstains Kopietz (1976) reported that immunofixation electropho- The earlier investigators used immunoelectrophoresis for resis in agarose had helped to clarify the phenotypes of some Gc typing, since this technique was the only one available samples which were originally typed by immunoelectropho- for several years. A number of efforts were made to type Gc resis. One serum typed as a 2-1 in agarose, but had looked in bloodstains by this procedure, but most of them were not like a "1-variant" of some kind by immunoelectrophoresis. very successful. More recent applications of electrophoresis Similarly, a specimen thought to be a possible 2-W by its and imrnunofixation detection techniques have been more immunoelectrophoretic behavior turned out to type as a Gc productive. 2-Ab in agarose. Immunofixation may be used in conjunc- In 1%3, Nerstrbm and Skafte Jensen reported that only tion with cellulose acetate membrane electrophoresis, as well the very freshest bloodstains could be typed for Gc using as with electrophoresis in gel media. Grunbaum and Zajac imrnunoelectrophoretic procedures. Peculiarly, serum stains (1977) described such a procedure, which needed only 20 could be grouped for several weeks. Vogt (1%3) in Prof. minutes of electrohoresis to achieve separation. This techni- Dr. Prokop's institute had similar results, although he could que could be used to type bloodstains up to 2 months old type stains up to about 10 days old made on glass plates. (Zajac and Grunbaum, 1978). A chloroform extraction Heifer and Bolkenius (1%6) found, however, that Gc technique was used to remove the hemoglobin degradation seemed to be a little more stable in stains made on glass than products from the bloodstain extract prior to electropho- in those on other substrata. There seem always to have been resis. Wraxall and Stolorow (1978) described an immuno- some difficulties with older bloods, or sera that have been frxation agarose electrophoresis typing procedure, which is transported over great distances (as is sometimes done in fully applicable to bloodstain typing as well. The procedure population studies), when immunoelectrophoresis is used is currently used in several laboratories that we know of, for the Gc typing. Problems awx5ate-d with stored, older and yields satisfactory results on stains up to a number of sera were discovered by Nerstrbm (1%3b) and Persson and months old. Ti& (1966). There is a distinctive decrease in the qual- Gc is not detectable in semen by radial immunodiffusion ity of the precipitin arcs in these samples. Nerstrem (1%3c techniques which would detect it in 2.5% solutions of serum and 1%4) was inwed by the fact that the Gc types in (Blake, 1976; Blake and Sensabaugh, 1976 and 1978a). If serum stains were more stable than in whole bloodstains, the serum level of Gc is taken to be 800 pg/mP, therefore, Gc and that old, hemolyzed blood gave typing problems that would have to be present in concentrations of less than separated serum did not. The lysed cells were thought to about U)pg/mP to go undetected. The serum concentration have an adverse effect on the Gc proteins, and studies of Gc may actually be lower than 800 pglrne (section showed that lysed leucocyte and thrombocyte material did 41.5.1). The Gc system is not, therefore, a useful genetic disturb the Gc pattern. A similar effect could be induced marker in semen. Gc cannot be detected in healthy urine by with proteolytic enzymes and products from yeast. Heifer immunoelectrophoretic analysis, but it can sometimes be and Bolkenius (1966) were able to type most of a series of determined in pathological (proteinuric) specimens (Nielsen bloods kept stored for two years, and about half the post et al., 1%3). Thus, while it is highly unlikely that Gc could mortem samples they attempted. They too found that senun be detected in urine stains, it is equally unlikely that urine stains could be grouped a little longer than whole blood contadnation would interfere with Gc bloodstain typing. Sourcebook in Forensic Serology, Immunology, and Biochemistry

41.7 Distribution of Gc Phenotypes- - in of 0.1). In most Asians, the frequencies are closer to those U.S. Populations seen in Europeans. Gcl subtyping is comparatively recent, and there is not very much data as yet. It appears, however, The data is shown in Table 41.2. The worldwide Wbu- 75-85% of European CIT ell. In BLackpop tion of the common Gc' and Gy alleles was dk-ussed by *ions, G~~Sis a much lower fdonof aeve (1973) and and (1969)' "' 15-Z.I.. I. Japanesepopulafions, the f&on is of about 0.25 to 0.30 in most European populations, and quite the order of 35%. a bit lower in most Black African populations (of the order Serum Groups-Gc

Table 41.2 Distribution of Gc Phenotypes in US. Populations

Fraquancy - Number (Percent1 Population -Total Reference CAUCASIAN New York. NY 122 Clew and Beam. 1961s New York. NY 86 Clove end Bearn. 1961b Southaastern GA 292 Blumberg et 01.. 1964 Boston. MA 407 Murray and Robinson. 1968 Tecumseh. MI 7.658 Schreffler et al.. 1971 California 4.488 Grunbaum et al.. 1978

Detroit. MI 503 Stolomw et 01.. 1979 Miami. FL 365 Stuver. 1979 Los Angeles. CA 109 Siglar. 1979 Southeastarn PA 110 Kueppen and Harpel. 1979

California 1.OH) Grunbaum et al.. 1980 NEGRO New York. NY 144 CIeve and Bearn. 19610 New York. NY 120 Cleve end Bearn, t961b Southeastern GA 231 Blumberg et al.. 1964 California 832 Grunbaum et al.. 1978 Detroit. MI 504 Stolorow et 01.. 1979 Miami. FL 339 Stuver. 1979 Los Anpales. CA 43 Siglar. 1979 Southeastern GA 219 Philadelphia. PA 273 Kueppars end Harpel. 1979 Combined 492 California 867 Grunbaum st 01.. 1980 HISPANIC California 1.417 Grunbaum et at.. 1970 Miami. FL 360 Stuver. 1979 Los Angeles. CA 102 Siglar. 1979 California 1.908 Grunbaum et el.. 1980 OTHER New York. NY (Chinese) 117 Cleve and Bearn. 1961s California and Hawaii (Asians) 3.043 Grunbaum at el.. 1978 California and Hawaii (Chinese end Japanasel 1.566 Grunbaum at 01.. 1980

1. Calculated from gene frequencies 9. Subtyping: 11. Population was "Chicano1 GA 1411W.41 IF. 40118.3) 1F-1s. 411.81 Amerindian: 0.5% ware "rare' 2. 0.4% were "rare" - 1s. 2119.6) 2-IF. and 512.3) 2-1s; 12. 210.5) were Gc 1-Y and 110.3) 3. One was Gc 1-Y PA 130447.61 1F. b2t21.21 1F-12. 1013.71 2s. - was Gc 2-Y 4. Subtyping: 312.71 IF. 19117.31 IF-IS. 51118.71 2-1F. and 1013.7) 2-1s: 37133.6)lS. 817.312-lF and 3212912-18 Combined - 271i55.1) IF. 98119.91 IF-IS. 13. Population was "Mexican" and many samples were from 5. 10 were "rare" 1412.8) IS. 72114.61 2-1F and 1513.01 2-1s: in PA. there were 612.2) Ab-IF. 311.11 Ab-IF Mexico City: 13 were "rare" 6. 2.4% were "rara" and 2i0.7) Ab-2: 14. 4% were "rare" 7. 18D.571 were Gc 1-Y. 310.6) wera Gc in GA. there were 311.4) Ab-IF and 15, 70 were "rare" 2-Y and 1 was "rare" l(0.5) Ab-2. 16. And see Shekr. 1970 8. 611.81 wera Gc 1-Y 10. 28 were "mn" Serum Groups-Tf

SECTION 42. TRANSFERRIN (SIDEROPHILIN)

42.1 Introduction In fie out of 420 Canadian Caucasian sera, Smithies (1958) Most of the acid-soluble iron in blood plasma is reversibly found a slightly faster protein called "B". Family studies in- associated with a special iron transporting protein, which dicated that B was conditioned by a third allele, BB. It was shuttles Fe3+ to marrow, and to organs such as the liver. soon established that the 8-globulin protein exhibiting this The presence of such a protein was suspected for decades. genetically controlled variation was equivalent to transfenin Starkstein and Harvalik (1933) localized it as a globulin pro- (Smithies and Hiller, 1959). Independent lines of evidence tein, and Vahlquist (1941) found that it had the electropho- based on independent experiments by Pert, Poulik, Allison, retic mobility of a @-globulin.Holmberg and Laurell (1945) and by Sutton and Bishop (communicated to Smithies and and Schade and Caroline (1946) independently established Hiller, 1959) all pointed to the 8-globulin-transfenin iden- that a specific protein in plasma was responsible for the tity. The locus was, therefore, named Tf, and the known iron-binding properties. They also established the physio- alleles TF, T~Dand TP.Harris et 01. (1958b) found two logical role of the protein, and recognized its clinical impor- more variants, in addition to those seen by Smithies and coworkers. The faster one was called "D2" (Smithies' "D" tance. Schade et al. (1949) called the protein "siderophiiin", "B2" and showed that it took up two Fe3+per mole, the uptake of becoming "Dl"), and the slower one was called one anion (HC0,-) for each iron being necessary for the (Smithies' "B" becoming "BIW).The subscripts were de- binding to take place. Ehrenberg and Laurell(1955) wried signed to classify the faster and slower (than C) bands ac- out magnetic measurements on Fe-transfenin which indi- cording to electrophoretic mobility. Giblett et al. (1959) then cated that the iron was femc, and ionically bound. Holm- found three new transfenins (as heterozygotes of C), CDo, berg and Laurell (1947) established that the protein bound CD, and BoC. In 1960, Harris et d. found a CD, in two copper ions as well, but that the biding was much weaker European familes. By this time, it had become apparent than with femc ions which would readily displace copper. that the nomenclature scheme which had been started was They suggested that the iron binding protein be called not going to be able to absorb the flow of new variants. "transferrid"' Parker and Beam (l%lb) found a Tf in Navajo Indians that migrated between Bo and B1. This one was named "B,,", 42.2 Genetic Variation and was observed as a CB,,. Two further variants were In 1956, Smithies and Poulik introduced two-dimensional found in New York Oriental populations by Parker and electrophoresis, the initial run being wried out on a fdter Beam (l%la). One was a B,D in a Japanese. The other, in a paper strip, which was then inserted into a starch gel for Chinese, ran very much like Dl, but rather than try to con- electrophoresis in the second direction, at right angles to tinue the numbering system, it was called "DChi". Some of the frrst. Further studies (F'oulik and Smithies, 1958) indi- the variants found subsequently were still given numerical cated several components in the @-globulinregion, the most subscripts, while others took on the names of the places or heavily staining one being third in its migration rate. This populations in which they were seen. The basic symbols band was designated "C" for that reason. In 1957, Smithies "C" for the usual Tf, "D" for the slower, and "B" for the reported that a &globulin had been found in the sera of two faster variants, were kept. Giblett (1%9) suggested dropping Black women from New York (out of 49 tested) and in the numerical subscripts and sticking to geographical designa- sera of 5 Australian aborigines (out of 23) which had not tions. Ultimately, as the chemical structures of the variants previously been seen. Two-dimensional electrophoresis was are worked out, a nomenclature desaiptive of the chemical necessary to see these differences. In most sera, @-globulins change can be adopted (Putnam, 1975d), as was done for called A, B, C, and E were observed, but in the sera just the hemoglobins (section 38). mentioned, a "D" component was found as well. This D References to some further variants are: B,,, seen in a had not been observed in several hundred sera from Cauca- Venezuelan family as CB,, (Arends et al., 1962); B*, seen sians. HorsfaU and Smithies (1958) extended these studies to as CBk and homozygous in New Guinea (La, 1%3); a larger sample of aboriginal sera, including samples from DAdelaide,seen in Australia and perhaps identical to Do members of families. The original sera had exhibited ap- (Cooper et al., 1%4); BAbti, seen in Greece, and ap- proximately equal amounts of components C and D, but a parently not clearly distinguished from B1 (Mmy et al., few were seen in the later sample that had only D. It was 1%4); DWlgan,seen in England (Glen-Bott et al., 1964); suggested that these 8-globulins were under the simple DFjd, seen in Finns, and perhaps the same as D2 Mendelian genetic control of a pair of alleles, called and (Seppda, 1%5; Seppiilii et al., 1%7); BLambert,perhaps the BD. The "C" and "D" type sera represented the same as B,, (Barnett and Bowman, 1968); DMadiga, homozygotes, and the "CD" sera were from heterozygotes. DMuhj, B,,- and B- seen in India (Rao et al., Sourcebook in Forensic Serology, Immunology, and Biochemistry

1979). There are several other variants as well, and further 42.3 Further Genetic Heterogeneity in characterization will be required in many cases to demon- Tf-Tf C Subtypes strate which of them are actually identical. Some of them are very rare, and serum is not available for the com- parisons. Variants are discussed by Kirk (1%8b), Bowman In 1978, Kiihnl and Spielmann (1978a) in Gemany, and, (1968), Giblett (1%9) and Putnam (1975d). Relative electro- somewhat independently, Thymann in Denmark demon- phoretic mobilities of the Tf variants are indicated diagram- strated that the common transferrin, Tf C, was resolved into atically in Figure 42.1. three patterns by isoelectric focusing on polyacrylamide gels The variants can be arranged on a scale of relative electro- (PAGIF'). Family studies revealed that these patterns phoretic mobility, as has been done by Sutton and Jamieson represented three phenotypes, called Tf C1, C2-1 and C2, (1972). They classified many of the Tf variants according to due to a pair of codominant alleles TPand TfC2. Thymann their relative mobilities on polyaayhmide disc gel elec- (1978) found the same polymorphism in Denmark. Ap trophoresis, setting the mobility of C = 0. This kind of parently, Kiihnl and Spielmann had first reported at a con- scheme provides a basis for comparing newly discovered ference (the 7th International Congress of Forensic Haemo- variants with older ones, even if one does not have access to genetics in Hamburg) that this was some kind of an Hp panels of sera containing variants, assuming the exact same polymorphism. She called the alleles Tf and Tf. Using electrophoretic procedures are followed. A representation Kiihnl and Spielmann's nomenclature, the German sample of this scheme is shown in Figure 42.2. In 1975, Rittner and showed TfC1 = 0.8195 and TfC2 = 0.1720, while the Rittner suggested a variation of this approach. They com- Danish one showed TfC' = 0.81 and TfU = 0.19. The TfC' pared a number of variants by high voltage electrophoresis of Kiihnl and Spielmann is the TS of Thymanu.Kiihnl and on agarose gels, and in longer runs could resolve most of Spielmann (1978a) showed that this was not a sialic acid ef- them. They wanted to use Tf B2 as the "reference variant", fect, for neuraminidase did not obliterate the three setting its relative electrophoretic mobility equal to +0.7 for phenotype behavior of the samples. The new Tf C subtypes practical reasons. The variants could then be designated ac- were quickly confmed by Stibler et al. (1979), who had cording to their mobilities, e.g. B 0.78, D 1.06, etc. This earlier noticed heterogeneity of Tf C upon isoelectric focus- nomenclature is analogous to that adopted for C3 variants ing (Stibler et d., 1978), and by Hoste (1979). In 1979, (section 45). This nomenclature scheme is also shown in Kiihnl and Spielmann (197%) found a third allele, which Figure 42.2. was called TfC3. Phenotypes conditioned by ththird allele The many population and family studies which have been were detected by a modified PAGIF procedure using a canied out are fully consistent with the variant forms being shallower pH gradient. The gene frequencies in the extended representatives of alleles at the Tf locus (e.g. Kirk et al., system were ~p= 0.795, TP= 0.155 and Tp = 0.042. 1964; Kurz and Ritter, 1972). At least two families have There are, thus, six subphenotypes: Tf C1, C2,C3, C2-1, been studied in which two non-C Tf genes occurred in C3-1 and C3-2. Family studies indicated the accuracy of heterozygous combination in some members. Beckman this genetic model. In 1979, Kiihnl et al. looked at a series of (1%2) found a B2DIcombination, the first in which "fast" Tf B and Tf D heterozygotes on PAGIF. In all CB or CD and "slow" variant types occurred together. Robinson et al. samples, only C1 or C2 was seen, but never both, suggesting (1%3) described an individual of B,,B2 phenotype. that the new series of Tf alleles are true alleles of those There are several reports of fdeswith Tf deficiency conditioning the B and D variants. If all these Tf genes are (Heilmeyer et al., 1%1; Goya et al., 1972). Other reports indeed allelic, then any of the variant alleles discussed in sec- have been discussed by Gitlin and Gitlin (1975). These tion 42.2 could be paired with TfC1, TfC2 or TfC3, increasing families suggest the possibility of a rare silent allele at Tf, the number of possible Tf phenotypes by three-fold. Trans- although the data are somewhat complicated, and other ferrin genetic variant nomenclature is due for some stand- genetic explanations are possible. ardization in view of the new findings.

Alb

Figure 42.1 Relative Electrophoretic Mobility of Some Tf Variants at Alkaline pH. Dotted line indicates position of the Common Tf C. Serum Groups- Tf

- B, (Atlanta) B 0.88 1 B 0.83 1 -/- B Seattle B 0.78 -- --B, ------B, = 0.7 -- - -\- B Lambert (B,,,) --6,

D Adelaide (DO) -/-1D Wigan ,- - D. (Do-,) D Montreal -DChi------___ D, = D Chi - D Raiford D Finland

- D 2 - D Indonesia - D, (Austin1

Figure 42.2 Arrangements of Transferrin Variants according to Relative Electrophoretic Mobility on (A) Polyacrylamide Gels (Sutton and Jamieson, 1972) and (B) Agarose Gels after High Voltage Runs (Ritner and Ritner, 1975). Sourcebook in Forensic Serologv, Immunology, and Biochemistry

42.4 BiochemicalStudies on differences in peptide fingerprints of a few of the variants. Transferrin Wang and Sutton (1%5) indicated that Dl differed from C by an Asp+Gly substitution, which was detected in chymo- 42.4.1 Structure of transfenin tryptic digests. This same substitution is found in Tf D, There have been many studies on the amino acid compo- samples from American Blacks or from Australian aborig- sition of human Tf, which are reviewed by Putnam (1975d). ines (Wang et al., 1%7). Wang et al. (1966) found that on There is strong evidence that the Tf molecule is a single the basis of tryptic digest patterns, Tf B2 differed from Tf C polypeptide chain (Greene and Feeney, 1%8; Mann et al., by a single GlyGlu substitution. A simple substitution of 1970; MacGillivray and Brew, 1975) of MW about 76,00& WArgwas reported for DChi by Wang et al. (1%7). It now 77,000 (about 630 amino acid residues). There are 15 or 16 appears (Sutton et al., 1975) that Tf Dl may have two intrachain disulfide bridges, and apparently no free -SH substitutions, Asp+Gly and Asn+Gly, while Dm has only groups. The molecule can be cleaved with cyanogen the one. Immunological differences between Tf C, Dl, DChi bromide to yield 9 fragments (see in MacGillivray and Brew, and D, were not detected by Wang et al. (1%8) using a 1975, and Putnam, 1975d), and a number of these have been radioimmune inhibition of precipitation technique. sequenced. Brune et al. (1978) reported the sequence of the Most authorities are inclined to think that the variants N-terminal peptide fragment (50 residues), which they said derive from one-step mutations resulting in single amino completed about a third of the total sequence. The acid changes. There are some serious diffxulties with such a N-terminal peptide fragment contained two intrachain view (Putnam, 1975d). It is hard to explain the large number disulfide bridges, between Cys9 and Cys48, and between of Tf variants, which appear to differ by at least a single unit Cys19 and Cys39. There is considerable similarity in the of charge, by single amino acid substitutions in a molecule composition of human and animal transferrins (Palmour that appears to have no subunit structure. It is possible that and Sutton, 1971; Graham and Williams, 1975). There is there are size differences, which are reflected in the electro- also quite a bit of structural homology in different segments phoretic mobility, but there is no evidence for significant of humm Tf C (MacGillivray and Brew, 1975), and between size differences. Perhaps more extensive structural differ- Tf and lactofenin (Metz-Boutique et al., 1978). ences do exist, although if these are different single amino Transferrin is a glycoprotein, and its carbohydrate struc- acid substitutions, double or multiple mutations might have ture has been worked out completely (Jamieson et al., to be invoked as an explanation. Deletions or additions are 1971). There are two identical carbohydrate chains per Tf possible; another possibility is that of repeating sequences molecule, attached through an internal GlcNAc residue to within Tf. Asn in the peptide chain.~achcarbohydrate chain has two 42.4.3 Metal biding properties N-acetyl neuraminic acid "ends", so that there are four in the complete molecule. The structure is shown in Putnam This subject is complex, and reviewed in more detail by (1975d) as well as in the original paper. Graham and Putnam (1975d). Each Tf molecule binds two iron atoms Williams (1975) compared the carbohydrate structure of (Schade et al., 1949), and these are Fe3+ (Ehrenberg and human Tf with that of the transferrin from several animal Laurell, 1955). Bonding is ionic and bicarbonate anion is species. The presence of terminal sialic acid residues in taken up for each ferric ion bound. Further, the binding is human Tf was recognized early (Parker and Bearn, 1%1c pH dependent. Tf can combine loosely with other metals, and 1962). Action of sialidase alters the mobility of Tf C in such as Cu (Holmberg and Laurell, 1947; Aasa et al., 1%3). stepwise fashion, yielding five different bands which appear Earlier work tended to suggest that the binding sites in Tf to differ from one another by a single unit of negative were independent and identical (e.g. Aisen et al., 1966). charge (Parker and Bearn, l%lc). This result shows that all More recent evidence indicates, however, that they are not four sialic acid residues are accessible to the sialidase. identical (Harris, 1977; Aisen et al., 1978). The sites are Sialidase treatment of Tf variants yielded the same kind of designated "A" and "B" (or "a" and "b"). Makey and behavior observed with Tf C (Parker and Beam, 1962). Seal (1976) showed that four transferrins, representing iron Similarly, neuraminidase treatment alters the pI of the Tf C free Tf (apo-Tf), Tf with Fe bound to site A, Tf with Fe subtypes (KiiM and Spielmann, 1978a). However, neither bound to site B, and Fez-Tf, could be separated by electro- the sialic acid nor the complete carbohydrate moiety appears phoresis in polyacrylamide gels containing 6M urea in to have anything directly to do with the polymorphism, Tris-EDTA-borate buffers at pH 8.4. It was not clear just which almost certainly reflects peptide chain compositional exactly why the forms could be separated in this way. These differences. There is evidence that the carbohydrate moiety results may help to explain the immunological differences has a biological role in Tf function (see in Putnam, 1975d). seen by Jager and Gubler (1952) and Kourilsky and Burtin (1%8). Leibman and Aisen (1979) took advantage of the 42.4.2 Structural differences among Tf variants ability to separate the forms in order to estimate their Much work remains to be done in this field, and it is made relative amounts in nonnal sera. Bmding was not random, more difficult by the size and internal complexity of the Tf nor was it determined by the relative binding strengths of the molecule in terms of amino acid composition and by the A and B sites. The more acid-labile and weakly-binding B rarity of many variants. Most of the information is based on site of Tf was predominantly occupied. Serum Groups- Tj

42.5 Medicolegal Applications is done in bloodstains at the present time. It is possible to The Tf system is used in some laboratories m disputed type Tf in some stains which are a matter of months old. parentage cases (Mauff et ul., 1975; Dykes and Polesky, The commoner variants can be detected on agarose gels, 1978). The exclusion probability is low in Europeans and m followed by immunofvtation detection, and Tf typing is American Caucasians (about I%), and about 5% in Black normally combined with Gc typing in the same gel. Americans. Chakraborty et al. (1974) quoted slightly higher Tf can be detected in a variety of human tissues (Mason values. The fmding that Tf C can be subtyped by electro- and Taylor, 1978). Transfenin can also be detected in sem- focusing techniques, however, and that the alleles are inal fluid but not on sperm cells (Blake, 1976; Blake and reasonably well distributed (at least m Europeans), greatly Sensabaugh, 1976). The minimum quantity of semen re- enhances the value of the system. The exclusion probabii quired for typing is about 50 pP compared with about 5 @ hcrgascs to about 13-15% in Europeans if Tf C is subtyped whole blood, since the concentration of Tf is 10 times lower (Thymann, 1978; Kiihnl and Spielmann, 1979a; Hoste, in semen (Blake and Sensabaugh, 1978). Tf typing might, 1979). Use of the Tf C subtypes is made more economical by therefore, be possible in seminal stains under favorable the fact that the Gc' subtypes (section 41.3) and the TF sub- conditions. types can be demmined in the same isoelectric focusing gel 42.6 The Distrlbution of Tf Phenotypes (Thymann, 1978; Hoste, 1979). in U.S. Populations in Turowska (1%9) noted that Tf could be typed fresh been bloodstains. There are a few laboratories in which Tf typing The data for American populations which have studied are given in Table 42.1. Table 42.1 Distribution of Tf Phenotypes in U.S. Population

Frequency - Number IPercentl Population -Total -C -Bl C -Ba c -CD, Note Reference CAUCASIAN

Southeastern GA 107 103198.31 1t0.91 211.91 1 Cooper et al.. 1983 Southeastern US 2.221 2.194198.8) 2t0.09) 1510.881 10l0.45) Roop et al.. 1988 Chicago, IL 101 101l1001 Shih and Hsia. 1989

Tecumseh, MI 7.854 7.580198.81 8311.1) 1110.14) Schreffier st al.. 1971

Greater Phlladelphla, PA 203 202199.51 110.51 2 Pakstls et 81.. 1978 198 193198.51 211.01 l(0.5)

Californla 801 788198.1) 3 Grunbaum et el., 1980

NEORO New York, NY 99 89189.91 919.1 1 4 Parker and Bearn, 1981a

Southeastern GA 133 120190.2) 1319.81 Cooper et al., 1983

Southeastern US 418 399195.51 1914.5) Roop et al., 1968

Chicago. IL 101 93192.1) 8t7.91 Shih and Hsla. 1969 Greater Phlladelphla, PA 184 151l92.11 1317.9) 2 Pakstis et a!.. 1978 189 158193.51 1015.91

California 502 467193) 3 Grunbaum et el.. 1980

OTHER New York. NY Chinese 118 109194) 5 Parker and Bearn, 1961a

California and Mexlco City Mexican 765 7421971

California and Hawaii 3 Grunbaum et at.. 1980 Asians 1.295 1.259197.2)

1. One was B,., 8, 3. The following numbers (percents) listed as "rare": Caucasian 1511.87): Negro 3516.971; Mexican 2313.011; Asian 38 12.78). 2. The sampling area included sections of PA and southern NJ. The two populations listed are separate sums of two members 4. One wes D, of twln pairs. Among the 169 members of the Black co-twin 2 5. 7 were CDChi. population, one was Dl. Serum Groups-Pi

SECTION 43. a,-ANTITRYPSIN (Pi) a,-antitrypsins represented by the two bands were immuno- 43.1 Introduction logically indistinguishable. The patient's three children had The principal protease inhibitors in human plasna are normal a,-AT patterns. When a similar case was found (Ax- a,-antitrypsin (alAT), a2-macroglobulin (a2M), intera- elsson and LaureU, 1%5), genetic and family studies in- trypsin inhibitor (Ian, antichymotrypsin (Achy; alX), an- dicated not only that the slow a,-AT variant was genetically tithrombi I11 (AT 111) and C1 activator (cl-Ina) (Laurel determined, but that the gene for it was allelic to that for the and Jeppsson, 1975). The most important of these are alAT previously observed aI-AT deficiency. and a2M,and a1AT is the subject of this section because of In 1%5, Fagerhol and Braend discovered a system of pro- the extensive genetic polymorphism it exhibits. tein zones in the "prealbumin" region, which exhibited Proteolytic enzyme inhl'biting activity in blood was recog- observable variation in low pH (4.95) starch gels. Five nized many years ago (Hahn, 1897; Camus and Gley, 1897). phenotypes were seen in the sera of 390 healthy Norwegian The complexity of the proteinase inhibitor system of plasma blood donors, and family studies showed that the system became more clear with the more recent studies of Grob was inherited, and under the control of three alleles. The (1943), ShSnan (1952) and Jacobsson (1955). Shulman name "Pr proteins" was suggested for the system. The locus (1952) was able to differentiate plasmin inhi'bition activity was designated Pr, and the alleles PrF, prM and * (for from the inhibition of trypsin or chymotrypsin. Jakobsson "fast", "medium" and "slow"). The phenotypes were (1955) recognized two trypsin inhib'i actM.ies in plasma, called MM,MS, SS, M and FS. It was soon realized that one associated with at globulins and the other with a, the "Pr system" proteins were not "prealbumins", but that globulins. The latter was much more plentiful. The a, they corresponded to the &,-AT proteins (Fagerhol and globulin associated trypsin inhiiitory activity was isolated Braend, 1% Kueppers and Beam, 1%6b), and Fagerhol by Schultze et al. in 1955, but it was not recognized as such and LaureU (1%7) suggested that, since these proteins by this group until later (Schultze et al., 1%2c), and was represented the major plasma proteinase inhl'bitors, the called "3.5s a, glycoprotein". It has also been studied by locus controlling their variation should be called Pi (for Pro- Poulik and Smithies (1958), who called it orl-globulin, Nor- tease inhibitor). The system exhl'bited a remarkable degree man (1958) who called it a,-antiplasmin, Bundy and Mehl of genetic polymorphism. By 1968, Fagerhol and Tenfjord (1959) who called it a,-trypsin inhibitor, and Burtin (1%4) could list seven alleles at Pi. Arranged in order of decreasing who called it a,,-globulin. Schultze et al. (1%2c) named the electrophoretic mobility of their products on acid starch protein "al-anti-trypsin", and it is this designation that has gels, these are PiF,Pi1, PiM,P~S,W,PiX and PiZ. A full persisted in the literature. The biochemical journals tend to description was given by Fagerhol (1%). An additional call it a,-protease inhibitor. Nomenclature is still something phenotype, MW,accounted for by a new allele m,was of a problem with this system, despite efforts to standardize found in two Spaniards by Fagerhol and Tenfjord (1%8). it (see further below). The slow moving variant originally described by Eriksson 43.2 Genetic Variation and Laurel (1%3) and Axelsson and Laurel (1x5) corre- sponds to the product of #,while the gene for a,-AT defi- 43.2.1 Multiple alleles controlling a,-antitrypsin ciency (homozygous serum having about 1&15% of the In 1%3, Laurel1 and Eriksson noted that the sera of nonnal a,-AT concentration) is H.Descriptions of the several patients suffering from pulmonary degenerative alleles h", PiF, Pzs and Piv may be found in Fagerhol diseases exhibited markedly reduced concentrations of (1967) and Fagerhol and Gedde-Dahl (1969). The latter a,-antitrypsin, and suggested that there was a correlation study also included family studies confirming the codomi- between the two things. The possibility of a genetically con- nant mode of inheritance of the various Pi alleles. Similar trolled 'inborn error of metabolism' was raised. Eriksson family studies were done by Kellennan and Walter (1970). (1%4) showed that the pathological condition was heredi- A number of additional phenotypes and vhtshave tary, behaving like a recessive, and that homozygotes for the been described. Some are better characterized than others, trait seemed to be most likely to have the emphysema. Fam- and family data are apparently not available for all of them. ily members presumed to be heterozygous had roughly half The ninth allele, hf,detected as MP,and of low frequency, the normal al-AT levels in their serum. These obmations was described by Fagerhol and Hauge (1%8). PiE and PiG were confirmed independently by Kueppers et al. (1%4) and were described by Fagerhol at an international meeting in others. Eriksson and Laurell(1%3) found an electrophoreti- 1971 (discussed by Cox and Celhoffer, 1974). Pi MW2 was cally slow al-AT band, accompanied by the usual band, in described by Gedde-Dahl et al. (1972), MY2 and ZY2 by the serum of a patient. The serum concentration was nor- Porter et al. (1972), Pi* seen as BM by Marti et al. (1973), mal, and each band accounted for about half of it. The and PiL in a Bantu population by Vandeville et al. (1974). Sourcebook in Forensic Serologv, Immunologv, and Biochemistry

Cox and Celhoffer (1974) described #, seen as MN,and Isoelectric focusing has revealed that the common pheno- they reviewed the variants known up to that time. type on starch gels, Pi M, can be resolved into at least six subtypes, accounted for by three different hM alleles which 43.2.2 Quantitative varietion and complete deficiency are comparatively well distributed in Europeans. The Several of the Pi alleles appear to be amchted with nomenclature of the new variants can be quite confusing to reduced aI-AT synthesis, in addition to coding for proteins the nonspecialist, in spite of the efforts of an international that are structural variants. These are Piz, Pip and hs. group to standardize it (see below). Association of deficient allele phenotypes with diseases is In 1976, Frants and Eriksson found three phenotypes discussed further below. In 1969, Kueppers found that the within Pi M, which they called MI, MlMn, and M2. The PiZ allele yielded a product electrophoretically ~~- alleles responsible were hM1and IF2.Another allele, W3, able from that of the common w,and that MZ and ZZ which was difficult to detect by conventional isoelectric people could be distinguished in this way. The frequency of focusing, was reported by Frants and Eriksson in 1978. The this allele was about 7% in 100 random blood donors from product of the PP3allele could not be detected very well by California. Talamo et al. (1973) found a 24 year old man conventional PAGE when it was paired with another hM with advanced pulmonary emphysema whose serum had no allele, but could be distiuguished when paired with a dif- detectable orl-AT by any technique. Members of his family ferent Pi allele. However, the introduction of so-ded had half the normal activity. The total deficiency phenotype "separators" into the gels along with the ampholines, a was called "Pi-", and the man was considered a homozy- technique called "separation isoelectric focusing" or gote for a Pi- allele. The half activity family members were "SIEF", enabled the differentiation of the products of all thought to be PM-,and their phenotypes were written three flalleles. The separators cause "plateaus" in the pH bdM/-" Or "M-" gradients, which can improve resolution in certain systems (Frants et al., 1978). According to Frants and Eriksson 43.2.3 Refinements of Pi typing methods, isoektrk (1978), the previously described Pi MI resolves to MI, MIM2 focusing, Pi M subtypes and other vruinnts and M2, Pi MlMn becomes M1M3, and Pi M2 resolves to A number of the Pi phenotypes can be detected by acid MIM, and M, in the new three allele system. Kueppers starch gel electrophoresis. Unequivocal characterization of (1976a) found one of these alleles, and called it IF' where some of the variants requires two dimensional crossed im- hM was the usual allele. Van den Brock et al. (1976) found munoelectrophoresis. Electrophoresis in the first direction is the same allele, and termed it WN.In 1978, Kueppers and carried out in acid starch gels, and that in the second direc- Christopherson described the other allele. Klasen et al. tion is done in antibodycontaining gels at 90° to the first (1977) also described the third common allele of w.Genz direction. Up to eight zones can be detected by the crossed et al. (1977) found all six phenotypes, and they called the immunoelectrophoresis technique, and the patterns differ alleles w, and 6VIC.The phenotypes were Pi Ma, according to phenotype. Agarose gel electrophoresis can be Mb, Mc, Mab, Mac and Mbc. The equivalencies between used for Pi typing as well, with protein staining or irnmuno- these different designations, framed in terms of the Pi Com- fixation for detection (Laurel and Persson, 1973; Jeppsson mittee recommendations, are: The Pi MI, MIM2and M2 of et al., 1979). A standard must be run alongside unknown Frants and Eriksson (1976) are equivalent to M, MMI and samples if simple protein staining is used to detect Pi zones Mi (Kueppers, 1976a). Ma, Mac and Mc (Genz et al., 1!377), (Laurel and Persson, 1973). and M, MMN and MN (van den Brock et al., 1976). The Application of isoelectrofocusing to Pi typing has shown committee recommended that these be called, respectively, that it provides a somewhat easier method for phenotyping MI, MtM2 and M2.The W'of Kueppers and Christopher- some of the variants than two-dimensional immunoelectro- son (1978) is apparently the same as that of Klasen et d. phoresis, and it has further revealed some additional genetic (1977) and of Frants and Eriksson (1978). The PP2 is also wiations in the Pi system. Procedures for Pi typing by iso- equivalent to the hfJ of Constans and Viau (1975), but not electric focusing on polyacrylamide gels have been described to the "PiN" of Cox and Celhoffer (1974). The MI, MIMl by Allen et al. (1974), Arnaud et al. (1975 and 1977) and and M1 phenotypes have been studied in the Japanese popu- Kueppers (1976b). Detection has been canied out by protein lation by Harada et al. (1977). staining, which may be accompanied by densitometric trac- Some additional phenotyes reported in the Pi system are ing, and by immunofixation techniques (Arnaud et al., MLamband MBaldwin(Joh~lson, 1976), Pi W3 Constantine 1977). Immunofdon can be done in the gel itself, or by (Khitri et d., 1977), Pi Nhampton (or Nham) (Arnaud et "printing" onto an overlaid cellulose acetate membrane al., 1978a), Pi B Alhambra (Yoshida et al., 1979), Pi T strip. Hercz and Barton (1978) described a Schiffs reagent (Kiihnl and Spielmann, 1979b and 1979~)~Pi disulfides (Pierce et al., 1976). frequency was dependent in certain ways upon the Pi alleles 5% Serum Groups-Pi present, and the phenomenon is apparently not yet fully variants imposed unreasonable restrictions on investigators, understood. and should not be adopted. Restricting the criteria to elec- trophoretic or electrofocusing mobilities was a mistake, he 43.2.4 Standardization of nomenclature and techniques said, because variants not differing in charge would be Pi phenotype identification is apparently not as easy to missed. Biochemical properties, including kinetic proper- master nor as straightforward as that for many other ties, amino acid analyses, etc., should be included too, he systems, especially when variant phenotypes are involved. said. It would also be helpful if standard electrophoretic and Some variants are more difficult to diagnose than others. isoelectrofocusing procedures were recommended, and all Special isoelectrofocusing techniques are required to be cer- established variants run in the standard systems, and the tain about the identity of the Pi M, phenotypes. In 1978, an results published. international committee met to consider the complexities of 43.3 Relationship of a,-Antitrypsin the Pi system, and their work is reported by Cox (1978) and Deficiency and Disease Cox et al. (1980). The committee recommended that the protein be designated a,-antitrypsin (alAT), and its genetic There is a well established connection between certain locus as Pi. Several reference laboratories have been kinds of degenerative lung diseases and the P? allele. established, and they try to maintain panels of variants Homozygotes for the gene, Pi ZZ people, have a high risk of against which "new" ones can be compared by various dif- developing chronic obstructive pulmonary disease (COPD), ferent techniques. In our part of the world, one reference which leads to emphysema. Similarly, Pi ZZ infants are at laboratory is maintained at the Hospital for Sick Children in high risk for developing neonatal hepatitis and cirrhosis of Toronto (Dr. Diane Wilson Cox), and another is at the the liver. This material is discussed by Kueppers and Beam University of North Carolina Medical Center in Chapel Hill (I-), Kueppers and Black (1974), Fagerhol (1976) and (Dr. A. Myron Johnson). Details are published in Cox et al. has been reviewed by Morse (1978). In homozygous defi- (1980). The committee established rigorous standards for cient subjects (ZZ), and less so in Z-heterozygotes, some of confmtion of variants as "new". They noted that several the liver cells accumulate a,-AT (see, for example, Lieber- different techniques must be used to compare a "new" man et al., 1972), and this leads to liver cell necrosis. Moa variant with known ones. In addition, they recommended investigators think that Pi SZ people are at higher risk for some standardization of the nomenclature. A laboratory COPD as well, but there is some controversy about the risk manual has been prepared under NIH sponsorship in this of MZ or MS individuals. The pathogenesis of the lung country (Talamo et al., 1978), and it contains the detailed disease almost certainly has nothing to do with trypsin, standard procedures. The papers should be read for the which rarely gets into circulation. a,-Antitrypsin can inhibit details. A few highlights are: The MI, M2 and M3 designa- other proteases, notably leucocyte collagenase and elastase. tions for the common subtypes of Pi M were retained, their These enzymes have been implicated in the production of alleles being PrM', hM2and ?F3;upper case letters will con- emphysema iesions, and their excess activity in a,-AT defi- tinue to be used to indicate relative position in isoelectric cient subjects may provide a basis for understanding the focusing gradients, and descriptive names can be appended relationship. There is much more to it, and more work will as needed; lengthy descriptive names can be abbreviated by be required before an understanding is achieved. For one using the fist few letters of the name, e.g. Pi bcincinthing, there is quite a bit of individual variation among ZZ ,ti ,ti = Pi ECin,Pi &hapcm= Pi Mcha, and so forth; the people with respect to clinical manifestations in lung or complete deficiency allele is called Wd.A number of addi- liver. Another thing is that a,-AT deficiency is implicated in tional variants are discussed in Cox et al. (1980). It is also only a small percentage of degenerative lung disease pa- clear from the report that unequivocal diagnosis of some tients. There is some evidence of the association of al-AT variant Pi types is not always an easy matter, wen for ex- deficient phenotypes with other diseases. Although the fre- perienced laboratories. They recommended against Pi typ- quency of PiZ is significant in many populations, and 2 to ing in ordinary clinical laboratories, suggesting instead that 5% of some European and American populations may be a few reference laboratories be set up for the purpose, each considered "at risk" on the basis of the preliminary data, serving a geographical area containing 5 to 10 million peo- there is still a question as to whether mass screening pro- ple. These larger labs would have the resources and volume grams are appropriate. These matters are discussed by of work to maintain complete sets of reference sera, and so Fagerhol (1976) and Morse (1978). Chapuis-CeIlier and forth. Amaud (1979) recently reported that the P? allele is prefer- Not everyone agrees completely with the committee's entially transmitted to offspring when it is present in the recommendations. Yoshida (1979) took exception to several father suggesting that P%anyingsperm enjoy a selective of them. He noted that the name "alpha-antitrypsin" had advantage of some sort. become widely used by clinical workers, but that the bio- chemical journals still preferred "al protease inhibitor". He 43.4 Biochemical Studies preferred the latter, since the ". . . . .protein is not an an- Some biochemical properties of a,-AT were mentioned at tibody against trypsin", and because that name is compati- the beginning of the section, in connection with the earlier ble with the locus name, Pi. He said further that the studies on its isolation and characterization. More recent guidelines for the identification and publication of new purification and isolation studies are reviewed by Laurell 597 Sourcebook in Forensic Serology, Imrnunologv, and Biochemistry and Jeppsson (1975). Schultze et al. (1%3) described the types of pi1,called I, and 12,were detected by electrofocus- purif~cationprocedures used at that time. a!-Antitrypsin is ing. Kueppers et al. (1977) described a woman with a Pi M an inhibitor of a number of proteolytic enzymes besides phenotype, but who was deficient in aI-AT. She had em- trypsin, including plasmin, chymotrypsin, thrombi (see physema, and her father had COPD. He had normal levels Rimon et al., 1%6), elastases and collagenases. The &erase of &,-AT, but her mother and two sibs had about 50% nor- activity of these enzymes is inhibited as well. The mech- mal levels. This family may have a variant "M", which is anism of inhibition involves some kind of complex forma- indistinguishable from the usual M, and the possibility of tion, but its exact nature has not been completely elucidated. piNuUcannot be ruled out in the family. Kahn et al. (1977) al-AT is unstable at low pH (less than 5) and loses its in- conducted studies on a number of normal, partially defi- hibitory capacity. The activity can be recovered, however, cient and fully deficient individuals, and the results indi- by titrating back up (Glaser et al., 1977). Aggregates form at cated that a,-AT contributes >W% of the total antitrypsin about pH 4 which retain immunological identity, but which activity of normal plasma. do not inhibit proteases. There is microheterogeneity Reviews of the Pi system may be found in Fagerhol(1%8 associated with al-AT regardless of the phenotype, and its and 1976), Giblett (1%9), Fagerhol and Laurell (1970) and biochemical basis is not very clear. Musiani et al. (1976) Lawell and Jeppsson (1975). isolated 8 fractions from Pi M sera on a preparative scale, and the fractions showed various distributions in a pH 4-6 isoelectric focusing gradient. These fractionation results will now have to be interpreted in terms of the now recognized 43.5 Medicolegal Applications Pi M subtypes. Hodges et al. (1979) isolated and character- No dim references to the application of the Pi system to ized the oligosaccharide chains of the al-AT molecule (from disputed parentage cases were found in the literature, Pi M people). There are two types of chains, a large one although a number of authors mention its obvious appli- with a complicated structure, and a smaller one. It appeared cability. The system is much more powerful if Pi M subtyp- that each protein contains one large and three small chains. ing is performed, and speciaked laboratories will probably Both kinds are attached to the peptide through be employing the system in this way. GlcNAc-Asn linkages. The large chain can have up to There has been little reported work on Pi phenotyping in three terminal N-acetyl-neuraminic acid residues, and the bloodstains. Frants and Eriksson (1979) indicated that they small chain has two. Therefore, there could be up to 9 ter- had obtained promising preliminary results on Pi phenotyp minal NANA residues in the intact molecule, if these relative ing in dried blood by isoelectrofocusing. The system would amounts are correct. be most useful if Pi M subtypes could be determined. As There are a few biochemical studies on the protease in- was noted above, difficulties can be encountered with many hibitor from specific different phenotype sera. Only in a few of the variant phenotypes. Stored sera tend to develop cases have the amino acid differences between variant and "storage" bands, which make the already complex patterns M molecules been worked out. The difference between Pi M more difficult to interpret, but sulfhydryl reagents alleviate and Pi S resides in a single amino acid (Owen et al., 1976; this problem appreciably. A study of the ability to determine Yoshida et al., 1977). Pi S has Val where Pi M has Glu, and Pi phenotypes in postmortem bloods was done by Conrad et this was clear in two completely different examples of the al. (1979). Sera stored frozen (- 20") retaiued their pheno- protein. Jeppsson et al. (1978) purified Pi M, S and Z typic characteristics quite well if the pH was 7.0-7.5. 86% of molecules, and said that they had detected a single amino the sera stored for 2-2% years, but only 30% of those acid difference in one cyanogen bromide peptide between M stored for more than 4 years, could be typed. There was and Z. M had Glu where Z has Lys. Yoshida et al. (1979) some decrease in the typability of sera that were not col- characterized their Pi B Alhambra mutant fully, and found lected immediately after death. two amino acid substitutions, LyAsp and GludAsp. a,-AT is found in genital secretions as well, where it is Hercz and Barton (1977) have purified Z protein, and found suspected of having some role in fertilization (Hirschhauser that it contained more glycine residues than the M protein, et al., 1972), and it is possible that the protein might provide but that it did not differ in carbohydrate content. They also a genetic marker in these fluids. The cervical mucus shows discussed some of the apparently conflicting results of vari- cycledependent varia!iom in a!-AT content, which are af- ous workers. Bauer et al. (1978) have isolated and studied Z fected by oral contraceptive therapy (Schurnacher, 1970). protein material from the liver of a Pi ZZ individual. It was aI-AT is found in semen at mean levels of 68-87 &mP, but homogeneous, and antigenically intact, but had no pro- not in sperm cells (Schumacher, 1970; Blake, 1976; Blake teinase inhibitor activity. There was no galactose or sialic and Sensabaugh, 1976 and 1978). Blake and Sensabaugh acid in the carbohydrate moiety and GlcNAc was reduced. (1978) estimated that about 100 fl semen would be needed Mannose was present in excess, however. to detect a,-AT, compared with about 5 pP whole blood. Arnaud et al. (1978b) studied a number of individuals They did not regard it as a very good prospect for typing in who had the Pi3 allele. It was found that this allele is in the forensic samples containing semen. It is not completely clear class of "deficient" alleles, in that people who had it whether the Pi phenotypes observed in senun are expressed showed reduced protease inhibitor levels. Further, two sub- in semen in the same way. Serum Groups-Pi

43.6 Distribution of Pi Phenotypes MZ,while 204 Negroes were %. 1To M, 2% MS and 1% MZ There have been very few studies in U.S. populations, and (Pierce el al., 1975). The gene frequencies observed in 188 in some cases only the gene frequencies were reported. Leav- Caucasians in San Francisco were very similar to those in St. ing aside Pi M subtyping for a moment, it is found that Pi M Louis (Kueppers, 1971). Kueppers and Christopherson is the common phenotype in most populations eed.In (1978) reported the frequencies of Pi phenotypes, including Europeans, the frequency is polymorphic, and is higher subtypes, in 240 Whites (Rochester, MN and southeastern in Spain, Portugal and the south of France than elsewhere. PA), and in 304 Blacks (southeastern GA and Philadelphia, The frequency of FM varies from about 0.2 to about 2%, PA). Among the Caucasians were: 98 (41%) MI, 14 (5.8%) and that of MZ is between about 0.2 and 5%. PiZ is more Mp, 2 (0.8%) M3, 51 (21.3%) MIMI, 39 (16V0) MIMs, 7 frequent in Caucasians, and relatively few non-M (2.9%) MZMs, 1 (0.4%) FMI, 2 (0.8%) IMr, 12 (5%) MIS, 6 phenotypes seem to occur in Africans, Asians, Fii,Lapps (2.5%) MS, 2 (0.8%) M3S and 6 (2.5%) MlZ. Among the and Greenland Eskimos. Negroes were: 248 (81.6%) MI, 1 (0.3Vo) M,, 15 (4.9%) In St. Louis, MO, Dew et d. (1973) reported MIMI, 29 (9.5%) MIM,, 2 (0.7V0) M2M3, 1 (0.3%) IMI, 3 @ = 0.9480, PIfl = 0.0027, Pii = 0.0012, h5 = 0.0344 (1%) FMI, 3 (lvo) MIS and 2 (0.7%) MIZ. The AML,PT2 and hZ= 0.0127 in 2,047 Caucasians. In another St. Louis and hM3 allele frequencies were, respectively, 0.64, 0.19 and study, 1,933 Caucasians were 90% M, 6.7% MS and 2% 0.11 in Whites, and 0.903, 0.028 and 0.054 in Blacks. Serum Groups-Gm, Km

SECTION 44. GENETIC MARKERS OF THE IMMUNOGLOBULINS-Gm, Km, Am AND Hv

44.1 Introduction 44.2.1 Tbe Gm factors The genetically determined antigenic markers which com- In 1956, Grubb found that Rh+ cells sensitized with prise the Grn and Km (and the Am and Hv) systems are some examples of incomplete anti-D were agglutinated by located on the immunoglobulins, the family of senun pro- the sera of some patients who had rheumatoid arthritis. Fur- teins which possess antibody activity. The classes and struc- ther, this agglutination could be inhibited by the sera of ture of the immunoglobulins were discussed in section some 60% of normal random donors. Siobservations 1.3.3.2. There are five classes of these molecules, each were made by Milgrom et al. (1956) and by Waller and possessing a different type of heavy chain. Immunoglobu- Vaughn (1956). The inhibiting property of normal serum lins have the general structure H2L2,where H stands for a was quickly shown by Grubb and Laurell (1956) to be in- heavy chain and L for a light chain. There are two types of herited as a simple Mendelian dominant. The responsible light chains, K and h, both of which occur in individuals, but factor was found in the gamma globulin fraction (now not in the same molecule. IgM is a polymer of the basic known as IgG), and it was called Gm(a). The active rheu- H2L2structure. matic sera contained an "anti-Gm(a)" which agglutinated Gm and Km are among the most complicated of the anti-D sensitized Rh+ cells, provided the anti-D IgG genetic marker systems, and they are very useN in forensic possessed the Gm(a) determinant. Normal sera having problems. Among the many excellent reviews of these Gm(a) on the IgG molecules reacted with the anti-Gm(a) in systems are Prokop and Bundschuh (1%3), Natvig and the active rheumatic sera, thus inhibiting its ability to agglu- Kunkel (1%8), Franklin and Fudenberg (1%9), Giblett tinate the sensitized Rh+ cells. Soon afterward, two other (1%9), Gortz (1%9), Grubb (1970), Mage et al. (1973), Nat- genetically determined, serologically detectable differences vig and Kunkel (1973), Ropartz (1974) and Giblett (1977). in the IgG were found using different reagent pairs (rheu- matoid serum agglutimtor and sensitizing anti-D), and they were called Gm(c) (Harboe and Lundevall, 1959) and Gm@) 44.2 Genetic Variation in the y Chains (Harboe, 1959). of IgG-The Gm System By 1%5, approximately 14 distinct Gm specificities had Some terminology arises in discussions of Gm that is not been identified, and nomenclature had become something commonly used in most of the other systems, and it will be of a problem. A great deal of new information had also introduced briefly here. Three degrees of specificity related become available about the structure and properties of im- to structural variation have been differentiated, based on munoglobulins around this time. In 1%4, the nomenclature . the antigenic properties of the immunoglobulin molecules of human immunoglobulins was standardized (W.H.O., within a given species (Giblett, 1%9). Zsotypc specificities 1%4). As a logical outgrowth of that work, a standardized are common to all members of a species, but differentiate nomenclature for the Gm and Km allotypes was recom- immunoglobulin molecules into classes and subclasses. mended in the following year (W.H.O., 1965; Ceppehi Allotypic specificities differ within a species, and reflect in- and many others, 1966). Numbers were assigned to the Gm herited variation of molecular structure. The polymorphic and Km -cities, and their use was recommended in- genetic marker systems (Gm, Km) represent allotypic stead of the letters that had been used. In the more recent specifcity differences, and one can and does refer to the nomenclature revision, however, letter designations and "Gm allotypes". Zdiotypic specificities are characteristic of their numerical equivalents are apparently equally accept- the products of single cells (or their clones). Myeloma pro- able (W.H.O., 1976). More will be said about the recent teins are a good example. The term haptotype is not used recommendations in appropriate sections below. too much in discussions of Gm genetics, but it could be. It is Almost 30 different Gm factors have been described. a very common term in HLA genetics. When a chromosome Some have been found to be the same as others, and in some segment carries closely linked genes that are part of the same cases, the specificities can no longer be tested because there system, and which tend to be inherited together as a unit, are no longer any reagents available. In a few cases, the rela- the gene makeup of one or the other homologous chromo- tionships of particular factors to the rest of the system are some segment is called a haplotype. In the Fisher-Race con- not very clear. The list of factors, and some of their ception of Rh genetics, therefore, one could speak of Rh equivalent names, is shown in Table 44.1. haplotypes. one could use the term in MNSs and The original anti-Gm antibody, now called anti-Gm(a), in Gm genetics, though it seems to be encountered most was found in the serum of a rheumatoid artbitis patient, often in discussions of HLA. and many of the subsequent examples of antibodies defining Sourcebook in Forensic Serologv, Ztntnunology, and Biochemistry

Table 44.1 Genetic Markers of the immunoglobulins

Racommendod Desiprution Other or Pravlous Daslpnatlona Chain Locatbn Alphameric -Numerlc Alphmmeric Numeric Allotvpic Markers of the y Chalns

Grubb. 1968 Herboo and Lundwall. 1969 Stelnborp and Wilson. 1%3: Gold et at.. 1965: Steinberg. 1%S Llmln and Kunkel. l%6. end 1Wb

Kunkel et al.. 1966: Natvip and Kunkal. 1967

~mlb~~(bOl Rowm ot ml.. 1963: van Lopham and Mananuon. 1907 Gmlbl. lbll. 1bY1 Hafboe. I%% ~~~~~z at 81.. 1-3: van Loghem and Martanlaon. 1%7 Gmlb3L IBetl Stelnbarp and Goldblum. 1966 Gmlb41 Stelnbafg and Goldblum. 1%6 ~mlb~l,IbSI Ropam at 01.. 1%): van Logham and Manenuon. 1%7 Em-IIke. is). k3l Stainbarp at al.. 1960 Gm-Ilke. Icl. le6l van Lophmm and Manansson. 1967 Gmlgl Natvlg. 1966 Gmlal Mananason ot 11.. 1-6 Gmld Manenason at at.. 1%6 GmlPal van Lophom and Grobbalaar. 1971: Stalnborp. 19?? GmlRavl

Markan whom amus I* unclamr andlor tor which raapants are no lowar avaHable

Gmlrl Gml7l Bnndeaw at el.. 1%1 Grnlel Gmial Ropam at al.. 1%2 Gmlpl Om191 Wallor at at., 1%3 R02. Rown 2 Gmlll) Ropana at mi.. 1167 RO3. Rouan 3 Gmi191 Ropmm at 01.. 1966

Sen Fnncisco 2 GmlZOl Klemwror at .I.. 1966: Ropam at .I.. 1966 Gmlvl GmI221+ Umln and Kunkol. 1967

Ropam at at.. 1%2 ROpom et aI. 1961. and l96lb Suinbm at at.. 1962

nGlmlal nGlmllI non-a Natvip ot 01.. 1%9 nGlmIz1 nGlmll71 nGlmIx1 nG3miWl nG3mll?l non-b0 Gaardner and Nmtvlg. 1372 nG3mlbll nG3rn(61 non-bl Gamrdnor and NatvIp. 1972 Natvip ot al.. 1%9 Kunkel at a!.. 1970 Kunkel et al.. 1970

Amlll. Am. Vves and Fundarbarg, 1969 AM121 ran Loghem at 01.. 1973

Irflll Roeam at at.. 1966 and 1968

Haaw chalns Ivarhbla ngion) of lgG. lgM and lpA Wang at .I.. 1978

- after W.H.O. I19761 + Equlvabnt to nGlmlsl = nGlmlll IRoparQ. 19741 Serum Groups-Gm, Km

Gm -cities have been found in such sera. These are htis, were described. Further examples had not been called "Ragg" antibodies (for Rheumatoid Agglutinating). found by 1970. Gm(l0) was described by Ropartz et al. Examples of various antiam of various qxcScities are also (1%3) as Gm(b9. The anti-Gm(l0) came from a healthy found in non-rheumatoid sera (Ropartz et al., 1960), Caucasian, and no further examples had been seen by 1970. especially in multiply transfused patients, women who have Grubb (1970) said that further studies would be needed to been pregnant, and in children. These antibodies are called show that Gm(l0) was different from Gm(13). Indeed, "SNagg" (for Serum Normal Agglutinating). The former Johnson et al. (1977) regarded Gm(l0) and Gm(13) as iden- are usually quite potent, but may have autoantibody activ- tical. Gm(l1) and Gm(12) were also found by Ropartz et al. ity, multiple specificity and they often show prozones. The (1%3), and called ~m(bB)and Gm(br), respectively. latter are usually of lower titer, but they are often mono- Gm(l1) has been called Gm(bo) (van Loghem and Mar- specific and do not show prozones. They are the preferred tensson, 1%7). Other examples of anti-Gm(1l) have been reagents for Gm typing. found. Gm(l1) and Gm(5) are usually found together in Gm(l), or Gm(a), was the fust specificity to be described Caucasians. Gm(12) is not clearly distinguishable from (Grubb, 1956) as noted above. Antisera to Gm(1) have been Gm(5), and they are considered identical. Gm(13) was the found in several hundred people. Hemagglutination inhibi- Gm(b3) of Steinberg and Goldblum (1%5). It has also been tion remains the best typing method, and the anti-D must called Gm(Bet). Further anti-Gm(l3) sera have been found. come from a Gm(1) person, preferably someone containing As noted above, Grubb (1970) said that Gm(13) and Gm(l0) as few other Gm factors as possible. It has occasionally been need to be more clearly distinguished. Gm(14) is the GmW) reported that Gm(1) can be detected by one reagent system of Steinberg and Goldblum (1%5). Anti-Gm(l4) sera are but not another, and there are reports of reagent systems apparently rare. Gm(l5) and Gm(16) were described as that appear to differ in the detection of Gm(1) in different Gm(s) and Gat) by Martensson et al. (1966). Some further racial groups. Gm(2), or Gm(x) was described by Hark examples of antisera to both have been found. Gm(17) is the and Lundevall (1959), and many additional examples of Gm(z) of Litwin and Kunkel (I%& and I!%&). It was anti-Gm(2) from rheumatoid and normal sera have been detected by a rabbit anti-Gm(l7) prepared by immunization found. Gm(2) is rare in Black populations. Gm(3) was with the Fab fragment of IgGl myeloma protein, followed defined by the serum of a 4 year old Black boy (Steinberg by absorption with Gm(- 17) immunoglobulin. Gm(18) is and Win, 1%3), and was called Gm(bw). It has aiso been the Gm (Ro~),or 'Rouen-2' of Ropartz et al. (1%7). An- called Gm(b2) (Steinberg and Goldblum, 1%5). Anti-Gm(3) tisera are apparently very rare. Gm(19) is Gm(Ro3) or has not been found in anyone else (Grubb, 1970). Gm(4) 'Rouen-3' of Ropartz et al. (1966). The antiserum was dif- was described by Gold et al. (1%5) who called it Gm(f). Ex- ficult to work with. Gm(20) was described by Klemperer et amples of anti-Gm(4) have been found in more normal peo- al. (1966), defined by an antibody from a rheumatoid ar- ple than rheumatoid arthritis patients. Discrepant results thritis patient. No further examples have been found. have been reported in Gm(4) detecting systems, depending Gm(20) was only found in Gm(1) people. Gm(21) is the upon the reagents used. There is considerable evidence to GmCg) of Natvig (1966). The fm antisera were human suggest that Gm(4) is not different from Gm(3) (Steinberg, Raggs, but anti-Gm(21) has been produced in animals. 1%5), and they are regarded as identical. Gm(5) was Gm(21) is almost always reciprocally related to Gm(5) and described by Harboe (1959) who called it Gm(b). It has been Gm(l1). Gm(22) is the Gm(y) of Litwin and Kunkel (1%7), called Gm(bi) (Steinberg and Goldblum, 1%5), and Gm(b9. and was defined by a rabbi anti-IgGl (Gm-1,4) myeloma Anti-Gm(5) antibodies have been found on a number of oc- protein. Anti-Gm(22) are apparently very scarce, if there is casions. Those which readily distinguish Gm(5) from still any available at all. Gm(23) is Gm(n) (Kunkel et al., Gm(-5) in Caucasians can give markedly different reac- 1%6), and was detected by an antiserum prepared in tions when used in other populations. This discovery has led primates against IgG2 myeloma protein. Gm(23) is the only to the delineation of a number of additional Gm factors. allotypic marker known on 162. Gm(23) can be detected Gm(6) was first called "Gm-like" by Steinberg et al. (1%0), by precipitation in gels, the first Gm specificity to be de- and later Gm(c) (Steinberg and Wilson, 1963). It has also tected in this way. The technique requires too much precious been called Gm(c5). A number of examples of anti-Gm(6) antiserum, however. No anti-D with Gm(23) has ever been have been found. Most of the antibodies have come from found, so a different technique was used (Natvig and Whites, many with rheumatoid arthritis, although the factor Kunkel, 1%7). 162myeloma protein with Gm(23) can be is mostly found in Blacks. Gm(7) was described by Brand- coated onto red cells using bisdiazotized benzidine, thus tzaeg et al. (1%1), and called Gm(r). Another anti-Gm(7) providing the "coat" for the test system. Tanned red cells has been found, but apparently most of the reagents have did not work very well (see sections 1.3.4.1 and 16.3). now been exhausted. Gm(8) was first described by Ropartz Gm(24) is apparently the Gm(cS) of van Loghem and et al. (1%2), and then named Gm(e). The original Martensson (1%7). Gm(25) appears to be identical with anti-Gm(8) is the only one that had been found up to 1970, Gm(13) and no longer treated separately. Thus, as noted and it came from a rheumatoid arthritis patient. Gm(9) was above in connection with Gm(lO), Johnson et al. (1977) originally called Gm@) (Waller et al., 1%3), and two ex- regard Gm(l0) = Gm(13) = Gm(25). Gm(26) was de- amples of anti-Gm(9), both from patients with rheumatoid scribed by van Loghem and Grobbelaar (1971) on the basis Sourcebook in Forensic Serologv, Zmmunologv, and Biochemktry of immune sera prepared in baboons. Steinberg (1977) G2m(n), and is the only known Gm specificity residing on described further studies on it. Gm(26) is also called Gm(u), IgG2. This nomenclature is extended to cover genetic and has been called Gm(Pa). Gm(27) is known as Gm(v) markers on other immunoglobulin heavy chains as well (see and Gm(Ray). Gm(28) was recently described by Rivat et al. below). (1978). A Gm factor called "Ll" was described by Blanc et Each subclass of IgG is thought to be under the control of al. (1976) in two families, one French and the other a separate genetic locus. Thus, if genes determining two or Algerian. It was transmitted with Gm(1) and Gm(17), was more Gm factors are present on a single chromosome, they always present in Gm(21) sera, and always absent in Gm(5) may be expressed together, separately, or not at all, on a sera. It is unclear whether it has yet been assigned a number. given IgG molecule. It will depend on the subclass of that Ropartz (1974) said that Gm(22) or Gm(y) had been found molecule. It may also be noted that myeloma proteins do to be equivalent to ~m(non-a),which according to the no- not possess the same phenotype as serum from the same per- menclature recommendations should be designated nGlm(1) son. In any given plasma cell, only one of the paired genes or nGlm(a) (see further below). for immunoglobulin synthesis on homologous chromo- somes is active. Immunoglobulins provide the only known example of autosomal allele exclusion in humans, i.e., only 44.2.2 Assignment of Gm factors to IgG subclasses one allele is active in a given cell. This phenomenon ap- Existence of isotypic subclasses of IgG has been recog- parently also occurs with the entire X chromosome in nized for some time. In certain neoplastic diseases, espe- females (see sections 1.2.4.4 and 33.1.4). In a Gm(1,3,5) cially multiple myeloma, immunoglobulins are synthesized Caucasian, for example, IgG myeloma proteins contain on- in excess. Urine may contain an unusual protein in these ly one, or none, of these determinants. The reason is that conditions, now known to represent immunoglobulin light Gm(1) and Gm(3) are at different locations in 161, and the chains, and called Bence-Jones protein. The IgG synthesized genes are on opposite homologous chromosomes, while that in multiple myeloma is unique in its high degree of for Gm(5) is in IgG3. homogeneity, as compared with the normal complex mix- ture of these molecules. The homogeneity of the myeloma protein is attributable to their synthesis by a highly selected 44.2.3 Isoallotypic markers of immunoglobulins-The population of cells, rather than by the sum total of all nonmarlcers immuno-globulin-synthesizing cells. The neoplastic cells Because myeloma proteins have been extensively used in producing myeloma proteins are regarded as being derived the study of immunoglobulin markers, it has been possible from a single clone. Because of the homogeneity of to defme a new class of genetic marker. Myeloma proteins myeloma proteins, and the fact that they can be obtained in represent only one subclass of I@, and as noted above, cor- large amounts, they are very important tools for immuno- relation studies on these proteins of subclass and Gm anti- chemists and immunologists. In 1%4, Grey and Kunkel gens have permitted assignment of Gm factors to particular made rabbit antisera to various myeloma proteins, and ab- subclasses. Antigens have been discovered which are shared sorbed them with other myeloma proteins. They were abl~ by different subclasses, but which exhibit genetic variation by subsequent immunodiffusion tests to distinguish four in only one of them. The immunoglobulin system thus subclasses of IgG, which they called We, Ne, Vi and Ge. shows two classes of markers. In one, a genetic event is Other investigators obtained similar results independently, related to a subclass-specific region, and two regular allelic but used different names for the subclasses. We now call the genes control a pair of markers, like Glrn(3) and Glm(17). In subclasses IgG1, IgG2, IgG3 and 164, according to the the other, a genetic event is shared by other subclasses, W.H.O. recommendations. The equivalent older names are reflecting the structural homologies preserved in subclass IgGl = We, y2b, C; IgG2 = Ne, 7%; IgG3 = Vi, y2c, Z; genes. Here, one may have a regular Gm marker behaving and IgG4 = Ge, y2d. All four subclasses are present in nor- as an allele in one subclass, like Glm(l), but where the anti- mal sera, though not in equal concentrations. The antigenic thetical marker is shared by other subclasses. These latter determinants which characterize them are, therefore, iso- are referred to as "nonmarkers" by Natvig and Kunkel typic. There is considerable structural simih@ in the pep- (1973), and are called "isoallotypic markers" by the tides derived from the four subclasses (Grey and Kunkel, W.H.O. Nomenclature Committee. The human non- 1%7). markers were detected by immunization of animals with Through studies of IgG myeloma proteins for both sub- myeloma proteins reflecting a partiah subclass. The first class and Gm -city, the Gm antigens have been as- was "non-a" (Frangione et al., 1%; Natvig el al., 1969). signed to the various subclasses of IgG on which they reside. Both the peptide and the antigenic marker were present in These assignments are now reflected in the formal all Glm(-1) IgGl proteins, and in all IgG2 and IgG3 nomenclature (W.H.O.,1976). Those antigens residing on regardless of type, but absent in IgG4. It is recommended by IgGl are denoted "Glm(l)", "Glm(2)", etc. Those on IgG3 the W.H.O.Nomenclature Committee that "non-a" be are called "G3m(ll)", "G3m(5)", etc. The letter designa- designated "nGlm(1)" or "nGlm(a)", as indicated in Table tions may be used as well, as indicated in Table 44.1. 44.1. There are several other nonmarkers as well. "Non-g" Gm(23) or Gm(n) is formally designated G2m(23) or was found in G3m(g) [G3m(21)]-negative IgG3 proteins, Serum Groups-Gm. Km and in all IgG2 (Natvig et al., 1%9). Antigens "non-bO" ten as the haplotypes, separated by a slash, e.g. Grnl.17;-21/ and "non-bl" [nG3m@O) and nG3m(bl)] are present on Gm3;2);5. IgG3 molecules which lack G3m@0) and G3m@l), respec- As mentioned above, various haplotypes tend to be char- tively, and also on all IgGl and IgG2 (Gaardner and Natvig, acteristic of certain racial or ethnic groups, and some factors 1972). The IgG4 class shows no regular genetic markers. are very seldom present in particular population groups. One antigen, however, called "4a" is present on some IgG4, One of the difficulties in population work is the rarity of whereas "4b" is present on all 4a-negative IgG4 (Kunkel et some of the testing reagents, with the result that a popula- a/., 1970). No IgG4 with both was found, but 4a is present tion may be tested only for certain allotypes for which anti- on all IgGl and IgG3, and 4b is present on all IgG2. Since sera are available to the investigators. Studies using antisera normal sera are positive for these "nonmarkers", they do for many specifiities are rare. Stedman and Wainwright not serve as genetic markers in the ordinary sense (hence, the (1979) summarized the factors and haplotypes that are rela- name "nonmarkers"), but they have been important as tively common in various groups: In Caucasians, Grn1J73I; markers in isolated subclasses of IgG. For example, Gml.2.17;21; Gm3;5.10,11.13,14. ad Gm3;23;5.10.11,13,14. In Michaelson and Natvig (1971) devised a procedure for sepa- Negroes, Gml.17;5.6.1124; Gml.17;5,6.10.11.14; Gm1,17;5,10.11,14; rating IgGl and IgG3 Fc fragments from normal serum, and Gml,17;5.10,11,13,14. and Gml.17;10.11.13.15; In Mongoloids, could use non-a and non-g as markers. Abel (1972) has Gm1.17;21; Gml.2.17;21. Gm1.3;U;5.10,11.13.14. ad Gm1,17; demonstrated the amino acid differences in IgG4 accounting 10,11913J5.16. Further information may be found in Johnson et for 4a and 4b. The IgG 4a protein has a Leu residue at posi- d.(1977). tion 309 which the IgG 4b protein lacks. A number of years ago, when Gm population studies were first being canied out, and the extensive biochemical data from studies of myeloma proteins was not available, 4.2.4 Gm genetics there were several ways of looking at the genetics. Giblett No other marker system, with the exception, perhaps, of (1%9) discd this point. Steinberg, for example, enter- HLA, shows such a high degree of variability in different tained at least for some time a conception of Gm genetics racial and ethnic groups. The formal genetics of the Gm that was analogous to Wiener's notion of Rh genetics (see system is complicated because of the structures of the genes. section 22.6.1) (e.g. Steinberg, 1%5). Such a model postu- Each subclass of IgG has its own cistron in the genome, and lates multiple alleles at a single locus, particular alleles being these are very closely linked to one another. Some of the able to code for proteins that contain one or several anti- marker and nonmarker genes are truly allelic, probably genic determinants. The alternative way of looking at it im- reflecting base changes at the same position of the structural agines the series of closely linked genes in the various sub- gene. Natvig and Kunkel (1973) call these pairs "homo- class cistrons, as outlined in the first paragraph of this alleles". Examples are Glm(f)-Glm(z) [Glrn(3)-Glm(17)], section. Glm(a)-Ghn(non-a) (Glm(1)-nGlm(l)] on IgGI, Glm(g)- Extensive studies of myeloma proteins of the various Glm(non-g) [Glm(21)-nGlm(2111 on igG3, and G3m(bO)- subclasses, and fragments obtained from them, have re- nG3m@O) and G3m@l)-nG3m(bl) on IgG3. Markers in sulted in the assignment of many of the markers to spedic different positions within allelic genes of the same cistron regions of the IgG chains-and thus, the genes determining are called "heteroalleles" by contrast. Because of the close them to sped% regions of the cistrons (see, e.g. Turner, linkage, they may be used as "allelic" markers in popula- 1976). In some cases, amino acid sequence studies have been tions, though. The formal genetics of Gm is discussed by carried out on the chains, revealing the chemical basis for Grubb (1970), van Loghem (1971), Natvig and Kunkel the antigenic differences. hsigmnent of the antigens to (1973) and Stedman and Wainwright (1979). specific regions of the chains is discussed by Natvig and As noted above, the W.H.O. Committee recommended Kunkel(1973), and well summarked by Stedman and Wain- nomenclature for the presence or absence of individual Gm wright (1979). The information is summarized in Figure factors. Ghn(1) indicates that the antigen is present; and 44.1. Because of the close linkage of the IgG subclass genes, Glm(- 1) indicates that it is absent. If a series of allotypic different genetic events can take place because of unequal markers is expressed, the designation is written in subclass homologous crossovers. Examples are deletions, hybridiza- order: e.g. Glm(1,2,3)G2rn(23)G3m(5,13,14).A haplotype tions and duplications. FamiIies have been studied in which is also expressed in subclass order, e.g. GmlJ-9;23;5,13.14.If apparent deletions are segregating, and Natvig and Kunkel G2m(U) were absent, the haplotype would be Gml.23; (1973) thought that this was a better explanation than silent -23;5,13.14, or Gm1.23;-;5,13,14. Phenotypes are written in sub- Gm alleles. Lefranc et al. (1976) reported on a deletion class and haplotype order, e.g. Gm(1,2,17,3;23), where the chromosome affecting IgG3. Kunkel et al. (1%9) have Glm subclass haplotypes are Gm1217 and Gm3, and G3m studied an example of a hybrid IgG chain, which is analo- antigens were not tested. Partial phenotypes may be written gous to the "Lepore" hemoglobins (section 38.2.3.5). There which indicate only the presence or absence of antigens is also evidence for duplication of the IgGl genes on one tested, e.g. Gm(1,2,3;-14). Stedman and Wainwright (1979) chromosome (Natvig et al., 1971a; Natvig and Kunkel, cover this material, and the other aspects of Gm in a brief 1973). The arrangement of structural genes on the chromo- but clear and understandable way. Genotypes may be writ- some is thought to be in the order IgG4-IgGZIgG3-IgG1. Sourcebook in Forensic Serology, Immunology, and BiochemMty

C~2 'H3 Chain f v v \ Position 115 226 334 446-COOH 17 n5 4a 1 2 nll I I I I I I ---- I I I I I

n5 4a n 1 nll I I I I ---- I I I I 3

n5 n21 4b nl nll I I I I I ---- I I I I

n5 n21 4b23 n1 nll I I I I I I ---- I I I I I

Figure 44.1 Distribution of the Genetic Markers on the lgG Subclasses CHI through CH3 refer to the constant homology regions. The "nonmarkers" are denoted nl, n5, etc. Serum Groups-Gm, Km

44.3 Light Chain Markers-The Km most Whites, roughly half the Blacks tested, and in 73% of System Japanese and 56% of Chinese. The numbers of people tested were small. Soon afterward, a second marker was Ropartz et 01. (]%la and l%lb) found an inheritable found by van Loghem et al. (1973), which they called property of serum which was detected in the same way as "A2m(2)", and it behaved as if its gene were allelic to that Gm antigens, but was independent. They called it "InV", for A2m(l). There are, thus, three A2m types: (1,-2). (1.2) and Iater "Inv(a)". An additional Inv factor, called and (-1,2). The frequency of the allele for A2rn(l) is high in "Inv(1)" was described by Ropartz et al. (1962). The "(1)" Caucasians, and much lower in Black and Oriental popula- in "Inv(1)" was apparently a lower case "L", but this can- tions. Van Loghem (1974) has discussed the properties and not be distinguished from a number one ("1") character in inheritance of the A2m system. The complete amino acid se- printing or typing. Steinberg et al. (1%2) descn'bed another quence of an d heavy chain from immunoglobulin factor called In*), and it behaved as though it were de- molecules of the A2m(2) allotype has recently been deter- termined by a gene allelic to the Inv(a) gene. Terry et al. mined (Toraiio and Putnam, 1978). (1%5) showed that the Inv factors were located on the K light AU the IgG markers described above reside on the con- chains of immunoglobulins, but not on the X chains. In the stant homology regions of the heavy chains. A marker was interests of wnsktency, the W.H.O. Nomenclature Com- recently ddbed on the variable regions of the heavy mittee recommended that the system name be changed to chains of IgG, IgM and IgA (Wang et ul., 1978). It was Td" Thus, as shown in Table 44.1, Inv(1) (lower case L) called "Hv(1)". or Inv(1) (number one) became Km(1); Inv(a) or Inv(2) became Km(2); and In*) or Inv(3) became Km(3). 44.5 Determination of lmmunoglobulin The antigens of the system are inherited by way of the codominant alleles Km', KmlJ and Km3 (Grubb, 1970). A Types number of populations have been tested with anti-Km(l), There are essentially only two ways of determining but the other antisera are apparently quite rare. The KmlJ immunoglobulin types. The first is serological, and is prob- allele is far more common than is Km'. In Caucasians, ably the most widely used. The second is immunoprecipita- almost all Km(1) are Km(1,2) if anti-Km(2) serum is used, tion using precipitating antibodies raised in auimals. and the figure is only slightly less for other populations. Precipitating anti-Gm antibodies can be prepared to some Similarly, over Wo of Caucasians and over 80% of antigens in rabbit., and in primates such as the rhesus Negroes who are Krn(1) are Km(1,3) if tested with monkey or the baboon (Hess and Biitler, 1%2, Alepa and anti-Km(3). Km(-1,-2,-3) people are known, and the Steinberg, 1%4, Litwin and Kunkel, I*, Yount et ul. , phenotype implies the existence of a Km- gene, an inhibitor 1967; Natvig et al., 1%8; Van Loghem and Grobbelaar, . of Km expression, or of another allele to which an an- 1971). tiserum has not yet been found. The serological method is based on antibody neutraliza- The amino acid substitutions in the K chains asociated tion (inhibition test). The presence of an antigen in a sample with the Krn allotypes have been worked out by meinet is indicated by its ability to prevent agglutination of red cells al. (1974). The residues which vary are 153 and 191. coated with -c IgG molecules which have the antigen Km(1,2,-3) has 153Ala, 191Leu, Km(-1,-2,3) has 153Ala, after incubation with the antiserum. The serological test, 191Val, and Krn(1,-2,-3) has 153Va1, 191Leu. Additional therefore, requires two reagents: the anti-Gm reagent (often information on Km may be found in Grubb (1970), Johnson called the "aggiutkator"), and an IgG incomplete anti- et d.(1977) and Stedman and Wainwright (1979). body, containing the Gm factor, which will sensitize the red cells without agglutimtiqthem (often called the "antl'body 44.4 Other lmmunoglobulin Markers coat" or simply the "coat"). Anti-D is customarily used as a The immunoglobulin marker called "Isf(1)" (Ropartz et coat, in part because of its availability, and in part because d., 1966) received its designation because the antiserum de- of its sufficiently higher titer. There can be variability in the fining it came from healthy donors from Sa.Francisco. It is behavior of both reagents of their pair, even though they apparently independent of Gm and Km, and resides on Ig- may appear to have the same specificity. The differences GI. It is clearly and simply inherited. Ropartz et a/. (1%8) between Ragg and SNagg agglutinators have been noted. found that its expression was agedependent in Caucasians The latter are preferable if available. There can be variabii- but not in Negroes. ity in the antibody coat as wen, and these reagents must be The fitst genetic marker on a heavy chain from a non-IgG carefully selected and standardized. Giblett (1969) noted immunoglobulin was independently found in IgA by Vyas that Rh+ cells with as many D sites as possible should be and Fudenberg (1%9) and by Kunkel et d. (1%9). LieIgG, employed (section 22.7.1). Some Gm specialists suggest that IgA molecules may be subclassified (into &types) according RIR, cells are preferable to R2R2cells. The semitized cells to antigenic determinants on their heavy (a)chains. The should be strongly Coombs positive. Giilett (1969) dis- genetic marker is asociated only with IgA2 molecules, and cussed both tube and tile techniques. Bore1 et al. (1%7) de- was fdcalled Arn(1) by Vyas and Fudenberg (1969) and scribed a microprocedure on microtiter plates. These Am, by Kunkel et al. (1%9). It is now called A2m(l) to be reagents are often very precious, and microtechnique is pref- consistent with the Gm nomenclature. A2m(l) occuned in erable wherever possible. A special technique was described Sourcebook in Forensic Serology, Immunology, and Biochemirtry in section 44.2.1 above for -cially coating red cells with be grouped specifically and reliably in stains. In some cases, appropriate myeloma proteins to detect a specificity for stains that had been stored for years could be typed, attest- which a proper anti-D coat could not be found. A clearly ing to the stability of the Gm antigens. Further studies on presented and very useful methods monograph has recently the typing of Glm(l), Glm(2) and G3m(5) were dedout been published by Kipps (1979). by Ducos et al. (1962 and 1%3a). In Germany, similar stud- 44.6 Medicolegal Applications ies were done by Fiinfhausen and Sagan (1%1), Fiinthausen et al. (1%2) and Sagan and Fiinfhausen (1965). They 44.6.1 Diioted parentage studied Glm(1) and Glm(2) determination in bloodstains on The Gm system has been employed in disputed parentage a variety of substrata. Even stains exposed to 100' condi- cases in specialized laboratories for quite some time. 'This tions for an hour (7 days old stains) could still be typed. In application is discussed in detail by Planques et al. (1%1), 1962, Prokop et a!. devised a rapid microprocedure for Gm Harboe and Lundevall (1%1), Prokop and Hunger (1%1), typing, which was applicable to bloodstain typing. Ducos el Prokop and Bundschuh (1%3), Ellis et al. (1973), van al. (1%3b) briefly reiterated the value of Gm typing. Loghem and Nijenhuis (1979) and %bring el d. (1979), Kobiela (1%3) confirmed that Glm(1) was reliably deter- among others. The value of Gm typing in parentage cases minable. Nielsen and Henningsen (1%2) also confmed depends on the number of spexScities which can be tested, that Gm factors could be detennined in bloodstains, as did and this varies to some extent from one laboratory to Lenoir and Muller (1%6) and Turowska (1%9). The factors another. In this country, the use of Gm in paternity testing were not detectable in older stains kept at higher humidity seems to be more common in the AABB Reference Labora- conditions, but the stains kept dry seemed to retain activity tories (Polesky and Krause, 1W.In the older papers, fewer well. In 1%4, Brocteur and Moureau reported that they had spe&cities were employed for the testing than in the more been able to sort out a car accident case, involving a deter- recent ones. mination who had been driving one of the cars, based in There is general agreement that the system is very useful, part on Gm typing of a bloodstain in the car, and com- and improves the overall exclusion percentages provided parison of the result with the Gm types of the occupants. A that the typing is carried out skillfully and that all the intri- lengthy and extensive study of Gm stain grouping was car- cacies of Gm and Km genetics are appreciated. The article ried out by Gijrtz in 1%9. The conditions of the inhition test by Sebring et d. (1979) provides excellent coverage of the were studied in detail, and he tested more than 950 blood- subject. They presented results on 925 cases in which Gm stains. Under favorable conditions, Glm(1) and Glm(2) typing was carried out using anti-Glm(l), (2), (3), (I?, could be typed using 0.1 mg of stain material, somewhat G3m(5), (101, (6),(241, (21), (151, (16) and Km(1). GZm(23) more being required for G3m(5). Stains which were still typing was employed to sort out a case involving an uncom- comparatively soluble were more readily groupable than mon haplotype. They said that the exclusion probability was those which had become insoluble upon aging. Lie other about 24Vo for Caucasians and 17070 for Negroes in this investigators, he emphasized the careful selection and country, using anti-Glrn(l), (2), (3) and anti-G3m(bl). The evaluation of reagents for the tests. The studies extended to figure would increase to about 33% for Black Americans if Glm(3), G3m(6) and G3m(10) as well, indicating that all Glm(17, G3m(21), (lo), (6), (24) and (15) were added. could be reliably determined. Gortz said that negative inhi- bition results had to be interpreted with great caution in 44.6.2 Gm and Km typing in bloodstains older, insoluble stains, since these might fail to inhibit the Efforts to group Gm antigens in bloodstains began about agglutinator even though the corresponding factor was pres- 20 years ago, and it is well established that reliable Grn typ- ent. A full paper on Glm(3) appeared in 1%9, emphasizing ing is possible with bloodstains. In practice, it appears that that information about the racial origin of stains could be Gm typing is limited to specialized laboratories that have obtained by combining Glm(3) and G3m(5) typing (Ducos et developed experience with the system. Km antigens can like- al., 1969). The Gm(-3,5) type was characteristic primarily of wise be detennined in bloodstains. Black people (cf. above in section 44.2.4). Stains as old as 15 Among the most extensive earlier studies on Gm stain typ- years could be typed. In 1971, Blanc and Gortz reported on ing are those of the group in Toulouse. Background for the the typing of Gm(Bet) [G3m(13)] in bloodstains. In this serum group systems was presented by Ruffie (1%1). In paper, they showed that typing results in sera and the corre- 1%1, Planques et al. presented their studies on the deter- sponding bloodstains were concordant for Glrn(l), (2), (3), mination of GIm(l), GIm(2) and G3m(5) in btoodstains. G3m(S), (6), (lo), (13) and for Krn(1) and (2). BIanc et al. Stains were deposited on a number of substrata, both ab- (1971) discussed the value of Gm phenotyping in blood- sorbent and nonabsorbent, and both Ragg and SNagg re- stains in terms of determining racial origin. Certain haplo- agents were tested. Dried blood on nonabsorbent substrata types and phenotypes occur in very much higher frequency were easier to work with. Nonspecific absorption of the in certain populations than others, as noted in 44.2.4, and anti-Gm was not observed, but they said that the SNagg an- careful selection of the factors to be tested can be informa- tisera gave more consistently complete inhibitions with tive. They also noted that only the positive identification of stains containing the corresponding Gm antigen than did an antigen should be used in the interpretation. Gortz et al. Raggs. There was no doubt that all the antigens tested could (1970) discussed the Gm typing technique and possible Serum Groups-Gm, Km sources of error. The agglutinator should be SNagg, and been typed in bloodstains many years old (Gortz, 1969; have a titer of 1:16 to 1:32. They seemed to think, too, that Ducos et al., 1%% Blanc et d.,1973). Budyakov (1%7) de- the coat reagents should be prepared in specialized tected Glm(1) in 5 year old stains regardless of exposure to laboratories. Most of the same types of problems encoun- often adverse environmental conditions. %milarly, Turow- tered with any kind of stain grouping are encountered in Gm ska (1%9) found that Glm(1) and Glm(2) could be typed in 5 typing as well. year old stains. Hoste et al. (1978) recently detected Km antigens can be determined in bloodstains. There are the Glm(l), G3m(10) and Km(1) types in 33 year old a number of reports on Krn(1) (Merli and Rouchi, 1967; bloodstains. Blanc and Gortz, 1971; Blanc et al., 1973; Khalap et al., The greatest difficulty with the Gm system is the availab'd- 1976), and on Km(2) (Blanc and Gortz, 1971; Blanc et al., ity of reagents. SNagg reagents and appropriate anti-D (or 1973). even other) coats for many of the specificities are not very Khalap et al. (1976) described a procedure for the typing common. Antisera can be developed in animals, but unless of Glm(l), Glrn(2) and Km(1) in stains. This procedure ap- there is more commercial interest in such reagents, they will pears in the MPFSL (1978) manual as well. They noted that probably continue to be rather hard to obtain for some woolen substrata can give "cloth reactions," i.e., non- time. Since the reagents are ordinarily precious, it is useful specific inhibition of the agglutinator. Khalap and Diva11 to develop systematic approaches to Gm typing, taking into (1979) reported on the typing of G3m(5) in bloodstains. The consideration the phenotypic distributions in the population antigen was reliably determinable, and they noted that the of interest, so as to be able to obtain the maximal discrimi- addition of G3m(5) to their routine testing protocol had ad- nation using the fewest reagents. Such a flowsheet for the ditional value in the interpretation of negative results with English population is given by Stedman and Wainwright Glm(1) and Glm(2). A substantial number of the samples (1979). tested in London (from British Caucasians) were Gm(-1,-2;s). If only Glm(1) and (2) were included in a test 44.6.3 Gm and Km typing in body fluids and tissues on a stain from such a blood, the negative results would Any tissue or fluid that contains immunoglobulins of the probably be uninterpretable. However, if G3m(5) is found, appropriate classes will contain the associated genetic the likelihood is much stronger than the Gm(-1,-2) result markers, but not necessarily in concentrations adequate for actually represents the type and is not a reflection of typing, particularly in stains. Nielsen and Henningsen (1%3) deterioration of the antigens. They noted that G3m(10) could not detect Gm factors in 23 seminal specimens. Klose could be used in a similar way. This matter is clearly discuss- and Schraven (1962) had detected Glm(1) in some specimens ed by Shaler (1982) as well, using casework illustrations. A from secretor patients, but reported to Nielsen and Henn- negative result for a factor can be interpreted as such if a ingsen (1%3) that they could not always detect it in semen positive result is obtained in the same sample for another from healthy donors. K-er (1%3) found Glm(1) in 9 of factor residing on the same IgG subclass. A very detailed 30 seminal samples. Davie and Kipps (1976) showed that discussion of Gm and Km typing procedures in forensic seminal Km(1) types match the type in serum from the same samples was given by Kipps (1979). Davie (1979) described a donor (31 samples). Special techniques were needed for procedure for Gm and Km typing on microtiter plates. grouping liquid semen (or saliva) samples because of their The immunoglobulin markers may represent the only ability to cause nonspecific aggregation of the test cells. A genetic marker system used in forensic serology which by freeze-thaw step, followed by centrifugation, prevented this their nature allow some control over the interpretation of effect in most cases. Many typing reagents perfectly suitable negative results. In general, failure to detect an antigen in a for senun typing were not very satisfactory for semen and bloodstain cannot normally be taken to mean that it was saliva, because of the low immunoglobulin concentrations never present. In the case of some Gm antigens, however, in these fluids. Jorch and Oepen (1977) reported that a the finding of one specificity can be used as a positive con- number of Gm factors and Km(1) could be detected in trol for the presence of the immunoglobulins in the stain, as semen if the quantity were sufficient, but that contamina- Khalap and Diva11 (1979) have noted. In this way, it may be tion by other body fluids which contained immunoglobulins possible to interpret negative reactions with more con- was a danger in medicolegal samples. It was considered fidence. Such positive marker control antigens are even bet- more likely that markers on IgGl would be detectable than ter if they reside on the same subclass of IgG molecules. One those on IgG3 in semen. Both nasal and vaginal mucus were wonders if the so-called nonmarkers, or antisera defining said to be possible sources of problems if they were found in particular subclasses (isotypic), could not be put to good use mixtures with semen. Blake and Sensabaugh (1978) noted as indicators of the presence of the IgG molecules contain- that there is about 50 times more I@ in blood than in ing the Gm factors being sought. seminal plasma, and that one would expect to require at Perhaps the most appealing property of the Gm and Km least the equivalent of 0.1 mf? of semen to detect an IgG system antigens for bloodstain work is their extraordinary marker. Davie and Kipps (1976) reported some limited suc- stability. No other genetic marker known, except for ABO, cess with seminal stains, but said that more sensitive tech- shows anything even approaching the stability of the Gm niques would be needed. antigens in dried blood. Various Gm and Km factors have In saliva, immunoglobulin levels are about 1000 times less Sourcebook in Forensic Serology, Zmrnunologv, and Biochernirtv than in blood (Waissbluth and Langman, 1971). Krimer tion must therefore be exercised if mixtures are suspected, (1%3) did not detect Glm(1) in normal saliva. Davie and and typing is to be attempted. Human inner ear fluid can be Kipps (1976) found that Km(1) types correlated in paired typed for Glm(1) and Krn(1) (Turowska and Trela, 1977). saliva and semen samples, but the saliva had to be treated by Tausch et al. (1977) studied the possibility of detecting freezing, thawing and centrifugation to remove its red cell Glm(l), (2), (3), G3m(10) and Km(1) m decaying blood and aggregating property. Only selected examples of anti-Km(1) organ materials. Serum remained typable the longest as a were found to be suitable for this typing. Some success was rule. Organ exudates were typable for varying lengths of reported with saliva stains, but increased sensitivity was time, from 1 to 8 weeks. Henke and Bauer (1980) recently needed in the test because of the relatively low immuno- reported that a number of Gm factors and Km(1) can be de- globulin levels. Km(1) was selected because it is not tected in the pulp material of human teeth. restricted to IgG, and might be expected to be present in Gm somewhat higher concentratons than Gm factors. Jorch and 44.7 Distribution of and Km Oepen (1977) found that Km(1) could be detected in saliva Phenotypes in Populations and saliva stains, but they had no success with Gm factors. There are a number of studies on various U.S. popula- Gm factors appear to be detectable in urine only if it con- tions, but they differ in the number of antigens determined. tains a pathologically high protein concentration (Nielsen et An older study by Blumberg et al. (1%4a) tested Glm(l), ai., 1%3). Sweat does not have levels of immunoglobulins (2), G3m(5), (6),Km(2) and Km(3) in both White and Black sufficient to permit Gm or Km antigen detection, but nasal populations of southeastern Georgia. The best overall refer- and vaginal mucus do have (Jorch and Oepen, 1977). Cau- ence is probably Johnson et d. (1977). Serum Groups-Other Systems

SECTION 45. OTHER SERUM PROTEIN GENETIC MARKERS 45.1 Serum Lipoproteins-The Ag, Lp This material has been thoroughly reviewed by Scanu et al. and Ld Systems (195) and by Osborne and Brewer (19n). The polymorphic serum lipoprotein antigens that are dis- cussed in the remainder of section 45.1 are awciated with 45.1.1 Introduction the low density lipoproteins (LDL). The lipoproteins are a heterogeneous and complex group of serum proteins. They are macromolecular complexes 45.1.2 The Ag system having reproducible lipid-protein ratios and stability in The first antigen of the Ag system was observed by aqueous solution. There are several ways of classifying lipo- Allison and Blurnberg in 1%1. There had been previous proteins, each based on a different separation criterion. The obsewations in animaln, and even in human beings, of the nomenclature is imprecise and sometimes ambiguous, and formation of precipitating antibodies by one individual there is no universal agreement on it as yet. In part, this sit- against the serum proteins of another in response to transfu- uation is due to the complexity of the systems, and in part it sion. No thorough or systematic studies had ever been done is due to the rapid expansion of knowledge in the field. An on these "isoprecipitins", however. The formation of such apolipoprotein is a homogeneous protein, containing no de- antibodies indicates serological variations in the serum pro- tectable noncovalently bound lipid. A lipoprotein is an apo- teins within the same species. AUison and Blumberg (1961) lipoprotein-lipid complex held together by noncovalent descnied a precipitating antibody in the serum of a patient forces. The lipid molecules are arranged such that there are at N.I.H. (Mr. C.deB.) who had received many transfu- no clearly defmed surface areas where polar groups shield sions. The antibody reacted with some but not all examples intramolecular hydrophobic regions from solvent. A lip of human sera from other people. The antigen in human protein panicle is an apolipoprotein-lipid complex, again serum that was reacting with C.deB. serum was called "Ag" held together by noncovalent bonds, except that here there (which just stood for "antigen"). The Ag factor was in- are extensive regions where polar groups of lipids form a herited in a simple Mendelian way, and Blumberg et al. surface to shield intramolecular hydrophobic regions from (1%2a) said that the gene determining the antigen should be solvent. These useful definitions were given by Osborne and cded AgA, and its allele Ag. AgAAgA and AgA~gpeople Brewer (1977) in their extensive review of plasma lipoproteh. would thus be Ag(a +), wbile AgAg ones would be Ag(a -). Lipoproteins have been cMied on the basis of (1) elec- Ag(a+) was shown to be associated with the low density (8) trophoretic mobility; (2) hydrated density; and (3) apolip- lipoproteins of serum (Blumberg et d., 1962b). In 1%3, protein composition. According to electrophoretic migra- Blumberg and Riddell described another precipitating anti- tion criteria, lipoproteins may be defined as those at the body in a patient's serum (the 'New York' serum)which de- origin (nonmigrating) and those migrating with pre-beta (a& fined an apparently different Ag factor, and this was called p and u (or a,)mobilities on paper or agarose. The migration Ag(b). It was thought to be conditioned by another gene, may be a little different on starch blocks (Scanu et d., A~B,and Blumberg (1963) suggested that this gene was 1975). Hydrated density is the most widely employed allelic to Ap, at least in some populations. Blumberg and cks%cation criterion. The relatively low density of these his collaborators continued to screen the saa of multiply proteins, compared with other serum proteins, is an advan- transfused patients for precipitating antibodies, and, by tage in separating them. They were originally dasifkd as: 1%4, they said that the antibodies an taken together defined chylomicrons (p < 1.006), very low density lipoproteins from 5 to 7 separate antigenic spedkities (Blumberg et al., (VLDL; p < 1.006), low density lipoproteins (LDL; I%&). laformation about the various antisera, and about p = 1.006-1.063). and high density lipoproteins (HDL; the frequencies of their corresponding an*, and their p = 1.063-1.21). Refinements of technique have resulted in relationships and inheritance, was not very detailed, how- the division of LDL into two classes and of HDL into three. ever. In addition, it was apparent by then that some of the Sodled very high density lipoproteins (VHDL;p = 1.21- antisera were polyspecific. The very early studies with 1.25) have also been recognized. Apolipoprotein composi- AUison were discussed by Blurnberg (1977) in his Nobel ad- tion studies of the varous density fractions has led to a dress. The study of antibodies in polytransfud patient sera cWcation of lipoproteins into families, based on the apo- led Blumberg and his collaborators off in a different direc- lipoprotein composition. The families are called Lp A tion once the "Australia" antigen had been discovered (sec- through Lp E, and each contains characteristic proteins or tion 50.2.3), and these studies culminated in the discovery of peptides, some of which have been better characterized than the infectious agent responsible for hepatitis B. Blumberg others. The specific peptide or protein constituents of Lp A shared the Nobel Prize for Physiology or Medicine in 1976 are called A-I and A-Ik those of Lp C are C-I, C-11, etc. for the hepatitis work. Sourcebook in Forensic Serologv, Immunologv, and Biochemistry

Hirschfeld and Blombiick (1964) described another anti- to that time. Red cells were "coated" with LDL of known Ag serum (the "L.L." serum) which reacted with about Ag specificity by a diazotization procedure. Corresponding 40% of Swedish sera, and had a peculiar relationship to anti-Ag sera would then give agglutination of the cells. This C.deB. serum. All C.deB.-negative samples were also kind of "coating" technique was introduced by Coombs et L.L.-negative, but C.deB.-positive sera could be either al. (1952) and discussed in section 16.3. It was further L.L.-positive or L.L.-negative. The relationship was similar discussed in section 44.2.1 in connection with the determina- to that existing between Gm(a) and Gm(x) (see in section tion of G2m(23) [G2m(n)] for which a suitable anti-D 44), and the -city in the L.L. serum was thus named "coat" was never available. Biitler et al. (1%7b) described Ag(x). C.deB. serum was found to contain not one, but an anti-Ag activity which was not precipitating, and could three different Ag antibody specificities. One of these, anti- be detected only by the passive hemasglutination technique. Ag(x), it shared with L.L. serum. The other two were named This antibody defined an apparently new Ag spet%city anti-Ag(al) and anti-Ag(z) (Hirschfeld et al., 1964). These called Ag(c). Further studies on Ag(c) and another new findings tended to simplify somewhat the conception of the specificity, Ag(e), were reported by Butler and Brunner Ag system (Hirschfeld, 1%8b). Some of the antisera shared (1%9). In 1%8, Contu found a serum ("R.M.") which ap- antibody specificities, and were polyspecific, and some of peared to define another Ag antigen, which he called Ag(m). the earlier complexities could be understood in these terms, He found that all "L.L. serumw-positivesera were Ag(m+) rather than in terms of relationships between the different while all "L.L. serum9'-negative ones were Ag(m-). antigens. The original so-called "Ag(a)". and its gene Although "L.L." serum was regarded as an anti-Ag(x), "A~A", disappeared in the revised interpretation. Contu thought that it was probably anti-Ag(x,m). Butler et A number of different isoprecipitin sera have been de- al. (1970a) described polyspecific antisera which contained, scribed, and sorting out the different speciilcities and their in addition to anti-Ag(a,) and anti-Ag(x), a new anti-Ag(g). immunological and genetic relationships has not always Butler et al. (1970b) described two sera containing anti- been an easy task. Until around 1964, there were really only bodies to yet another specificity, Add). Boman (1971) de- three fairly well characteIized antisera, the C.deB., the New scribed an antiserum whose activity was detected by passive York, and the L.L. ones. C.deB. was an anti-Ag(al,x,z). hemaggluthation technique. This was not an anti-Ag(x), The L.L. serum was considered to be anti-Ag(x), and pos- but its relationship to the other factors is not clear. sibly the only monospecific reagent of the three, since the The complexity of the Ag system, and the difficulty in New York serum was suspected of being more than just understanding the relationships between the antigens, can be anti-Ag(b) (Blumberg et al., 1%4b). In 1966, Geserick et al. accounted for in several ways. Antibodies are apparently found an "anti-Ag@)" antibody in a patient in Berlin who found only in the sera of people who have received many had been transfused over 200 times. It reacted with about transfusions. Many of the antibodycontaining sera have 93% of German sera. Hirschfeld et al. (1%6) described several antibodies in them. In addition, anti-Ag sera are another serum ("B.N.") which appeared to define a new comparatively rare. Geserick and Dufkovd (1%7) found specificity called Ag(t). It seemed that B.N. serum was a an anti-Age) [anti-Ag(t)] in the serum of a patient who had monospecific anti-Ag(t). Then, in 1%7, Hirschfeld and received 205 transfusions. After 12 additional transfusions, Bundschuh compared the Berlin example of "anti-Ab(b)" an anti-Ag(x) developed in the individuals's serum. Vierucci (the "New York" serum was no longer available) and the et al. (1966) reported screening 80 sera from multiply "B.N." [anti-Ag(t)] serum, and said that they were iden- transfused patients, and finding anti-Ag activity in 6 of tical. Another serum, called "C.P.", was described by them. Hirschfeld (1%8b) said that about 60 reagents had Hirschfeld et al. (1%7). It reacted with an antigen in about been described by the time that review was written. There 90070 of Italian and Swedish sera, and was thought to be are technical problems and variations assodated with Ag defining a new antigen, A&), whose determiuing gene, typing as well. It is difficult to "standardize" the test proce- AgY, appeared to be allelic to A@. Two other sera sent in by dure because of variations in the strengths of antisera, and other workers for evaluation appeared to be anti-Ag(y) as perhaps because of differences in the reactivity of the same well. Data in a number of populations were consistent with lipoprotein antigen in different sera. Hirschfeld (1%8b) the assumption that A@ and AgY were allelic (Hirschfeld conducted extensive studies on 28 different isoprecipitin sera and Okochi, 1%7), and examples of anti-Ag(x) and with a panel of 462 sera from unrelated individuals. Par- anti-Ag(y) were found in the Japanese population (Okochi, ticular antisera do not always give the same results with the 1967). Butler et al. (1%7a) examined eight serums with anti- same panel serum in repeated tests over the course of time, Ag activity. Six had anti-Ag(x), and in two, an additional although the great majority of repeated determinations were antibody defining a new specificity called Ag(r) was found. concordant. The "L.L." serum, for example, tested with One serum appeared to have a monospecific anti-Ag(r), and 108 panel sera in 536 determinations gave deviant results in another, a monospecific anti-AgOI) as well. In 1966, Biitler 15 instances. Some series of immunodiffusion plates give and Brunner described a passive hemagglutination tech- better results than others, and nonspecific precipitin zones, nique for typing Ag qxdicities. It was sensitive, and of- formed around the antigen wells, can sometimes cause prob- fered a number of advantages compared with the double lems of interpretation. Incubation time is another variable diffusion technique which had been employed exclusively up that is difficult to optimize because it is dependent on which Serum Groups-Other Systems reagents are used, and the amounts of each, the distance tisera were so rare. For this reason, and perhaps others, the between the wells, and the temperature. Pol-c system has been used very little in medicolegal work. The reagents can contain antibodies with different optimal in- Ag(x)-Ag(y) pair, which is believed to be conditioned by a cubation times. There can be differences in the reactivity of pair of alleles at a single locus, has been employed as a reagents at different times during storage as well. Sera kept marker in paternity cases in the United Kingdom (Brad- at -20" sometimes showed negative results after having brook et al., 1971). According to Lee (1975), the PE for a reacted with the same antiserum when they were fresh. In a falsely accused White father would be about 14% using few other cases, sera previously determined to be negative A&~,Y)testing. with a particular antiserum reacted with it after being stored frozen for 2 years. These tkhnical factors obviously have an 45.1.3 The Lp system effect on the results obtained with particular reagents, and In 1%3, Berg reported that he had detected a new, the subsequent conclusions that are drawn about the Ag genetidy controlled serum lipoprotein antigen in humans. system. Serum produced in rabbits by imrnmidon with human Much of the evidence (Morganti et al., 1970; Biitler et al., LDL, and then absorbed with various human sera to remove 1971; Hirschfeld, 1971) tends to support a genetic model for the "anti-LDL" activity, retained a precipitating activity the Ag system that postulates four closely linked loci con- against about 35% of sera from unrelated donors. The an- trolling a total of eight antigens. AgXIY can designate the tigen being detected was called "Lp(a)", where "Lp" stood locus at which AgX or Agy alleles may be found. The other for "lipoprotein". Reactors were designated Lp(a +), and three loci are A~~I,~,AgGg and AgtJ.This model permits 16 nonreactors were Lp(a-). The gene postulated as being different chromosomal arrangements, which can theoreti- responsible was called LP, and it behaved as a mendelian cally give rise to 136 genotypes and 81 phenotypes. Not all dominant. Further genetic studies (Berg and Mohr, 1963) in- of them have been found. In addition, AgZ is never present dicated that the genetic model was probably correct. The in the absence of Agal (Hirschfeld, 1971). The four closely hypothetical allele of LPwas designated Lp.In one fami- linked loci model is not the only possible one. A single locus ly, however, an Lp(a- ) x Lp(a -) mating had produced for the Ag system with multiple alleles is conceivable, if each an Lp(a+) child. While there was no evidence of il- allele produces a product with four different antigenic deter- legitimacy, it could not be ruled out either. Lp(a) was dif- minants, or factors. These two models are closely analogous ferent from any of the antigens of the Ag system (Berg, to the Fisher-Race and Wiener conceptions, respectively, of 1%4a). Problems could be encountered in typing Lp(a), and Rh (section 22.6.1). Hirschfeld (1971) has noted that, in Berg (1%4b) discussed an albumin-B-lipoprotein precipita- either case, antibodies are regarded as being monospecific tion which he said could be a source of misinterpretation in (simple) while each Ag chromosome produces four different reading irnrnundiffusion gels. Lp(a) was not very stable to adtigens which are inherited as genetically nomegregaihg storage, or to freezing and thawing, and Berg (19%) noted units ("complex antigens"). He therefore calls these models that it should be typed in fresh serum. Protocols for prepar- "simplecompiex". One can imagine other kinds of models ing rabbit anti-Lp(a) were given by Berg (lwa), and he also in which the chromosome produces only one antigen with a found (1%5b) that the antibody the rabbits made was a 7s single determinant, and where anthdies are complex, i.e., y-globulin. Additional family and population studies (Berg, they can react with the product of more than one Ag 1966) were consistent with the proposed genetic model. chromosome. If both antigens and antibodies are allowed to Wassenich and Walter (1968) reported on another family, be complex, even more involved models can be constructed however, in which an Lp(a- ) x Lp(a -) mating had pro- (Hirschfeld, 1971). While it is convenient to think of Ag duced three Lp(a+) children (out of a total of ten). The Lp system genetics according to a model having four closely locus is apparently not closely linked to Ag (Berg, 1967). linked loci, there are undeniable problems with it. Apart In 1964, Bundschuh found that "anti-Lp(a)" produced in from those mentioned above, it is not clear where Ag(m) fits horses behaved differently from the rabbit reagent. Two into the picture. Biitler et al. (1971) said that Ag"' might be precipitin hes were produced against Lp(a+) sera, one an allele of AgX and AgY. Biitler et R (1974) have reported showing immunologic identity with Lp(a) and one showing an additional qxcXcity called A&), thought to be condi- nonidentity. Prokop and Uhlenbruck (1969) discussed these tioned by an allele Agh.This allele was said to occupy a fifth studies in more detail. The horse antiserum was thought to closely linked locus, and its hypothetical partner was desig- be detecting a different but related antigen, called Lp(x). All nated A$]. Giblett (1977) brought up the interesting idea Lp(x+) sera were Lp(a +), but not conversely. The relation- that the five Ag loci, assuming the model is correct, might ship between Lp(a) and Lp(x) is not very clear. Berg (1%8) have a correlation with the five classes of LDL, in a manner tested the horse serum as well, and confirmed the results of somewhat analogous to the relationship between the Grn an- the German workers. tigens and the IgG subclasses (section 44). Sirnons et al. (1970) partially pwed the Lp(a) lipopro- The Ag system was reviewed by Biitler (lWa), BiitIer et tein. They said it differed from LDL and HDL in amino al. (1971) and Hirschfeld (1968b and 1971). Allison (1%3) acid composition, but was similar to the former in lipid noted that the Ag system was not a particularly promising composition. They further said that the protein had a char- prospect for forensic blood identification because the an- acteristic mobility upon disc electrophoresis, and that this Sourcebook in Forensic Serology, Immunology, and Biochemistry technique might be used for Lp typing. Utermam and dried blood on fdter paper. He optimized the pH and buffer Wiegandt (1970) found that they could not characterize concentration of the buffer used to make the immunodiffu- Lp(a +) sera by gel electrophoresis. Rittner (1971) reported sion gels, and then tested a number of samples. The antigen electrophoretic variation in the lipoproteins using disc elec- was detected in 20 of 21 Lp(a+) samples, and the iustability trophoresis. This appeared to be genetically controlled, but of Lp(a) was noted. its relationship to Lp is not very clear. Seegers et d. (1%5) also described an inherited electrophoretic variation in the 45.1.4 The M system 8-lipoprotein pattern, and this too may be related in some Berg (1%5c) found an isoprecipitin in the serum of a way to Lp. multiply transfused boy which reacted with about 42% of In 1970, Harvie and Schultz found that Lp(a- ) sera con- the sera of unrelated people. The antigen was awxiated tained measurable quantitites of Lp(a) when concentrated. with LDL, and was called "M(a)". It was not related to the They regard the Lp polymorphism, therefore, as being a Lp system, and reacted best at lower temperatures. Its con- quantitative genetic trait. Utennann and Wiegandt (1970) trolling allele was called Lda, which behaved as a Mendelian interpreted their electrophoretic dtsas being consistent dominant in family investiguions. Berg and Reinskou with Lp(a) as a quantitative trait. Enholm et d. (1971) (1967) found that the anti-M(a) was an IgG antlibody. Berg found that serum Lp(a) levels varied over a very wide range (1971a) said that the oriwsenun had later been found to (about %fold), and this finding would be consistent with contain both anti-Ag(x) and anti-M(a), but that mono- quantitative variation. Albers et d (1974) measured Lp(a) s-c anhfor M(a) had been found. Another an- concentrations by radial irnrnunodiffusion, and did not tiserum had also been found which appeared to react with detect bimodality in the concentration distriiution. They the product of the dele of Lh. concluded that the variation was consistent with the poly- genic inheritance. Sing et d. (1974) and Schultz et d. (1974) canied out quantitative studies on Lp(a) in serum using a 45.2 Complement Components sensitive radioimmune inhibition technique. They found that all individuals had Lp(a), but that the concentrations The complement system was discussed briefly in section were distributed bimodaly. They imagine that L@, the 1.3.5. Genetic variation has been found in a number of the dominant allele, determines one distribution of Lp(a) con- components of the complement system, and both quantita- centrations, while the allele determines another distribution tive and structural variants have been reported. Comple- with a lower mean value. Fisher et d. (1975) have found that ment component variants are typed by electrophoresis or by human LDL seem to exist m several different MW ciasses, isoelectrofocusing, and may be detected by protein staining, and that the classes possessed by a given individual are immunofmtion, or by functional detection overlays, de- under genetic control, apparently at a single locus. The rela- pending upon the component. A recent review of this mate- tionship of this fmding to the Lp system is not yet clear, but rial may be found in Hauptmann (1979), and other reviews it may be related to the concept of Lp(a) as a quantitative are Cooper (1W8), Hobart and Lachman~~(1976) and Gitlin genetic variant. The studies of Sing et d. (1974) and of and Gitlin (1975). The complement component deficiency Schultz et al. (1974) indicated that there is a slight overlap in states, which are under genetic control, were reviewed by the Lp(a -) concentration range and that of Lp(a +) people. Agnello (1978). In this view, the Lp(a+ ) and Lp(a- ) phenotypes are not unambiguously related to genotype. This model would help 45.2.2 C2 Component to explain some peculiarities of the system, the most C2 is a single chain protein of approximate MW 102,000. troublesome of which is the occasional observation of Genetic variation in this component was detected by Hobart Lp(a+) children from Lp(a -) x Lp(a -) mgs. and Lachmam (1976), and independently by Alper (1976). The Lp(a) protein has been isolated in a number of differ- There are two or three prominent bands and several addi- ent ways, and characterized to some extent (Enholm et d., tional minor bands in the C2 patterns of most individuals. 1W1; Utermann and Wiegandt, 1969; Dubany and Mod- C2 patterns are usually determined by polyacrylamde gel lec, 1976; Desreumaux et al., 1977a and l977b). The Lp(a) isoelectrofocusing (PAGIF), and detected by functional lipoprotein is closely related to LDL in some respects, but is techniques, using an agarose overlay containing sensitized apparently not identical with them. Upon storage or red cells and a CZdeficient serum. Alp (1976) called the detergent treatment, the Lp(a) preparation may dissociate common pattern "C2-C", and it accounted for about 96% into LDL, albumin and Lp(a) apoprotein. of the samples. The rarer variants are of two kinds, acidic The Lp system has been reviewed by Biitler (1%7), Berg and basic, and were designated "C2-A" and "C2-B". (1968 and 1971a) and Cooper (1978). There have been These were accounted for by three alleles, @, C2A and almost no reports on applications of Lp to forensic or medi- aB. Meo et al. (1977) studied this polymorphism further. colegal problems. In view of the complexities of Lp(a), They used the designation "C 2-1" for the common type, many of which are not yet completely understood, it is prob- and "C 2-2" designated the rare basic type. The rare acidic ably not a very suitable system for forensic work. Haferland type would be "C 2-3". The responsible alleles are C?' (1%5) did studies on the detection of Lp(a+ ) in 24 hour old (= @, a2(= C2B) and Cz3 (= @). Olaisen et al. Serum Groups-Other Systems

(1978) confirmed these findings, and they used the designa- conditions, with C3 S and C3 F1defined as having unit tions of Meo et al. (1977). C2l was found to be rare in the mobility. Thus, fast phenotypes have designations like F,,, Norwegian population as well. C2 deficiency is the most F,.,,etc., and the slow ones are similarly called S , SO.a, common deficiency of a classical complement component. etc. A heterozygote having one aFand one gene The deficiency is inherited and quite rare. In view of the would have the phenotype C3 FS0.4. Phenotypes represent- fmding of structural variation at the C2 locus, the deficiency ing the expression of at least 22 rarer alleles had been found states are now usually attributed to homozygosity for a rare by the time the Bonn conference was held. Dr. Rittner's silent allele CZOmemperer, 1974). The C2 locus is linked to laboratory was designated the C3 Reference Laboratory at the HLA locus (Fu et al., 1974; Alper, 1976; Meo et al., the meeting, and an updated report of new variants was 1977; Olaisen et al., 1978). published for 1973-1974 (Rittner and Rittner, 1974). The nomenclature and other standards adopted at the June, 45.2.3 C3 Component 1972, meeting are sometimes referred to in the literature as C3 is a pivotal protein in the complement system. It oc- the "Bonn conventions". C3 mobility is very sensitive to the curs in relatively high concentrations in human serum, and it concentration of Ca++ in the buffers. This cation has the can be detected comparatively easily by simple staining for effect of decreasing C3 mobility and, hence, of making the protein after high voltage electrophoresis. For these reasons, C3 bands easier to visualize by simple protein staining. The it is perhaps the best studied of the complement component Bonn convention recommended that C3 typing be done on polymorphisms. agarose gels in buffers containing 1.8 mM Ca lactate. Other The genetic variation in C3 was detected by Wieme and variables in the typing procedure were discussed by Kiihnl Demeulenaere (1967) using agarose gel electrophoresis. The and Strobel (1974). polymorphism was described by Azen and Smithies (1%8) In 1969, Rose and Geserick observed genetic variation in and independently by Alper and Propp (1968). Azen and a serum protein which they designated "Pt" (for "post- Smithies (1968) used high voltage starch gel electrophoresis transfertin"). There were three phenotypes, accounted for to separate the proteins, and found 6 phenotypes, which by a pair of alleles, and the variation was seen only in aged could be attributed to four alleles. Only three phenotypes, 1, serum samples. Before too long, it became apparent that the 2-1 and 2, accounted for by a'and 02,were fairly com- "Pt" system was intimately related to C3 (Geserick and mon. Alper and Propp (1968) used prolonged agarose gel Rose, 1973). Pt A is related to C3 F, Pt AB to C3 FS,and Pt electrophoresis to detect the variations. They found 8 B to C3 S. C3 is converted to C3a and C3b during comple- phenotypes, and could account for them on the basis of ment activation in vivo, and C3b can be further converted to three certain and 2 probable additional alleles. The common C3c and C3d by senun factors. It appears that C3c is a ma- variants were designated C3 F, F-S, and S, where "F" and jor storage fragment of C3 in serum. The socalled "Pt" "S" stood for "fast" and "slow". The fastest variant was system variants are apparently equivalent to the common C3 called C3 F, and the slowest, C3 St. The other variants were variants, which are preserved in the C3c fragments (Alper, designated according to their relative mobility taking C3 F, 1973; Mauff et al., 1974; Hauptmann, 1979). The conver- and C3 S, as unity. They were C3 FO,, F,, and Tech- sion of C3-*C3c is inhibited by 45.6 mM Na2EDTA (Patzelt nical refinements for C3 typing have been described (Azen et al., 1977). The common C3 variants are evident in the C3c et al., 1%9; Teisberg, 197Oa). Family and population studies fragment (the "Pt" system), but rarer variants cannot be by a number of workers have amply confirmed the codomi- readily typed using the Pt typing system. If antigen-aatibody nant inheritance pattern, and many additional variauts have crossed electrophoretic techniques are used, C3 variants can been described (Azen et al., 1969, Teisberg, 1970b, 1971a, be detected more readily in C3c fragments as well as in 1971b and 1971~;Brannestam, 1971; Bronnestam et al., native C3 (Schwamborn et al., 1976). C3 apparently does 1971; Dissing and %rensen, 1971; Segers et al., 1974; Seger not cross the placental barrier, and fetal C3 corresponds to and Salmon, 1977; Schlesinger et al., 1979). C3 S is more the type of the fetus and not to that of the mother (Propp common in most Caucasian populations (as frequency and Alper, 1%8; Azen et al., 1969). Reviews of the C3 poly- about 0.8) than C3 F. The @ allele has a very high fre- morphism have been given by Alper (1973), Seth and Seth quency in the Lapps (Bronnestam et al., 1971; Teisberg, (1976), Hobart and Lachmann (1976) and Hauptmann 1971b). A possible association of aFand rheumatoid ar- (1979). thritis has been suggested (Bri5nnestam, 1973), but no The C3 polymorphism has been applied to disputed association of the C3 polymorphism with Graves disease parentage problems in a number of countries (Teisberg, could be detected (Pepper and Farid, 1979). 1972; Farfiud and Walter, 1973; Spielmann, 1973; Hoste et The nomenclature and recommended typing methodol- al., 1977; Geserick et al., 1980). The probability of exclud- ogy for C3 is based upon agreements reached by an interna- ing a falsely accused western European Caucasian father tional panel of specialists convened at Bonn in June of 1972 with C3 is about 13%. No references to C3 typing in blood- (Rittner, 1973). The common C3 variants are designated C3 stains were found. F and C3 S, and the responsible alleles BFand as,respec- tively. Variant types are designated according to their elec- 45.2.4 C4 Component trophoretic mobility on agarose gels under a specitied set of C4 is a glycoprotein of Mw about U)6,000,consisting of Sourcebook in Forensic Serology, Immunology, and Biochemistry three polypeptide chains. Normal serum contains a C4 bind- the two locus hypothesis. Hauptmaan (1979) said that ing protein which can form stable complexes with C4. This O'Neill and collaborators had recently reported finding Ch complex formation is prevented by EDTA or heparin, how- and Rg antigens on the red cells of a homozygous C4 defi- ever, and EDTA must be incorporated into buffers used for cient individual, but absent in the serum. This finding ap- C4 polymorphism studies if EDTA or heparin plasma is not pears to indicate the Cha and Rga may be controlled by a available (Hauptmann, 1979). locus independent of C4. The relationship between Ch/Rg Genetic variation in C4 was described by Rosenfeld et al. and C4 will require further study. Hereditary C4 deficiency (1%9) and studied further by Bach et al. (1971). They is quite rare, but most workers now attribute it to a rare studied EDTA-plasma by antigen-antibody crossed electro- silent allele C40 in the one locus model, or to the genotype phoresis employing an anti-C4 serum for irnmunofixation f"f"SSin the two locus model. detection. Ten different patterns could be distinguished, but Olaisen et al. (1979) have recently found that the C4 locus the underlying genetics was not very clear. Teisberg et al. is duplicated, at least in some people. They suggested a (1976) investigated C4 patterns using high voltage agarose change in the nomenclature for the C4 polymorphism to gel electrophoresis, and irnmunofvtation detection. They de- take this fact into account. In addition, they do not appear tected three common phenotypic patterns and one rarer one, to regard the model proposed by O'Neill and coworkers, in and family studies indicated the involvement of three co- which C4 is duplicated on all chromosomes but has a high dominant alleles at an autosomal locus, which were closely frequency of silent alleles at both loci, as proven. linked to the HLA locus. The common phenotypes were C4 F, S and FS, accounted for by @ and Another 45.2.5 C6 Component phenotype, FM,was attniuted to heterozygosity of C& Polymorphism of C6 was detected by Hobart et al. (1974) with a rarer allele v.These studies were extended (Teis- using isoeiectric focusing and functional detection with a berg et d., 1977), and an additional allele ~4~1was found. specific C6 hemolytic assay system. Three common pat- Mauff et d. (1978a) confumed these findings in a German terns, called A, AB and B, could be accounted for by a pair population, and gave a modified electrophoretic procedure of codominant alleles C6A and c@. Some rarer patterns enabling easier detection of the F1 types. In addition to the were thought to be due to the expression of a less common common F, FS and S types, FlF, FlS, Fl, MF, MS, MF1 allele, called C6R. They said that C6R might not be a single and M phenotypes were observed. allele. The common types of C6 are fairly well distributed in Recent studies by O'NeiU et al. (197&, 1978b and 19%) western Europeans, and the inheritance pattern has been have resulted in a different picture of C4 genetics. It is clear confumed (Hobart and Lachmann, 1976; Rittner et al., that C4 is closely linked to HLA, especially to HLA-B and 1979). C6 is closely linked to C7, the locus controlling the Bf, but there is no clear association with GLO Veisberg el seventh complement component, but it is not linked to HLA d., 1977; Mauff et ad., 1978a). The C4 locus is thus ap- (Hobart and Lachmann, 1976; Kagan et al., 1979). In- parently within the HLA complex. O'Neill et 01. suggest that herited deficiency of C6 has been described, and is at- C4 phenotypes are controlled not by codominant alleles, but tributed to homozygosity for a silent allele C69 (Glass et al., by two separate but closely linked loci. In addition, they 1978). Ritter et al. (1979) and Mauff et al. (1979) have have shown that the "red cell" antigens Chido (Cha) and recommended incorporation of C6 testing into paternity in- Rodgers (Rga) are in fact distinct antigenic components of vestigations. The probability of excluding a falsely accused C4. C4 F is said to be controlled by one locus, with alleles F man is about 18% in western Europeans. and f" determining the presence or absence, respectively, of the fast bands. Similarly, C4S is controlled by a second 45.2.6 C7 Component locus having alleles S and 9.Cha is believed to be a compo- Hobart et al. (1978) found three C7 phenotypes by iso- nent of C4 S, and Rga of C4 F, because C4 F people (Et?S$ electric focusing. There was one homozygous type, C7 1, or Ff"99) are always Ch(a- ), while C4 S people (FfPSS or and two apparently heterozygous types, C7 2-1 and C7 3-1. f"f"W) are always Rg(a- ). The C4 antigenic determinants Three codominant alleles, C7', CP and CT, were postu- are associated with the C4d part of the molecule (Tilley et lated to explain the types. In one family, a 2-1 x 3-1 mat- al., 1978). Hauptrnann (1979) has noted that known varia- ing had produced a 3-2 child. They noted that C7 is closely tions in C4 FS patterns would be consistent with the predic- linked to C6. tion of the four different genotypes for phenotype C4 FS in the two locus model. There are characteristic associations of 45.2.7 CS Component certain HLA-B and Bf types in individuals of C4 F and C4 S Raurn et al. (1979) found genetic polymorphism in C8 us- type. The levels of C4 show variation according to pheno- ing PAGIF. Two common alleles, C8* and @, give rise to type as well, C4 F and C4 S individuals having significantly three common phenotypes. C2tA is more common, having lower levels than C4 FS ones (O'Neill and Dupont, 1979). frequencies of about 0.65 in Blacks and Whites, and about Awdeh et al. (1979) have recently shown that antigen- 0.7 in Orientals. A rarer dele, (XA',is most frequent in antibody crossed electrophoresis shows different and Blacks (about 0.05), very rare in Caucasians, and it was not distinguishable patterns in FS,PS, FsO,FSso and FPS types, seen at all in Orientals. C8 deficiency is attributed to a silent thus allowing their discrimination, and lending support to (CP)allele. C% is not linked to HLA . Serum Groups-Other Systems

45.2.8 Properdin factor B ('50 In connection with studies on Lp(a) (section 45.1.3), Berg Properdin factor B was discovered (and named) by produced an antiserum in rabbits which, after absorption, Pillemer et al. (1954). Properdin is a name that has been reacted with some but not all human sera. The precipitates given to several factors which can activate complement by stained for protein, but not for lipid, and the antigen was, an alternative pathway to the classical one (section 1.3.5). therefore, not a lipoprotein. The antiserum had been pro- Thus, properdin factor B is a C3 proactivator. The protein duced by immunization with the serum of a single Lp(a-) was isolated by Haupt and Heide (1%5) and called "fit-gly- female donor. Reactions of the antiserum strongly indicated coprotein 11". It was also isolated by Boenisch and Alper that the antigen might be controlled by an X-linked gene. (1970) and called "glycine-rich-B-glycoprotein" or "GBG". The antigen could also be localized to the macroglobulins, There was a suggestion in the work of Boenisch and &per and the system was named "Xm" to indicate the X-linked (1970) that the protein might exist in multiple molecular macroglobulin protein antigen. The antigen was called forms. Alper et al. (1971 and 1972) described the polymor- Xm(a) and the gene controlling its presence X& (Berg and phism of "GBG", and suggested that the protein had some Bearn, 1%6a and 1966b). The antigen was shown to be lo- relationship to the complement system. The "F',"FS" and calized in aZM, and extensive linkage studies were carried out "S" (for "fast" and "slow") types were common in all with other known X markers (Berg and Bearn, 1%8). Xm populations tested. The locus controlling the protein was mapped fairly near "deutan" and a little farther from Gd. named Gb, and the alleles G~Fand GbS. A phenotype deter- Cooper (1978) said that the limited supply of anti-Xm(a) re- mined by a rarer allele G~FIwas seen in Blacks, and one agent had been exhausted, and that no further examples had determined by another rarer allele G@' was seen in Whites. been found. It was then realized that GBG was identical to factor B of In 1969, Kasukawa et aI. found an isoprecipitii in a the properdin system (Aiper et d., 1973) as well as to a pro- chronic hepatitis patient who had never been transfused. It tein isolated by mtze and Miiller-Eberhard (1971) that had reacted with about 30% of random sera in Japan, and the been called C3 proactivator. The locus has been re-named antigen, which was said to be an al-globulin, seemed to be Bf, and the common alleles are BFand Bp. The less com- inherited in a simple Mendelian way. Another example of mon alleles described by Alper et d. (1972), GbF1 and GbS1 the antiserum was then found by Leikola et al. (1972), and are now called BJF' and BfSO.'. By 1978, there were seven they showed that the antigen resided in the atM fraction. more alleles. An internationally agreed upon nomenclature The antigen was named "AL-M", and it was inherited as an for Bf types was adopted (Mauff et al., 1978b), and a autosomal dominant. Reference Laboratory headed by Prof. Dr. Mauff was set In 1974, Gallango and CastiUo reported another poly- up at Cologne. Variants other than F, S and F1 are now morphism in azM. This one was detected by immunoelectro- designated "F" or "S" (depending upon whether they are phoresis in the sera of a Venezuelan Mestizo population "fast" or "slow") with a relative mobility value listed after using anti-azM sera. The types were distinguished by electro- the upper case letter. The reference distance in this case is phoretic mobility upon irnmunoelectrophoresis. There were S+F1. Kiihnl and Spielmanu (1978b) have done population apparently three homozygous types, A, B and C, and three studies on Bf in the German population. Weidinger et al. heterozygous types AB, BC and AC. Genetic data indicated (1979) presented evidence for a silent allele, By, in one of that the types could be accounted for by three codominant the families in their material. alleles at an autosomal locus, aNA,ad@ and a@. The The Bf locus is closely linked to HLA (Ah, 1974; Ritt- autosomal inheritance pattern indicated nonidentity with ner et al., 1975), and it probably lies closest to HLA-B. the Xm system. Identity with AL-M seemed to be excluded There are striking linkage diseqdiiria between HLA-B and by the fact that no "negatives" had been observed. Mroueh Bf alleles. As noted above, the C2 and C4 structural loci are and Adham (1970) assayed semen for a2Mlevels, and found closely amxiated with Bf in the HLA complex. measurable quantities in 11 out of 34 samples. Con- centrations in the 11 ranged from 30 to 130 mg/lOOmP with a mean of 70.4. There was no correlation between a2Mlevel 45.3 a,-Macroglobulin (ar,M)-Xm, and oligo- or azoospermia. AL-M end a,M az-Macroglobulinis one of the major protease inhibitors 45.4 a,-Acid Glycoprotein of human serum (Laurel1 and Jeppsson, 1973, as noted in (Orosomucoid) section 43.1. The molecule was purified and characterized a,-Acid glycoprotein is a plasma globulin characterized by by Hall and Roberts (1978). It had a MW of 718,000, and a high carbohydrate content, a large number of sialyl probably consists of 4 polypeptide chains of MW about residues and a very acidic PI. The protein occurs in the so- 185,000. These derMW units are cleaved to yield 85,000 called "seromucoid" fraction of plasma, and is the fastest MW fragments upon interaction with a variety of proteases. moving serum protein component on starch gel electropho- The serum concentration of a2Mis dependent upon age and resis using borate buffers at pH 8.6. One of the first puxified sex, mean values being of the order of 175 mg/lO(hP for preparations of the protein was described by Weimer et d. men and 206 mg/100 mP for women. The value is 2-3 times (1950). At that time, it was referred to as the "MP-1 com- higher in small children. ponent" . Schmid (1953) obtained a crystalline preparation Sourcebook in Forensic Serology, Immunology, and Biochemistry of &]-acidglycoprotein. The purified protein had a MW of The detailed structure is apparently still not completely about 40,000, and about 45% of its weight is attniutable to clear. Subunit structure has been proposed for Cp (see in the carbohydrate content. The primary structure of crl-acid Poulik, 1%8), but there is also evidence to suggest that the glycoprotein has been completely solved (Schmid et d., molecule consists of a single polypeptide chain (Poulik and 1973), and upon close analysis, revealed an extraordinary Weiss, 1975). Kingston et al. (1979) determined the sequence number of amino acid substitutions in different examples of of a lengthy segment of the molecule (159 amino acid the protein. 21 different residues, out of 181 in the complete residues), and appeared to favor a single chain structure for molecule, have been found to be substituted. The sequence the intact protein. This segment did not contain any of the also showed structural homology with the immuno- carbohydrate. globulins. The exact function of Cp is still not settled altogether. In 1%4, Schmid et al. found that al-acid glycoprotein ex- The protein exhibits oxidase activity with a number of arti- hibited electrophoretic heterogeneity, and suggested that ficial substrates. Cp is usually detected by its oxidase activity this behavior might have a genetic basis. Further studies after electrophoretic separation. Uriel (1958) devised such a were carried out on desialyzed material, and indicated that detection procedure with pphenylenediamine as substrate. the three different patterns which could be observed were The protein is also an effective "ferroxidase", catalyzing the result of a pair of alleles (Schrnid et d., 1%5). Because the conversion of Fez+to Fe3+ .It has been assigned a name of its molecular properties, the protein shows considerable and number by the Enzyme Commission on this basis: ce- microheterogeneity upon electrophoresis, in addition to the ruloplasmin = ferroxidase; iron(II):oxygen oxidoreductase; macro polymorphic patterns. This microheterogeneity is E.C. 1.16.3.1. The ferroxidase activity is one of the three even more apparent if isoelectric focusing is used as the functions of Cp that have been assigned physiological separation technique (Gordon and Dykes, 1972). significance, the others being copper transport and detoxica- The crl-acid glycoprotein phenotypes were studied by irn- tion, and the maintenance of copper homeostasis in tissues. munofixation electrophoresis on agarose gels by Johnson et The functions are not necessarily mutually exclusive. At the al. (1969). Further family and population studies, extending present time, a number of investigators think that the fer- the work of Schmid et d. (1%5), were conducted to roxidase activity of Cp is its most important function. Fer- establish the genetic basis for the polymorphism. The rous ions are oxidized by Cp to ferric ions, which are then phenotypes were called SS, SF and FF (where "S" and "F' incorporated into transferrin (section 42). Goldstein et al. stand for "slow" and "fast"). These are accounted for by a (1979) have shown that Cp can also act as a scavenger of pair of autosomal codominant alleles called Ofi and OF superoxide radicals, thus mimicking the action of SOD (sec- (the protein is also called "orosomucoid"). The 09 fre- tion 37.4) in this respect. quency in U.S.Caucasians (n = 220) was 0.36, and varied Molecular heterogeneity was observed in Cp by a number from about 0.33 to 0.57 in other populations. The polymor- of the earlier workers, such as Uriel (1958), Deutsch (1%0), phism is thus fairly well distributed, and may well become Morel1 and Scheinberg (1%0) and Hirschman et d. (1%1). another valuable serum group system marker. A genetic basis for the variation was considered, but Poulik Sayce and Rees (1977) reported a series of experiments on and Beam (1%2) indicated that it would be difficult to inter- seminal orosomucoid. There appeared to be two different pret the genetic -cance of the heterogeneity. Other ex- and distinguishable irnrnunologicd reactions using anti- planations, such as polymeridon and denaturation caused orosomucoid serum. These experiments weere discussed by methods of preparation or storage, were considered more briefly in section 10.13.2. The relationship between these likely. immunologically distinguishable forms and the serum poly- Qualitative genetic variation in Cp is now known, how- morphic types, if there is any relationship, is not clear. ever. A possible genetic variant was described by Martin et The properties of crl-acid glycoprotein have been discussed al. (1%1) and more funy by McAlister et al. (1%1). The in detail by Schmid (1975). The biological role of the protein variant form was detected by vertical starch gel electropho- is not yet fully clear. resis at pH 8.6. It was faster than the usual Cp (called "Cp 1") and was called Cp 1-F. It was easier to detect, they said, 45.5 Ceruloplasmin (Cp) in pH 9.1 buffers. The variant Cp was transmitted through Copper is an essential trace element in the nutrition of four generations of a family as a dominant trait. In spite of many animals, including human beings. Most of the copper large scale Cp typing in a number of laboratories, however, in human serum is bound to an crz globulin protein (Holm- this variant was not encountered again (Poulik and Weiss, berg and Laurell, 1947). Cp has a MW of about 150,000 and 1975). can bind 8 atoms of Cu per mole. The isolated protein has a Schreffler et d. (1971) described three genetically deter- strikingly beautiful sky blue color, which is often mentioned mined forms of ceruloplasmin, called Cp "A" (the fastest), in discussions of it. The name "ceruloplasmin" was given to "B" (intermediate) and "C" (the slowest), which could oc- the protein by Holmberg and Laurell (1%). Cp has been cur alone, or in combinations. Five phenotypes were seen: extensively purified, and a crystalline preparation was ob- A, AB, B, AC and BC. These phenotypes were attributable tained by Deutsch (1960). The detailed properties of the pro- to three codominant alleles at an autosoma1 locus, C', tein are described in the review of Poulik and Weiss (1975). C'and @. Cp B is the common phenotype. CpA oc- Serum Groups-Other Systems curred in Caucasians but was more frequent in Negroes, biochemical lesion as the inability to synthesize Cp, but while Cpc was not seen in Cauasiam. These phenotypes evidence is conflicting. Studies on "Wilsonian" Cp as com- were detected by starch gel electrophoresis in borate buffers, pared to that from nonnal individuals (e.g. Neifakh et al., pH 9. Shokeir and Schreffler (1970) described two addi- 1972) have not yielded a clear set of differences. Some have tional variants in the sera of American Blacks, Cp New suggested that the disease may result from homozygosity for Haven (Cp NH) and Cp Bridgeport (Cp Bpt). Cp NH mi- different genes in different populations, or perhaps from grated between Cp B and Cp C, and was due to an allele of different modifying genes. @ and Cp called Cp Bpt was seen in a Black cT. 45.6 Transcobalamin il (Tc II) family, and migrated between Cp A and Cp B. Its genetic relationship to the other forms was not as clear. had a Vitamin BIZbinding and transport in serum are mediated higher frequency in American Blacks than did CpC.Shokeir by two types of transcobalamins, called 1 and 11. Tc I is (1971) compared the biochemical and immunological prop- about three times larger in MW than Tc I1 (Hall, 1969). erties of Cp A, AB, AC, ANH, BNH and BptB. AU the Tc II has been purified from human serum and charac- forms were of similar molecular size, and charge differences terized to some extent. Allen and Majerus (1972) obtained were not attributable to differences in sialic acid content. No an affmity purified Tc I1 that had a MW of 53,900 by immunological differences were detected with an anti- ultracentrifugation, and bound one Vitamin BIZper mole human Cp, but there were some differences in the inhibition of protein. It dissociated in SDS to yield two peptides, and of oxidase activity by cyanide or azide. McCombs et al. did not appear to contain carbohydrate. Li~ldemanset al. (1970) have described another variant, Cp Galveston, (1979) also purified Tc 11 by affmity chromatography on detected by slab polyarrylamide gel electrophoresis in pH cyanocobalamin-Sepharose. The MW of the native mole- 9.1 buffers. The variant protein was seen in serum and in cule was about 37,000, but a faint band of MW about ascites fluid, and although an additional allele was sus- 29,000 could also be seen on SDS-poiymyhide gel elec- pected, family studies could not be carried out. trophoresis. The preparation was heterogeneous by isoelec- One, and possibly two, new variants were described by tric focusing. Haus et al. (1979) have found that human Tc Buettner-Janusch et al. (1973) in the Malagasy population I1 seems to be distinctly different immunologically from the of Madagasw. They found that Cp B was the common protein in other species. phenotype, as in other populations. Examples of Cp NH, In 1975, Daiger et al. found polymorphism in Tc I1 by Bpt and Galveston containing phenotypes were observed polyacrylamide gel electrophoresis and autoradiographic among the 405 sera. A previously undescribed phenotype, detection using radiolabelled Vitamin B1, (Daiger et al., called Cp 'Tananarive' (or Cp 'Tan') was observed as 197%). There were some 7 different patterns, with 2-4 A/Tan and Tan/Tan in this population. No B/Tan hetero- bands each. They were stable in a given person and in- zygotes were seen. The failure to find B/'Tan types might herited. Four alleles at an autosomal locus were postulated mean that the allele determining 'Tan' was not codominant to explain the types. Tcf and Tc' were quite common, while with @, but it could also mean that the sample of the pop- Tcl and TC2 were rare. Tcl bands were the fastest migrating, ulation typed simply did not include a person of this type. and Tc' bands, the slowest. Heparinized plasma gave very Sera were observed that typed as 'Cp O' by nonnal testing poor results, perhaps because heparin bii these proteins. procedures. If these were subjected to prolonged activity Similar observations on the polymorphism have been made staining, however, a weak band, which was named 'Cp X', by Fdter-Schrijder and Hitzig (1977) and Frhter-Schriider et eventually appeared. The band appeared to represent Cp af. (1979a and 1979b). They too used polyacrylamide gel and not some other plasma protein, but its relationship to electrophoresis and radiolabelled vitamin BI2 autoradiog- the other Cp variants was not completely clear. raphy for typing, and could distinguish eleven phenotypes, Genetically controlled quantitative variation in Cp is three of which were fairly common. These were called Tc I1 known, the most noted manifestation of it being Wilson's 3-3, 1-3 and 1-1. They were accounted for by two common disease. The first complete description of this comparatively alleles TcP and TcIP at an autosomal locus. Three addi- rare disorder was given by Winin 1912. The disease has tional alleles, TcP, and TcIP and TcIP had lower frequen- been called hepatolenticular degeneration, since it is charac- cies. Family studies were consistent with the five domi- terized in adults by deterioration of the cerebral lenticular nant allele explanation. Occasional families with an in- nuclei and by progressive cirrhosis of the liver. It is also herited Tc I1 deficiency have been described (Hitzig et al., characterized by greatly decreased levels of serum Cp (hypo- 1974), and deficient children usually suffer from megalo- ceruloplasminemia). The major biochemical feature in blastic anemia. Two unusual Tc II patterns m patients with Wilson's disease is the disruption of normal copper metabo- anemias have been described as well (Frilter-SchrMer et a/., lism. The defect is inherited as an autosomal recessive. 1979a) Win's *patients have audied quae men- 45.7 Thyroxine Binding sCiobulin sively, but a full understanding of the molecular pathology does not appear to have emerged as yet. Gitlin and Gitlin (TBG) (1975) and~oulikand Weiss (1975) discussed the subject in Throxine in human plasma may be bound to three dif- detail. There has been an inclination to regard the primary ferent proteins: thyroxine-binding prealbumin (TBPA), Sourcebook in ForemhFIcSeroIogv, Zmmunology, and Biochemistry thyroxine-binding globulin (TBG), and albumin. TBPA was pressure of blood, fatty acid trausport and sequestration, first isolated by Schultze et al. (1962d) and designated and transport of bhbii. Albumin was extensively re- "tryptophan-rich prealbumin" because its electrophoretic viewed by Peters (1975). mobility was faster than that of albumin at pH 8.6. The pro- Serum albumin has been extensively pded and charac- tein has since been more thoroughly characterized. The terized. It is a simple protein, containing one polypeptide amino acid sequence has been worked out (Kanda et al., chain and no carbohydrate. Some fatty acid is apparently 1974), and there are only two Try residues per polypeptide associated with even the purest examples of the protein. chain of 127 amino acids. The x-ray structure of TBPA at a Human albumin has a MW of 66,248 (calculated from the resolution of 2.5A has also been published (Blake et al., amino acid composition). Its sequence has been completely 1974). TBPA can bind the retinol-biding protein (RBP), determined (Brown, 1975), as has that of the bovine protein the -c vitamin A canier in plasma, on a one for one (Behrens et al., 1975). Both sequences may also be found in mole basis. The TBPA exists in a tetrameric structure for Peters (1975). There are 17 intrachain disulfide bridges, the binding of RBP or thyroxine, and complexation with creating a structure with 9 loops. Albumin is well known for TBP apparently has no effect on thyroxine binding (Put- its ability to associate with a great number and variety of nam, 1975b; Cooper, 1978). Cha.racte&ics of retinol- small molecules, including cations, anions (especially fatty binding protein are further discussed by Putnam (1975b). acid, but including hormone and amino acid), drugs and The majority of protein-bound thyroxine appears to be organic dyes. Albumin is a good antigen, and anti-albumin associated with TBG, the thyroxine-binding capacity of is surely a major constituent of the "anti-human serum" TBPA apparently being secondary to its RBP-binding func- prepared in animals (section 16.1). Atassi et uf. (1979) have tion (Cooper, 1978). Thyroxine binding to albumin is non- recently reported that the bovine protein has fie antigenic specific, and apparently not of much physiologic signifi- sites. cance (Putnam, 1975b). Strictly speaking, albumin is not polymorphic in most TBG has not been characterized as well as TBPA, in part human populations. It does exhibit a large number of struc- because the former is present in serum at significantly lower tural variants, most of which are exceedingly rare. A few concentrations (1-2 mg/lOOrd) than that of the latter variants reach significant frequencies in isolated popula- (10-40 mg/100 mP) (Putnam, 197%). Giorgio and Tabach- tions. Albumin variants have been reviewed at length by nik (1%8) developed a purification scheme for TBG from Gitlin and Gitlin (1975). Structural variants are of two human plasma, resulting in a preparation that was homoge- types. One of these is represented by the group that is said to neous by starch gel and polyacylamide gel electrophoresis as give rise to "paralbuminemia", "bisalbuminemia", or well as by ultracentrifugation. The purified glycoprotein "all~albuminemia'~.The tenns are essentially equivalent in had a MW of 58,000. most of the literature, and mean simply that more than one Familial quantitative variation in TBG has been reported. albumin band (usually two) is seen upon electrophoretic Nicoloff et al. (1%4) first noticed the near absence of the separation. The other type is represented by albumins which protein in six members of a family whose members had no tend to dimerize. Quantitative variation is known, and is evidence of Thyroid disease. Nikolai and Seal (1%6 and represented by markedly low albumin levels, usually termed 1%7) observed TBG deficiency in a second family, and "analbuminemid"' could show that the characteristic was X-linked. Other Genetic variation was first noted by Nennstiel and Becht pedigrees showing this same behavior have been noted, and (1957) and by Knedel(1958). They observed two albumins in in one family the TBG deficiency was linked to the Xga serum by electrophoresis, undoubtedly the reflection of blood group (Grant et af., 1974). Shane et af. (1971) found heterozygosity for a variant structural gene. A large number an inherited elevated level of TBG in four generations of a of variants have since been described, especially by Weit- family. The characteristic was X-linked, and other families karnp and collaborators. Normal albumin (the usual kind) is are apparently known which show it as well. often called "A". Many others are named after places. Un- Daiger (1976) described an electrophoretic variant of til the variants can be studied structurally, it will be difficult TBG in Black and Oriental populations. The variant gene to know exactly which ones are really different. Weitkamp occured in about 15Vo of American Blacks. The variant et af. (1%9) described eight distinguishable "types" of locus is apparently X-linked as well, but it is not clear albumin, but they said that each "type" might be a collec- whether it is identical to the locus controlling the quan- tion of a number of different variants. In 1973, Weitkamp titative variations. et al. carried out extensive studies on 23 variants. The most frequent variant among Europeans is called "B", but even it is quite uncommon. Weitkamp et af. (1973) said that the fre- 45.8 Albumin quency of albumin variants may be as low as 1 in 2,000 in Serum albumin is the most familiar and most plentiful of Europe. Several electrophoretic typing systems must be used the serum proteins. Serum nodycontains 42 It 3.5 g/P to distinguish all the different variants. Fine et uf. (1976) ex- of albumin (with a range of 35-50. Albumin represents amined 24 cases of albumin variants in France, and 16 of about 60% of the total serum protein. It has been assigned them were type B. Efremov and Braend (1964) saw only one numerous functions, including maintenance of the osmotic variant in 1,015 Norwegians, and no variants were found by Serum Groups-Other Systems

Persson et al. (1971) in Greenland Eskimos. Some albumin In 1%8, Haupt et al. found a family in which two sibs variants have been found only in isolated populations (Lie- had no 6,-glycoprotein I and whose parents had about half Injo et al., 1971; Frohlich et al., 1978). People with familial the normal amount. Cleve (1%8) then canied out a popula- analbuminemia have been descnied (Bennhold and Kallee, tion study using radial immunodiffusion technique. 94% of 1959), but are very rare. Apart from the family studies by people had concentrations ranging from 16-30 mg/100 mP Bennhold and Kallee (1959), Gitlin and Gitlin (1975) said with a mean of 21, while the remaining 6% had concentra- that only 10 additional cases had been seen. It is not clear tions of 6-14 mg/100 mP with a mean of 10 in their serum. what the exact nature of the defect is in these cases, since The distniution was sharply bimodal in these initial data, "analbuminemics" do make a small amount of albumin. In and it was proposed that P2-glycoproteinI concentration was addition, the known cases may not all have the same genetic controlled by a pair of codominant alleles called BgN and basis. The albumin locus is known to be linked to Gc BgD at an autosomal locus. ~g~Bg~people have the higher (Weitkamp et al., 1%6 and 1970). concentration range, while BgNBgDpeople have the lower Since albumin is not significantly polymorphic in most one. "N" and "D" stood for "normal" and "deficient". populations, it does not provide a useful genetic marker The two sibs of Haupt et al. (1968) with no detectable pro- system for medicolegal problems. Nevertheless, Reinskou tein were presumably uncommon BgDBgD homozygotes. (1%8b) reported that one of two putative fathers in a pater- BgD is thus probably a silent allele. Various nongenetic fac- nity case had an albumin variant which had been inherited tors like sex, age, pregnancy and disease affect the concen- by the child. The man had also passed a Gc2 gene to the tration, however. One family in the material studied by child. The case illustrates the value of rarities when they do Cleve (1%8) did not fit the model. Cleve and Rittner (1%9) occur in a case, and are detected. studied 88 families with 213 children for this characteristic. In 9 families, one parent and about half the children ap- peared to be BgNBgD.But a few families did not fit the 45.9 P,-Glycoprotein I (Bg) model. Parents with nonnal serum concentrations can have This protein was isolated from human plasma by Schultze children who have intermediate levels. Koppe et al. (1970) et al. (1%1). It may be recalled in passing that this group of studied an additional 49 fdes, and again, a few excep investigators isolated a "P2-glycoprotein 11" (Haupt and tions were found. Atkin and Rundle (1974) looked at 381 Heide, 1%5), which was later recognized as being identical people in the British population, and found almost the same with factor B of the properdin system (section 45.2.8). BgN frequency as in the German population (about 0.94). Heimberger et al. (1%4) described a number of the charac- They also found one deficient person. It is assumed by most teristics of the molecule. Liu and Putnam (1975) have re- investigators that the basic genetic hypothesis is correct, and ported similar studies more recently. The MW is about that most of the variation is controlled by a single Bg locus. 40,000, and the molecule appears to be a single polypeptide The situation resembles that of Lpa in some ways (section chain about 300 amino acids long with 8 or 9 dimhide 45.1.3), except that the codominant nature of the Bg alleles, bridges. It occurs in nonnal plasma at concentrations of in contrast to the dominance of Lpa, makes the variation 15-30 mg/100 mi'. It has no known function. somewhat easier to interpret (Cooper, 1978). TheHLA System

SECTION 46. THE HLA SYSTEM AND THE HUMAN MAJOR HISTOCOMPATIBILITY COMPLEX

46.1 Introduction scribed another polymorphism independent of FOUR. It In his Nobel address in 1930, Landsteiner drew a parallel was called "LA" (where "L" stood for "leukocyte" and between hemolytic reactions caused by the transfusion of "A" was supposed to indicate the first locus). From these blood-group incompatible blood and skin graft rejections. begidngs, an understanding of the HLA system has The two phenomena, he said, ". .. .are basically related emerged over the past 15 or 20 years. The evolution of and rest on chemical differences of a similar kind". developments in this field has been characterized by an im- The immunological system is fairly similar in all verte- pressive and very productive degree of international brates, and vertebrates reject foreign skin transplants as well cooperation and agreement. In 1980, Dausset and Snell as react against foreign particles or fluids by the formation shared the Nobel Prize for Physiology or Medicine with of specific antibodies. Rejection of "foreign" skin gmfts Benacerraf, for their work on the "genetically determined may be acute (within weeks), or chronic (within a few weeks structures of the cell surface that regulate immunological or months). In species able to reject acutely, it has been reactions" (Marx, 1980). Gorer, who died in 1x1, would possible to identify a small region on a single chromosome probably have been included in the group, had he lived. in which numerous genes controlling immune reactions are The HLA and genetically amciated systems constitute located at closely associated loci. This genetic region is the most intricate and complex array of polymorphisrns yet called the "major histocompatibility region", or "system", dm'bed. The impressive degree of international coopera- or "complex" (MHR, MHS, MHC). Mammals are in- tion and agreement that has characterized the developments cluded among those vertebrates having a MHC. In animals in HLA serology has surely simplified the nomenclature capable only of chronic rejection, the responsible genes are problems, and provided order in what could otherwise have distributed throughout the genome. Even in acutely reject- been a chaotic nightmare of complicated data. Even given ing species, which have a MHC, however, there are addi- the relatively organized history of the system, the complexi- tional genetic loci whose products are asociated with ties of trivial nomenclature, workshop nomenclature and chronic rejection, and these are distributed throughout the official nomenclature, along with the recognition of such a genome. large number of different specificities, can be quite The pioneering work in the recognition of bewildering to the nonspecialist. Many of the antisera con- major histocompatibility regions was done by Gorer and tain antibodies to multiple spedlcities, others contain cross Snell and their collaborators beginning in the 1930's. Their reacting antibodies, and it has taken time to recognize and work was with mice, and resulted in the definition of the understand all these facts. Not all the spedicities are de- H-2,or major histocompatibility system, in the mouse. Ma- tected by the same methods either. Several different meth- jor histocompatibility complexes are now recognized in the ods have been used to recognize and type the lymphocyte- mouse, rat, chicken, guinea pig, dog, rhesus monkey, chim- defined antigens especially, and the different methods do panzee and in human beings (Albert and Scholz, 1978; not always give exactly concordant results. The sheer extent Giitze, 1977). The H-2 system in the mouse has been an im- of the HLA system, however, makes this systern the best portant model in many respects for the studies responsible genetic marker system yet described. This value has already for unravelling the MHC in humans. been exploited in parentage investigations, but there is little In 1952, Dausset and Nem found antiidies in a mul- work as yet on the determination of HLA antigens in dried tiply transfused patient which agglutinated the leumcytes of blood. some, but not all other people. The presence of leucoag- No review as brief as this one can do justice to the history, glutinins in multiply transfused people was found to be a development and complexities of HLA. Many excellent more general occmence pausset, 1954). In 1958, Dausset reviews are available, however, including those of Mayr described the fim antigen in what was to become the HLA (1969), Walford (1%9), Amos and Ward (1975), Albert system. It was called "Mac" (today, HLA-A2). A second (1976). Albert and Giitze (1977), Gotze (1977), Mdler (1!377), leucocyte polymorphism was found by van Rood (1%2). He Albert and Scholz (1978), Bodmer (197&), Bender (1979) called the responsiile locus "4" (or "FOUR"), and the two and Perkins (1979). Beginning in 1%4, a series of eight in- supposed alleles "4a" and "4b" . Recognition of these ternational conference/workshops has been held periodical- -cities was achieved primarily by statktical genetic ly, at which the specialists have exchanged information and analysis of many antibody-containing sera which had been reagents, and have reached agreements about the status of tested against panels of leucocytes from selected, unrelated the HLA system. A nomenclature committee made up of in- donors. Using similar methods, Payne et al. (1964) de- ternationally recognized specialjsts, and functioning under Sourcebook in Forensic Serology, ~mmunology,and Biochemistry the auspices of the World Health Organbation (WHO) and tisera against them are polyspecXc, although bese defini- the International Union of Immunological Societies (IUIS), tions are operational. Operationally defined anfigens have has met after the last few of these conferences, and set forth frequently been found to be of a compound natFe as more the current standardized nomenclature. The proceedings of refined analysis with more and different antiser& and dif- the eight conferences, held at Durham, Leiden, Torino, Los ferent cell panels, became possible. The revisi* in the Angeles, Evian, Aarhus, Oxford and again in Los Angeles, HLA nomenclature over the years largely rew these (Histocompatibility Testing, 1964, 1%5, 1%7, 1970, 1972, changes in the operational definitions of the antigens. 1975, 1977 and 1980) provide an accurate picture of the pre- The involvement of cell surface antigens in tdplant re- vailing information about HLA during its evolution and up jections was clearly shown in animals, and these antigens to the present time. were known to be present on a variety of tissues and on leucocytes. van Rood et al. (1964) and Dausset et al. (1%5) 46.2 Definition and Function of HLA canied out studies on the relationship of the leukocyte an- Specificities tigens to skin transplantation survival. These studies have been Detailed genetic characterization of the H-2 region in the extended by van Rood et al. (1%6) and by many mouse has come about largely through the use of inbred others, and it was soon clear that HLA represents the major strains of animals developed for the purpose by the pion- histocompatibility complex in the human species (Bach and eering investigators. Human populations are, however, Amos, 1%7). hlghly outbred, and contain a large number of haplotypes 46.3 Genetics and Nomenclature of representing a large number of recombinations. Accord- the Serologically Defined HLA Loci ingly, population and statistical genetic methods have played a major role in the analysis of the human sero1ogical 46.3.1 HLA-A and HLA-B Antigens data. Thus, although there are clear analogies between As noted in 46.1 above, it was clear, at least by 1964, that human and mouse MHC genetics, different methods have there appeared to be two different genetic loci controlling been required to arrive at a detailed understanding of them. the lymphocyte surface antigens. These were orighally It became clear early in the development of HLA serology called "4" ("FOUR") and "LA" and both were multiple that leukoagglutinating, and later cytotoxic, antiies are allelic. The antigens controlled by the multiple alleles were found in the sera of pregnant and parous women (Payne inherited in a simple Mendelian codominant fashion (e.g. and Rolfs, 1958; van Rood et al., 1958). A substantial Payne and Hankel, 1961). Dausset et al. (1%5) proposed a percentage of pregnant or parous women turn out to have in unifying genetic model in which these two loci were seen as their sera lyrnphocytotoxic antibodies against the HLA an- part of the same system (called "Hu-1", but later known as tigens of the child which were inherited from the father "HLA"). Population and family studies indicated that (Wolf, 197 1; Escolar and Mueller-Eckhardt, 1971; Nymand these two loci were autosoma1 and closely linked (Dausset et et al., 1971; Bertrams et al., 1971). In the first years of HLA al., 1%5; Bodmer et al., 1%6; Ceppellini et al., 1%7). The research, it was not possible to find two antima which gave first unequivocal report of crossing over between the LA exactly identical reaction patterns against defined panels of and FOUR locus genes was that of Kissmeyer-Nielsen et a!. donor cells. This behavior was recognized as being in- (1969). dicative of an extraordinary antigen diversity and structural The Nomenclature Committee in 1967 combined the complexity, but it also made the task of sorting out the FOUR and LA loci into a system that was called "HL-A". specificities more difficult. At the beginning, large numbers The different alleles were assigned numbers, in the sequence of cell samples form unrelated donors were tested against of their discovery or of their final definition. Rapid develop- large numbers of different undefined sera. The resulting ments in the field resulted in a large number of newly dis- reaction patterns were then analyzed by a complex series of covered antigens, and of antigens whose status was not im- 2 x 2 comparisons of pairs of antisera, in which each an- mediately clear. In 1970, an important new agreement was tiserum is eventually compared with all others. These reached. Most of the newly discovered antigens, which had analyses, done in a number of different laboratories, were a variety of local and unsystematic designations that were made more manageable with the help of computers. Details quite confusing, were given provisional HL-A numerical of the 2 x 2 comparison method will be found in Cavalli- designations. The provisional designation was indicated by a Sforza and Bodmer (1971). Briefly, such an aaalysis is "w" (or "W"), which stood for "workshop", according to designed to show Mtyor dissimilarity between Bender (1979). The "w" designations were chosen so that members of a pair of antisera, and combinations of these the conversion to a recognized specificity could be ac- analyses leads to defmitions of groups of sera which are complished simply by omitting the "w". In 1976, the highly associated. Associations between sera indicate that Nomenclature Committee set forth the rules which, with they share antibodies directed against the same antigen. Cell minor modification, still form the basis of HLA nomen- samples that react with all the sera of a highly asochted clature (WHO-IUIS Terminology Committee, 1976). The group are inferred to possess the "antigen" corresponding system was to be called "HLA" (the dash in HL-A was to the "antibody" common to all the sera in the group. In dropped). The genetic loci of the system would be desig- this way "antigens" can be defined even when the only an- nated by upper case letters, A, B, C, D, etc., as necessary. The HLA System

The "A" locus would thus be called "HLA-A", the "B" For example, an antiserum produced by immunizing an locus "HLA-B", and so on. Antigens would continue to be HLA-A2,A3,B8,B12 recipient with HLA-M9A3,B7,B12 designated by numbers, assigned by international agree- cells reacted with all HLA-B7 positive cells (as expected), ment. Provisional specificities would continue to be indi- but also reacted with cells positive for HLA-B27 or Bw22, cated by the prefur "w" or "W". These would continue to even though the immunizing cells did not carry these anti- be assigned so that "promotion" to a fully recognized speci- gens. Absorption of this antiserum with cells positive for ficity could be achieved by dropping the "w". A lower case HLA-B27 or Bw22, but negative for B7, removed all the ac- "w" was preferred for the provisional designation, but the tivity. It was thought for a time that this phenomenon was upper case "W" was permitted because it was realized that due to overall biochemical hilarity of the antigens, but the . most computer output devices are incapable of printing idea is contradicted by the existence of anti-HLA-B7 sera lower case letters. The loci previously called "LA" (first which cannot be absorbed by HLA-B27 or Bw22 cells. This locus) and "FOUR" (second locus) would henceforth be kind of cross reactivity was found to be a generalized pheno- called 'HLA-A" and "HLA-B", respectively. For histo- menon among certain groups of antigens, e.g. HLA-Al, rical reasons, specificities belonging to HLA-A and HLA-B A3, All, or HLA-A2, A28 at the HLA-A locus, and are numbered jointly, so that there is no overlap in numbers HLA-B7, B27, Bw22, or HLA-B5, Bw35, Bwl5, B18 at the between them. Thus there are antigens HLA-A1, HLA-A2 HLA-B locus. The patterns of cross react^ were seen not and HLA-A3, but there are no "HLA-Bl", "HLA-B2" or only in antiserums resulting from planned immunhtions, "HLA-B3", and so forth. There are certain criteria for the but in those obtained from unselected multiparous women recognition of -cities, and for the recognition of provi- as well. The groups of cross reacting antigens, it may be sional specificities. These have been somewhat refmed as noted, always involve the products of the same locus. Tim- time has gone along. Each major workshop has resulted in tion experiments with antisera directed at a group of cross the recognition of some new provisional specificities, and reacting antigens indicate that most of them have one the conversion of previously provisional spedicities to Nly "main" specificity that is relatively high titered in com- recognized ones. parison with the activity toward the other spec$%Aies. This By the time of the sixth workshop in Aarhus in 1975 behavior has led to the subdivision of some antigenic (Histocompatibility Testing, 1973, there were 8 fully recog- specificities into what Bender (1979) called "subtypes". The nized HLA-A specificities and 12 additional "w" or provi- terms "broad" and "narrow" qxdicity are also used in sional ones. There were eight specificities at HLA-B and 12 this context. Thus, HLA-Aw23 and Aw24 are regarded as provisional ones (WHO-IUIS Terminology Committee, "subtypes" of HLA-A9. Another way of putting it would 1976). In the next report (WHO-NISNomenclature Com- be to say that A9 is the "broad" qmifkhy,while Aw23 or mittee, 1978) 10 established and 10 provisional specificities Aw24 are "narrow" ones. Similarly, HLA-A25 and A26 are at HLA-A, and 12 established and 21 provisional ones at subtypes of HLA-A10. The divisions of HLA-A9 into HLA-B were listed. Most recently (WHO-IUIS Nomen- Aw23 and Aw24, or of A10 into A25 and ,426, are referred clature Committee, 1980), there were no changes in the to in the literature as "splits", and there are quite a number HLA-A locus antigens. At HLA-B, there were nine new of them known at HLA-A and HLA-B. The nomenclature provisional specificities, HLA-Bw55 through Bw63. rules allow for specification of the broad specificity in It should be noted that the HLA-A, HLA-B and HLA-C parentheses next to a narrow one, e.g. HLA-Aw23(9). (see below) antigens are defined principally by serological reactions, and are sometimes referred to as "SD" 463.3 Formal genetics and chromosomal locabtion ("serologically detected" or "serologically defined") anti- times. Many population and family studies have been carried gens. The loci have been called SD1, SD2, and SD3 at out on the HLA system (e.g. Svejgaard et ul., 1970; Mayr, In section 46.4 we will briefly discuss HLA antigens that 1971 and 1977; Colombani and Degos, 1972; Spielmann et were defmed originally by other than serological reactions. al., 1974; Greenacre and Degos, 1977). A child inherits an HLA hapiovpe from each darent. The haplotype is, essen- 46.3.2 Cross reactWy of antisera and "splits9' tially, the series of HLA genes on the chromosome that is in- The phenomenon usually called cross reactivity (the term herited, and consists of an HLA-A allele, an HLA-B allele, is not altogether accurate according to Albert and GWe, and one allele each from the additional loci to be discussed 1977) has been a major problem in fully understanding below. The sum of the two inherited haplotypes is the in- HLA serology. Cross reactiViry was recognized by Svej- dividual's genotype, although it is not always possible to gaard and Kissmeyer-Nielsen in 1%8, and is a fairly general discern the genotype from the phenotype, since one pheno- phenomenon among certain combinations of HLA anti- type may be dictated by a number of different genotypes. If gens. When immunization experiments were done in which the paternal chromosomes are designated P/P' and the the donor differed from the recipient for only one known maternal ones M/M1, four types of children are possible: HLA antigen, it was observed that the dtingantiserum P/M, PM', P'/M and Pf/M',where P, P', M and M' reacted not only with cells positive for the antigen that was represent haplotypes (or chromosomes). If the HLA-A, -B different (and to which the antibodies were expected to be and -C loci are all considered, up to six different alleles directed), but also with cells positive for other antigens. could occur in a person, e.g. HLA-M,A3,B12,Bw35,Cw4, Sourcebook in Forensic Serology, Immunologv, and BiochemUry

Cw5, representing the haplotypes HLA-A2, B12,Cw5 and dere et al., 1975; Bijnen et al., 1976), however, (about 0.8%) HLA-A3,Bw35,Cw4. Haplotypes cannot be determined that once linkage disequilibrium is established, it will take from phenotypes without doing family studies. Typing many generations to reestablish equilibrium again. results often yield information about five, four or even Pious and Soderland (1977) estimated the mutational rate fewer alleles, and it is not possible to distinguish between for the HLA-B locus, and said that their data were consist- homozygosity for an allele, and the presence of a ent with most of the variants having arisen by mutational serologically undefined allele (the latter often called a events. "blank"). In a case, for example, of an HLA-A2,A9,B12 In 1970, Lamrn et al. found a hint of linkage between phenotype, the genotype could be A9,B12/A2,B12 or PGM, and HLA. Somatic cell hybrid studies then enabled A9,B12/A2,BX or A9,BX/A2,B12, where BX represents a Jongsma et al. (1973) to assign PGM, to chromosome 6. serologically undefined allele at HLA-B. The probable HLA,PGM, and SOD-B were then shown to be syntenic by genotype of the person could be calculated on the basis of further somatic cell hybrid studies (van Someren et al., haplotype frequencies. The situation is reminiscent of Rh 1974). Mayr et al. (1975) confirmed the PGM3:HLA system genetics (section 22). Suppose the HLA-A2,A9,B12 linkage. A peculiar inversion allowed the absolute assign- person in the example above were a mother with three ment of HLA to chromosome 6 by Lamm et al. (1974). children, all of whom had inherited an HLA-A9,B12 Thus, the human MHC,which includes the HLA loci, is on haplotype; this result would show that she had an chromosome 6, and is linked to PGM, as well as to GLO HLA-A9,Bl2 chromosome, but it would not distinguish and Bf (Weitkamp, 1976) and to a number of the poly- between the genotypes HLA-A9,B12/A2,B12 and HLA-A9, morphic complement component loci (section 45.2). Albert B12/A2,BX. In surveying populations, and even families, and Giiue (1977) said that the likely order for all the loci therefore, statistical methods must often be used to estimate clustered on the short arm of chromosome 6 is PGM,. . .. . genotypic and haplotype frequencies. There are fairly com- GLO. . .. C2. .HLA-D. .Bf. .HLA-B. .HLA-C. .. plex methods for estimating gene frequencies (e.g. Yasuda HLA-A, with C4 somewhere near Bf or HLA-B. and Kimura, 1968) as well as comparatively simpler ones (e.g. in Albert and Giitze, 1977; Bender, 1979). AU the 46.3.4 The HLA-C locus methods give generally similar results. Sandberg et al. (1970) found an antiserum which detected As with other blood group, enzyme and serum group an antigen that would not fit into the two locus model. A polymorphisms, HLA gene frequencies are very differently third locus, called "AJ", was postulated and was soon distributed in different racial and ethnic groups. Summaries proven to exist (Solheirn and Thorsby, 1973; Mayr et al., may be found in Albert and Gotze (1977) and Bender 1973). It has also been called the third serologically defined (1979). Among the most frequent HLA-A alleles in Euro- locus, or "SD3", but is now known as HU-C. The pean Caucasians are HLA-A2, Al, A3 and A9, in African HLA-C locus is closely linked to HLA-A and -B, and Blacks, AWN is frequent along with A2 and A9, while in crossovers between HLA-A and -C alleles and between Japanese, A9 is very frequent with A2 and All somewhat HLA-B and -C alleles have been reported (Liiw et af., 1974; less so. Similar differences are seen in the alleles at other Hansen et al., 1975; Bijnen et al., 1976; Waltz and Rose, loci. The most common Caucasian haplotypes are HLA- 1977). Al,B8, A2,B12 and A3,B7. The third locus antigens have been studied in part by a Many of the HLA haplotypes occur much more fre- special technique called "capping" (Taylor et al., 1971). A quently than would be expected on the basis of the gene fre- number of cell surface proteins, including HLA antigens, quencies for the alleles involved (e.g. Hiller et d., 1978). In can, after binding to specific antibodies in the absence of the case of two loci like HLA-A and HLA-B, linked but complement, redistribute to form aggregates on the cell sur- separable by recombination, it is expected that large face. They may then form a "cap" at the cell pole numbers of recombions between the loci will eventually (capping). The aggregated surface proteins may then be bring the alleles into random association with each other, taken up by the cell, or shed to the medium. Lymphocytes i.e., into "linkage equilibrium". The situation that is actu- . treated in this way show a temporary loss of the antigen in- ally found in some haplotypes, where associations between volved. The f~ndingby Bernoco et al. (1972) that HLA certain alleles differ significantly from what would be ex- specificities can be individually capped provided a clever pected on the basis of random association, is called "linkage method for the study and characterization of these antigens. disequilibrium". Liage disequilibrium can be found at At present, there are 8 alleles at HU-C (WHO-NIS any pair of linked loci. Some alleles at the closely linked loci Nomenclature Committee, 1980). They are designated determining salivary polymorphic proteins (see in section HLA-Cwl through Cw8,and the first five are considered 47) exhibit this phenomenon, for example. Details of link- fully established specificities. Because of the close associa- age disequilibrium analysis may be found in CavaUi-Sfona tion of the complement component polymorphic loci C2 and Bodmer (1971). The reasons for the dhquili'bria are and C4 with the HLA locus region, and the possibility of unknown, and a number of different explauations are pos- resulting ambiguity and confusion in the nomenclature be- sible. The recombination frequency between HLA-A and tween the alleles of C2,C4 and HLA-C,the committee has HLA-B is sufficiently low (Svejgaard et al., 1971; Belve- recommended retaining the "w" designations for all The HLA System

HLA-C alleles for the time being. Of the antigens defined known HLA loci. Families were soon found (Amos and thus far, HLA-Cw3 and Cw4 are the most frequently occur- Yunis, 1971; Yunis and Amos, 1971; Mempel et al., 1972) in ring in Europeans. whose members the HLA types and MLC behavior pro- 46.4 Lymphocyte Defined Antigens-A vided convincing evidence for a separate "MLC" locus, closely linked to HLA-B. Further HLA Locus Recognition that the MLC tests were detecting antigens As has been noted, the HLA-A, -B and -C locus alleles coded for by a separate locus led to efforts to devise and antigens were defined by serological reactions, primarily methods for identifying different individual allelic products. by the lymphocytotoxicity test (discussed further below) MLC stimulation is a measure of difference, but not of the using antibodies directed at the specZcities. The antigens exact type of difference, between the antigens of the cell coded for by these alleles have thus been referred to as populations. Individuals can be selected, however, who are "serologically defined" ("SD"). homozygous for certain MLC alleles. Their cells, homozy- Another group of antigens belonging to HLA, but de- gous typing cells (HTC),do not provoke stimulation in one- tected by different methods, has also been found. These way MLC if the responding cells carry the same MLC allele. were detected by cellular reactions involving lymphocytes, If a difference exists, however, a stimulation reaction is and are referred to as "lymphocyte defmed" ("LD"). The seen. The HTC technique was developed by Mempel et al. distinction may soon be mainly methodological, since it ap- (1973), Dupont et al. (1973), JBrgensen et al. (1973) and pears that the "LD" antigens can be detected serologically van den Tweel et al. (1973). Another procedure for typing now. MLC determinants was developed by Sheehy et al. (1975) When lymphocyte suspensions from unrelated individuals and Sheehy and Bach (1976), among others, and is called are mixed and incubated in culture, the cells may enlarge "primed lymphocyte typing" (PLT). This method is based and transform into dividing blast cells (Hkschhorn et al., on the observation that stimulated T lymphocytes kept in 1%3; Bain et al., 1%4). The dividing cells must replicate culture for a matter of days will give a strong secondary (or their DNA, and the lymphocyte stimulation reaction can "second set") stimulation response when stimulated by cells thus be followed by monitoring the incorporation of radio- possesing the same MLC determinant as the stimulatii labelled DNA precursors (e.g. 'H-thymidine). One of the cells in the original culture ("priming" cells). two cell populations can be inactivated by treatment with Following the Aarhus workshop (Histocompatibility radiation or mytomycin so that they cannot divide and pro- Testing, 1975), the nomenclature committee designated the liferate, but can still stimulate the untreated cells in the "MLC" determinant locus as "HLA-D" (WHO-IUIS Ter- culture to do so. This f~ndingprovided the basis for a one- minology Committee, 1976). The locus had previously been way or unidirectional stimulation test (Bach and Voynow, designated variously as "MLR-Sl", "LD-1" and 1966). A two-way mixed lymphocyte culture could, there- "MLC-1". Six specificities were provisionally recognized at fore, be analyzed as the sum of the two one-way reactions. that time owl through Dw6). There are presently 12 The rationale for mixed lymphocyte culture studies was the HLA-Dw specificities (WHO-IUIS Nomenclature Com- finding (Bach and Hjrschhorn, 1W)that the degree of blast mittee, 1980). Further infonnation on cellular typing may be transformation observed in the mixed culture was related to found in the reviews by Albert and Gotze (1977) and Bender the degree of incompatibity for transplantation antigens, (1979), and in Bach et al. (1973, van Rood et al. (1977) and and an in vitro measure of transplantation incompatibility Bradley and Festenstein (1978). was clearly desirable. The test was developed (Bach et al., It was found by Ceppellini et al. (1971) that the MLC 1967) into what is now called the "mixed lymphocyte reaction can be inhibited by certain HLA antisera, and this culture" ("MLC"). Absence of stimulation in this pro- observation provided the beginning of a serological def~- cedure is interpreted to mean identity of the "MLC" an- tion of the B4alloantigens. The antibodies in the HLA tigens in the two kinds of cells. At first, MLC was thought antisera causing the inhibition are not HLA antiiodies, to be just an alternative way of typing HLA antigens, because they cannot be absorbed by appropriate platelets. because Bach and Amos (I%T observed that the lympho- They can be absorbed out by appropriate leucocytes, cytes of HLA-identical siblings did not stimulate each other however, and eluates from these leucocytes are also active in MLC, while those of HLA-nonidentical siilings did so. It (Revillard et al., 1973; van Leeuwen et al., 1973). These an- was soon found, however, that the cells of unrelated HLA- tibodies have been better characterized by van Leeuwen et identical individuals stimulated each other in MLC al. (1973), and are detecting serologically antigens on B (Kissmeyer-Nielsenet al., 1970; ScheUekens and Eijsvoogel, lymphocytes that are identical or very similar to the "MLC" 1970; ScheUekens et al., 1970; and others). These observa- determinants. These antigens are also referred to as "Ia" tions could be understood in two different ways: (1) that the antigens, by analogy to the findings in the mouse. Sera con- HLA antigens were still "heterogeneous", i.e., that avail- taining strong HLA antibodies are often found to contain able typing serums did not allow for complete type deter- strong Ia-antibodies as well (Jones et al., 1975). The com- mination; or (2) that the stimulation in MLC was not being plex subject of human Ia antigens has been reviewed by brought about by HLA antigens as such, but by products of Wernet (1976), Barnstable et al. (1977), Bodmer (1978b) and one or more "MLC" genes at a locus closely linked to the Winchester and Kunkel(1979). It is not yet completely clear Sourcebook in Forensic Serologv, Immunology, and Biochemistry whether the same polymorphic antigens are being detected tivity using large panels of highly selected cells of well de- by the cellular (HTC and PLT) techniques, and the Ia- fined specificity. The well known cross reactivity of HLA serological teas. The WHO-IUIS Nomenclature Committee antisera, and the fact that many serums contain a number of (1978) has assigned the symbol "DR" (for "D-related") to different antibody specificities, can cause serious difficulties the HLA-D antigens detected serologically. The HLA-DR of interpretation in inexperienced hands. As one example, designations correspond to the relevant HLA-D designa- the cross reactivity of HLA-B7 and HLA-B27 can lead to tion. At present, there are six fully recognized HLA-DR typing errors (Joysey and Wolf, 1978; Larsen, 1979). A specificities, and an additional four provisional ones tissue type cannot be reliably diagnosed until it has been (WHO-IUIS Nomenclature Committee, 1980). confmed by a battery of well authenticated antisera. Like- 46.5 HLA Testing and Typing wise, antisera must be thoroughly evaluated against large panels of cells of selected known specificity. Panels for Procedures evaluating most antisera can be smaller in number if they Methods for HLA typing are unlike any of the methods are highly selected (Kissmeyer-Nielsen and Kjerbye, 1972). used for the typing of blood or serum groups or polymor- Since around 1%5, the NIH Wational Institutes of phic enzymes. I-ILA antigens are routinely typed on Health, Public Health Service, U.S. Jkpartment of Health leucocytes, and HLA typing generally tends to be done in a and Human Services] Serum Bank has been a major coor- relatively few specialized laboratories. Typing of HLA-A, dinating center for the exchange of HLA antima. Catalogs -B and -C antigens is carried out fairly universally using of these reagents are produced periodically, and the sera are some variation of the complement mediated lymphocyto- available to quawed investigators (see in Amos and Ward, toxicity test on peripheral blood lymphocytes. Special pro- 1975). Many exchanges of serums and cells take place cedures which were briefly discussed in section 46.4 are used among specialized HLA testing laboratories as well. These for the defintion of HLA-D and -DR spedicities. exchanges, and the fairly frequent international workshops, The microdroplet lymphocyte cytotoxicity test introduced have helped considerably in the standardization of typing by Teresaki and McClelland in 1%4 has become universally reagents, techniques, and nomenclature. Because of the accepted as the method of choice for HLA antigen typing. relative scarcity of thoroughly evaluated reagents, and the The principle of the test lies in the fact that lymphocytes complexities associated with accurate HLA typing, this containing a particular antigen will, after reaction with work will probably continue to be restricted to a relatively antibody to that antigen followed by the addition of small number of speciaLized laboratories. It mybe noted complement, exhibit a cytotoxic reaction detectable by the that the microdroplet lymphocyte cytotoxicity test is now penetration of the cell by an added dye. Test results are applicable to the determination of some HLA-D specifici- evaluated microscopically. If the lymphocytes being tested ties as well (Teresaki et al., 1978a). lack the antigen to which the antisenun is directed, there is no cytotoxic reaction, and the added dye is not taken up by 46.6 Biochemical Studies on HLA the cells. This procedure can be employed using very modest Antigens amounts (pl) of cells, antisera, and complement (rabbit The HLA antigens are membrane-associated, and the serum is usually the complement source). The procedure has usual problems connected with efforts to dissociate and been refined and modified over the years (Mittal et al., 1%8; study membrane bound proteins have faced biochemists Teresaki et al., 1978a). A detailed review of the develop- engaged in HLA antigen characterization. Solubilization ments will be found in Teresaki's Philip Levine Award lec- and isolation of these molecules has been carried out using a ture (Teresaki et al., 1978a). variety of techniques and extraction media, including mech- There are a number of technical problems eedwith anical disruption, hypotonic lysis, freezing-thawing, various HLA typing that must be appreciated if correct results are to salt or proteolytic enzyme (especially papain) treatments, be obtained. These problems are discussed by Teresaki et al. and detergent treatments. The development of continuous (1978a) and by Joysey and Wolf (1978). In addition, the lymphoid cell lines derived from normal donors has contrib- complexities and idiosynchrasies of HLA system antigens uted to the biochemical studies by providing relatively large must be taken into consideration. Skill and experience in amounts of genetically uniform cellular material. Reviews lymphocyte typing, and the use of carefully and thoroughly of the biochemical work may be found in Albert and Gotze evaluated reagents, are both essential. (1977), Albert and Scholz (1978) and Barnstable et al. Efforts to standardize the lymphocytotoxicity tests used (1978). for routine HLA typing have been quite successful, al- There are conflicting reports in the literature about the though there are some variations in procedure in different carbohydrate content of purified HLA antigens. Some of laboratories (Teresaki et al., 1978a, Ray, 1979). Small dif- the solubiition techniques apparently yield preparations ferences in technique can significantly affect the sensitivity which lack detectable carbohydrate. The consensus appears of the test, however (Joysey and Wolf, 1978). to be that there is some carbohydrate awxiated with the na- The biggest problems in HLA serology probably have to tive antigens, but its exact composition and the mode of its do with the antisera. Antisera must be procured, and then attachment to the protein moiety have yet to be established. exhaustively evaluated in terms of specificity and cross reac- Isolated HLA antigen molecules have a general structure TheHLA System consisting of one heavy and one light chain. The chains are resemble mouse and guinea pig Ia antigens. Snary et al. not covalently bound to one another. The heavy chain is the (1977) have presented a model for the structure of these one coded for by the HLA locus gene, qd contains the molecules with which the data of Klareskog et al. (1977) ap alloantigenic portion of the molecule. The MW of the heavy pear to be consistent. The molecule is a glycoprotein con- chain varies from 34,000 to 55,000 depending on the method sisting of two polypeptide chains of MW about 33,000 and used to solubilize the antigen. Strominger et al. (1977) 28,000. The chains are not covalently linked, and both of presented a model of the native structure of HLA-A and -B them have associated carbohydrate. The 33,000 peptide is antigens based on studies in cultured lymphocytes. The suggested to be the one coded for by the HLA-DR genes, N-terminal end of the heavy chain is the one that protrudes and accordingly, the one containing the dotypic site. Syn- to the outside of the cell membrane. This protruding part of thesis of the 28,000 peptide might well be controlled at a the chain appears to represent a large portion of the whole completely different locus. molecule. A hydrophobic portion of the heavy chain was postulated to occupy the membrane itself, with a compara- 46.7 Medicolegal Applications tive short portion of the chain (the C-terminal end) pro- truding to the inside of the cell membrane. There are two in- 46.7.1 Disputed parentage trachain disulfide bridges in the heavy chain. Amino acid The use of the HLA polymorphism in disputed parentage composition studies on a number of solubilized HLA an- represents its only significant medicolegal application at the tigens from various HLA-A and -B types indicate consider- present time. The extraordinary extent of the polymorphism able simihrity. It appears that the amino acid residue at and its widespread distribution in populations make HLA position 9 is locus-specific (Phe in the HLA-A products; the most singularly powerful system for parentage investi- Tyr in the HLA-B ones), but the nature of the differences gations. This fact may be appreciated by examining Table giving rise to the alloantigenic specificities have not yet been 18.1. The probability of exclusion of a falsely accused father cMied. Most of the data suggests that the carbohydrate using HLA typing is very high, and values ranging from moiety is not involved in determining antigenic structure or about 80010 to over 90% are quoted by various authors. The specificity. PE is dependent, of course, on how many specificities can The light chain (MW 11,600) of the HLA-A and -B mole- be typed, and on the distribution of haplotypes in the pop cules is associated with the N-terminal end of the heavy ulation in question. The system is so powerful in making chain, according to the structure proposed by Strominger et distinctions in the population because of the large number al. (1977). The light chain is apparently not involved in of haplotypes and their widespread distribution in the determining doantigenic specScity, and the light chain has population. With 17 alleles at HLA-A and 28 at HLA-B, been identified as 82-microglobulin. 8, microglobulin is Bender (1979) said that 17 homozygous and 136 heterozy- found in small amounts in various body fluids and was first gous A types, and 28 homozygous and 378 heterozygous B isolated and purified by Berggird and Beam (1%8). It has types were possible. Since the antigens are inherited as 100 amino acid residues and a single -de bridge formed haplotypes, it must be considered that there are then 476 dif- by Cys residues at positions 25 and 81. There is no carbo- ferent A,B haplotypes which allow for about 113,500 com- hydrate in the purified preparation. There is some sequence binations! The most frequent phenotype occurs in only homology of 8,-microglobulin with the constant region of about one of 100 persons. Bender (1979) also said that at the IgG heavy chain (Smithies and Poulik, 1972). 8,-micro- least two-thirds of the German population have two dif- globulin synthesis is controlled by a gene on chromosome 15 ferent A and two different B alleles (a "full house" in labo- (Goodfellow et al., 1975; Zeuthen et al., 1977). and its struc- ratory jargon). tural gene is completely separate from the HLA complex Application of HLA typing to parentage cases has been locus, therefore. practiced for some years, and application of HLA to pater- Although there are fewer detailed studies on the HLA-C nity cases is discussed by Mayr (1971 and 1974), Soulier et antigens than on the -A or -B molecule., the data indicate cd. (1974), Spielmann and Seidel (1974). Spielmann et al. that at least the general structure is very similar. (1974), Speiser (1975) and Miller, in AABB (1978). Calcula- Berg (1971b) reported the curious observation that there tions of PE were discussed by Mayr and Pausch (1975), and are amino acid compositional similarties between HLA an- the principles underlying calculation of the probability of tigens, the Lp(a) protein and the LDL of serum (section paternity where no exclusion is obtained were given by Mayr 45.1). Charlton and Zmijewski (1970) found that HLA-A7 (1972). Hummel and Conradt (1978) noted that very low people had a soluble HLA-A7 in their plasma which oc- probability of paternity values could be obtained in some curred as a low density lipoprotein. Orr et al. (1979) recently cases, even when the PE value for exclusion of a falsely ac- noted that an 88 amino acid residue fragment of the cused father was high, and they tended to take the view that HLA-B7 heavy chain showed signifit structural homol- the probability of paternity calculation was the only mean- ogy with the constant domains of immunoglobulins. ingful one in a given case. The structure of the HLA-DR antigens (Ia-antigens) is In this country, Dr. Teresaki has been a leader in the ap- somewhat different from that of the HLA-A, -B and -C plication of HLA typing in paternity cases (Teresaki, 1978; molecules. The data thus far indicate that the DR antigens Teresaki et al., 1978b). If ABO and Hp are typed in addition Sourcebook in Forensic Serology, Immunology, and Biochemistry to HLA, probabilities of paternity are at least 95% in the used in the tests. Newall (1979) evaluated two different ver- non-exclusion cases. In a number of states, blood grouping sions of microlymphocytotodcity inhibition for the typing evidence is not admissable unless it is exculpatory (i.e. of HLA-A2 and B5 in dried bloodstains of varying age. The results in excluding the putative father), but this situation is two-stage extraction-absorption test was not found to be changing somewhat as members of the legal community very satisfactory. Better, more reliable results were seen with become more aware of the present status of blood testing a one-stage procedure. Antisera varied in their ability to give (e.g. Seider, 1980). Lee (1975) surveyed 24 countries, and unequivocal typing results, and stains from different per- found that HLA typing was used in paternity cases in just sons that contained the same antigen gave differing degrees under half of them. In this country, it is one of the seven of inhibition, especially with older stains. Stains up to 10 systems recommended by the joint AMA-ABA Committee weeks old could be typed correctly, however, if three dif- (Joint AMA-ABA Guidelines, 1976), along with six blood ferent antisera were used in each test. Hodge et al. (1979 and group systems (ABO, Rh, MNSs, Kell, Duffy and Kidd). 1980) recently investigated HLA-A1 typing in 4 cm long For some reason, this committee chose to overlook isoen- bloodstained threads using inhibition of microlym- zyme and serum group systems altogether. Polesky and phocytotoxicity procedures. Three anhwere used in Krause (1977) found in their survey of American labora- parallel. Most stains could be typed correctly even in stains tories that about 17% of AABB referene laboratories could over 100 days old. StiU, a few false positive and negative do HLA testing, but less than 2% of other laboratories typings were observed in the samples. The well known surveyed had this capability. HLA-A1-A11 cross reaction could be seen in The applicability of HLA typing in some uncommon HLA-A2,All stains with one of the anti-HLA-A1 sera situations has been described. Speiser et al. (1974) discussed known to be cross reactive with HLA-All. While these in- the situation in which the putative father could not be itial results are encouraging, more groundwork remains to tested. Teresaki (1978) had a case in which the mother could be done before this powerful polymorphic system can be ex- not be tested. In both situations, exclusions could be ob- ploited in bloodstain typing. tained. Two very rare cases of so-called superfecundation Hillman and Shaler (1981) have recently explored pro- have recently been diagnosed by HLA typing. In both, cedures for the recovery of leucocytes from dried blood- fraternal twins were shown to have different biological stains. Using special extraction techniques, relatively high fathers CTeresaki et al., 1978c; Bertrarns and Preuss, 1980). recoveries are possible. The leucocytes thus isolated from bloodstains might enable HLA typing to be canied out 46.7.2 HLA Antigen typing m dried blood much more readily than is possible with whole bloodstains. Only a few studies have been wried out on bloodstain Such leucocytes can be used for cytologic sexing studies (sec- typing. Rittner and Waiyawth (1974) obtained a high per- tion 48). and for the determination of genetic markers that centage of correct results using an inhibition of lymphocyto- are expressed in white cells but not in red cells. toxicity test. They then (1975) compared rnicrolymphocyto- It may eventually be of medicolegal interest that HLA an- toxicity and microcomplement Man procedures on tigens appear to be present in semen (Singal et al., 1971; bloodstain material, and concluded that the latter would be Singal and Berry, 1972) and on sperm cells (Halim and better suited. A number of -cities could be reliably Festenstein, 1975; Festenstein et al., 1977). detected, but relatively large amounts of dried blood were Salivary Polymorphic Proteins

SECTION 47. POLYMORPHIC PROTEINS OF HUMAN SALIVA 47.1 Introduction was covered in section 11.2. Pini Prato (1970) noted two Saliva contains a variety of proteins and enzymes, many alkaline phosphatases in human saliva following poly- of which it shares with other body fluids and tissues. Some acrylamide gel electrophoresis. The relationship of these of the antigens, enzyme and serum group system proteins isozymes to the other human alkaline phosphatases (section that exhibit genetic polymorphism and occur in saliva as 37.2) is not yet clear. Detection of the PGMI isozymes in the well as in other tissues and fluids have been discussed else- cellular fractions of saliva has been reported (section where in this book. Soluble ABH and Lewis blood group 27.4.3), although PGM should probably not be regarded as substances (sections 19.8.1, 19.8.3.1 and 20.4) are found in a salivary enzyme as such. The immunoglobulin markers of saliva as well as in many other body fluids and tissues. Lie- the Gm and Km system can be detected in saliva, and this wise, the antigen Sda (25.8) can be found in saliva. Some im- matter was covered in section 44.6.3. munological studies which included identification of some Several systems of salivary proteins and enzymes, which of the proteins of saliva were dixussed in section 11.4. have not yet been discussed, exhibit genetically controlled Saliva contains a few proteins which appear to exhibit in- variation by electrophoresis or isoelectric focusing. The rest herited variation (presence or absence) that have been de- of section 47 is devoted to a discussion of these systems. tected by their reactions with various lectins. One of these, Most of the work is comparatively recent, and only a hand- which was called "L", was detected by its reaction with a N of laboratories appear to be engaged in these studies. lectin from Lotus tetragonolobus (section 19.8.3.1). The most comprehensive review of this material was given In 1%4, Niswander et al. detected a component in saliva by Azen (1978). I am indebted to his review in helping to by immunoelectrophoresis and reaction with a rabbit anti- organize some of the material in this section. A useful and serum raised against human parotid saliva. It was called comprehensive review of methods employed in studying "SC," (for "salivary component" migrating most anodally salivary polymorphism will be found in Tan and Teng by immunoelectrophoresis at pH 8.6). The component (1979). migrated in the "albumin" position, but was not albumin, 47.2 Polymorphic Salivary Protein nor was it identical to or associated with ABHLea. It did not Systems occur in serum. It was inherited, but appeared to be subject to post-translational modification, and the inheritance pat- 47.2.1 Acidic protein (Pa system) tern was not fully clear. The relationship of SCl to the pro- Polymorphism in an acidic protein in saliva (as against teins discussed in section 47.2 is not clear. Two more saliva the basic ones of the Pb system dixussed below) was de- antigens were detected by Balding and Gold (1973). They scribed by Friedman et al. (1972), and studied further by were studying bacterial hemaggluthim from Clostridium Friedman et al. (1975). The Pa protein, which is either pres- botulinum types C and D. It was found that some but not all ent [Pa( +)I or absent Pa(-)I was detected using acid urea salivas from different people would inhibit these reactions. starch gel electrophoresis, and a stain designed to detect The antigen inhibiting the type C agglutinin was called CbC, argninine-rich proteins. Other electrophoretic systems were and that inhibiting type D activity was called CbD. The used to study Pa further, and to compare it with other characteristic was inherited, and the responsible genes, salivary proteins. Pa can be typed in submandiiular or called salCbC and salaD, respectively, had frequencies of parotid saliva, but something like half the whole saliva about 0.73 and 0.27 in a Caucasian population. In 1975, samples were found to be unsuitable for typing, even if Wiener and Moon described an antigen called "Cl" on red frozen immediately after collection in preparation for cells and in saliva. The antigen was defined by a lectin lyophilization. Saliva preparations are usually frozen, and prepared by absorbing extracts of the seeds of Clerodendron lyophilized, and the lyophilizate later reconstituted for elec- trichotomum Thumberg (from Korea) with selected group 0 trophoretic study (Friedman et al., 1975). The locus con- red cells, and was said to be asociated with the ABHLe trolling the Pa protein was designated Pa, and the protein macromolecule. was clearly inherited as an autosomal dominant as indicated Saliva contains a number of enzymes, the most important by studies on a number of families. Friedman et al. (1975) of which for forensic purposes is probably amylase (at least call the phenotypes Pa(+) and Pa(-) and the genes Pa* at the present time). The use of amylase as an identification and Pa-. Azen (1978) calls the phenotypes Pa 1 and Pa 0, marker was discussed in section 11.3. The polymorphism of and the genes Pa1 and Pd, respectively (because there is the salivary amylase (AMYI), as well as that of pancreatic now a "Po"').Pa( -) x Pa( -) rnatings did not give rise to amylase (AMY*) was discussed in section 37.3. The use of any Pa(+) children. Gene frequencies for Pa- (Pa1)were salivary alkaline phosphatase as an identif~cationmarker approximately 0.21, 0.14 and 0.42 for American Whites, Sourcebook in Forensic Serologv, Imnnmologv, and Biochemistry

American Blacks and Orientals, respectively. In 1977, Azen ucts. The other X protein is always associated with a prod- described a rare variant of Pa, called Pa 2. Pa 2 is a separate uct of Prl. These findings led Azen and Denniston (1974) to protein which is seen on the typing gels, and was accounted suggest that there were at least two additional alleles at the for by a rare allele Pd. The finding of the P42 accounts for Pr locus, called Pr' ' and W'.The data also suggested that the change in nomenclature noted above. the gene locus responsible for the Pa 1 protein, Pa, and the Friedman and Merritt (1975) partially purified the Pa Pr locus genes have undergone occasional recombination. (Pa 1) protein, and found that it was rich in Pro, Glu and In addition, all Pr 2' individuals are Pa 1, and most Pr 1' Gly, low in Thr, and that it had no Tyr or Cys. The pI was people are Pa 0, but an occasional Pr 1' individual is Pa 1. 3.9-4.5. Azen and Denniston (1974) and Azen (1977) found, This suggests a ~ig~cantlinkage disequilibrium between however, that Pa 1 was a disulfide-bonded dimer, using Pr and Pa. There is now no doubt about the close linkage disulfide bond splitting agents and 14C-iodoacetamideto between the two loci (Yu et al., 1978). identify the products. Friedman and Merritt (1975) men- Azen and Denniston (1974) found another pair of poly- tioned that there were some assodated acidic proteins in morphic proteins, called "Db" (for "double band"). These, saliva, called "Pa II", to distinguish them from "Pa I", the like Pr, were detected by negative staining with dimethoxy- polymorphic Pa. benzidine after electrophoresis on polyacryiamide gels. The double bands, one of which is between Pr 2 and Pr 3 and the 47.2.2. The Pr and Db systems and their relation to Pa other of which runs behind (cathodal to) "X", are either The Pr system polymorphism was originally described by present or absent. The phenotypes are denoted Db+ and hen and Oppenheim (1973). The proteins exhibiiing this Db -.The locus controlling the variation is called Db, and polymorphism had previously been characterized to some the gene determining Db+ is dominant. Approximately extent by Oppenheim et al. (1971). Four proteins made up 12% of Whites, 56% of Blacks and 7% of Chinese were the system, and were detected by alkaline slab polyacryla- Db +.Db is very closely linked to Pr (Azen and Denniston, rnide gel electrophoresis using parotid saliva which had been 1974; Friedman et al., 1975; Azen, 1977). concentrated 5-10 fold by lyop~onand reco~ution Extensive studies on the linkage relations of Pa, Pr and in gel buffer. The proteins are visualized by means of Db have been cmied out by hen and Demon (1974), acid5ed 3,3'-dimethoxybenzidine.The proteins were nega- Friedman et d. (1975), and Yu et al. (1978 and 1980a). The tively stained, i.e., the background changed color while the evidence suggests that there are three separate, but closely protein zones did not. More contrast could be obtained if a linked, loci, Pr and Pa being closer than Pr and Db. second staining step was done with a peroxide solution. Ap Definite recombinations have been seen in the family parently, some of the DMB precipitated in the gel in the first material of Yu et al. (1978), thereby proving the existence of step, everywhere except at the protein zones, and could then separate loci. Azen (1978) said that the Pr locus had three react with the peroxide in the second step to give a brown definite alleles, Pr', Pr" and W,that the Pa locus had PaO, background. The basis for this unusual staining reaction is Pa1 and P42, and that Db had Db+ and Db- .A reasonable not known (Azen and Oppenheim, 1973; Azen, 1978). Three (but not yet proven) order for the loci is Pa. . .Pr.. .Db patterns could be distinguished, and family studies showed (Yu et al., 1980a). that these could be accounted for by two alleles, called Prl and P?. The Pf gene was responsible for the protein bands 47.2.3 Basic protein Qb) called Pr 1 and 3 (originally I and III), while P13 was respon- Polymorphism in basic proteins of parotid saliva was de- sible for Pr 2 and 4 (origbdy I1 an rv). Frequencies of the tected by Azen in 1972, using acid urea starch gel electro- Pr' allele were about 0.73 in Whites, 0.8 in Blacks and 0.84 phoresis and staining for argining-rich proteins. Three pat- in Chinese. terns are seen, and called 1, 2-1 and 2. The proteins show The Pr proteins are especially rich in Gly, Glu and Pro asymmetric patterns in the homozygous types, and there is and have pI in the range of 4.09-4.71. PI 1 has a MW of variation in band intensity in different samples of the same 12,300 and Pr 3 of 6,100 by hentation ultracentrifuga- type. Pb 1 homozygotes show four bands, called "a", "b", tion (Oppenheim et al., 1971). Immunological evidence sug- "d" and "e", while Pb 2 homozygotes show only a "c" gests that the Pr proteins separable by Mectric focusing band. The letters represent the order of the bands on the are very similar if not identical in antigenic structure (Fried- plate. The patterns can be explained by a pair of codomi- man and Karn, 1977). nant alleles, Pbl and Pb2, at an autosomal locus (Azen, Azen and Denniston (1974) studied the Pr proteins more 1973). Pbl frequency is about 0.84 in Blacks, but over 0.99 closely, and found that two additional proteins at the so- in Whites; the Pb system is thus polymorphic principally called "X" position (slightly slower than but close to Pr l) among Blacks. could be distinguished by changing the pH or gel concen- The Pb proteins have been purified and partially charac- tration for electrophoresis. One of these proteins had a terized (Peters and Azen, 1977). The MW ranged from disulfide bond, and is now known to be identical to Pa 1 5,800 to 7,200, and the proteins are extremely basic (pI >9.5). (hen, 1977 and 1978). Some people have the "X" proteins Approximately 45% of the amino acid residues are the basic whiie others do not. One of the X proteins is.usually found His, Lys and Arg ones. The proteins contained no car- associated with P? products, and only rarely with Prl prod- bohydrate, and lacked Cys, Pro, Thr, Val, Met and Try. Salivary Polymorphic Proteins

There is evidence from the biochemical and amino acid Homozygous types could not be distinguished from the composition and sequence studies (Peters and Azen, 1977) heterozygous condition in which one of the alleles was GP that the polymorphism is due to a combination of allelic dif- (e.g. GPGP and GPGP people type as G1 1, and so forth). ferences and post-translational modifications involving pro- Studies on 41 families with 146 children were completely teolysis and deamidation. There is probably a single amino consistent with the postulated mode of inheritance. Studies acid difference (Glu vs Gln) in the primary gene products. on informative families strongly suggested linkage of GI This model helps to explain the peculiar asymmetric band- with Pr and Db, thus placing it in the Pa-Pr-Db region. ing patterns in the homozygous types. Peters et al. (1977) Products of GI show evidence of strong linkage disequili- found a larger MW protein in parotid saliva, called "post- brium with those of Pr, Db and Pa. Biochemical studies on Pb protein", which most closely resembled the Pb "e" band the G1 1 and GI 4 proteins indicated similarity in amino acid protein in MW. It was immunologically cross-reactive with composition, and similarity to the composition of Pr 1. The an antiserum raised against the Pb proteins, and was first polymorphic forms of G1 proteins showed microheterogene- thought to be a possible precursor of the Pb proteins. It is ity due to differences in size and charge, and the polymor- now believed that this is not the case, however (Azen, 1978), phism seemed to be accounted for on the basis of MW dif- and this protein is probably determined at a different genetic ferences in primary gene products. locus. 47.2.4.2 Pm. In 1977, Ikemoto et al. described another Despite a considerable amount of effort by the Wisconsin polymorphism in parotid saliva using a modification of the group, the Pb proteins have not been assigned a physiolog- acid urea starch gel electrophoretic system described by ical function thus far. Azen (1972 and 1973). The Pm protein migrates in between Pa and Pb proteins (the designation "Pm" stood for "sali- vary parotid middle band protein"). The Pm protein is 47.2.4 Other polymorphic salivary proteins either present or absent in a given individual, and its pres- 47.2.4.1 The major parotid salivary glycoprotein (Go. ence or absence can be determined in whole saliva, suggest- In addition to the acidic proline-rich proteins which have ing that it is one of the more stable of the salivary parotid been shown to exhibit genetic polymorphism (Pa, Pr and Db proteins. Family studies indicated that the protein is simply systems-see above), parotid saliva contains various basic inherited as a Mendelian dominant, and the Pm locus is proline-rich proteins. Unlike their acidic counterparts, many autosomal. Pm is apparently not linked to Pa or Pb. In a of them are glycosylated. Earlier studies (e.g. Friedman et sample of 195 Japanese, the Pm+ frequency was about al., 1971) have shown that they are heterogeneous, differing 0.38. 20 Chinese were also typed, and although the number in size, charge and MW,and there was speculation that this is small, Pm+ was about 0.3 by my calculations. Azen and variability might have a coherent genetic basis. In 1979, Denniston, in a personal communication to Yu et al. Azen et al. carried out extensive studies on the major (1980b), have found that Pm is closely linked to Pr and Db. glycosylated protein in parotid saliva, and showed that it ex- 47.2.4.3 Ph. In 1979, Ikemoto et al. reported on the hibits discrete, genetically controlled molecular variants. polymorphism of a further protein in parotid saliva, Parotid saliva was extensively studied using a polyacryla- detected by SDS-polyacrylamide gel electrophoresis mide gel electrophoretic system and acidic (pH 2.4) buffers. (Ikemoto et al., 197%). This protein differed from Pb, Pa, Fairly high gel concentrations were required (routinely 9% Pr, Db and Pm, and was designated "Ph", for "salivary and sometimes 13.5%). Thick (6 mm)gels were employed, parotid heavy protein", because it appears to have a larger and sliced prior to staining for carbohydrate. The optimal MW than the other parotid proteins (this observation had resolution of the bands was obtained with samples that had been previously reported by these authors in the Japanese been subjected to neuraminidase treatment. Other staining literature). The MW of the Ph protein was determined to lie overlays served to distinguish the proteins of the Pa, Pr and between 68,000 and 155,000 by SDSpolyacrylamide gel Db systems from those under study here. electrophoresis. Ph, like Pm, is either present or absent, and Eleven phenotypes of the major glycosylated proteins the Ph + condition is simply inherited as a Mendelian domi- were observed, five of which appeared to represent homozy- nant at an autosomal locus. Among 218 Japanese subjects, gosity at the controlling locus. The system was termed Ph+ frequency was about 0.026 with Ph- about 0.974. Ph "Gl", and the homozygous phenotypes Gl 1, 2, 3, 4 and 0. was not associated with Pr, Db or Pm, but was associated In addition, heterozygous phenotypes 1-2, 1-3, 14, 2-3, with Pa. Ph was a glycoprotein, and the authors said that its 24, and 34were seen. There are four band positions in the relationship to G1 would require further study. system used, and they represent (in order from origin to 47.2.4.4 Sal I and SaI II. In 1974, Balakrishnan and cathode, in the direction of the run) the 4, 1,2, and 3 bands. Ashton described two polymorphic proteins in parotid G10 has no bands. Five codominant alleles at an autosomal saliva, detected by polyacrylarnide gel electrophoresis at locus account for the phenotypes, and they were called GP, pH 8. They said that this work had been reported at the 4th GP, GP, GP and GP. Gene frequences seen in 143 Whites International Congress of Human Genetics in Paris, and were GP = 0.742, GF =0.04, GP = 0.155, Gl' = 0.017 that these proteins did not correspond to the Pb proteins of and GP = 0.046, while in 82 Blacks they were GI' = 0.459, Azen (1972 and 1973). The polymorphism was detectable in GP = 0.05, GP = 0.337, GI' = 0.044 and GP = 0.11. whole or in parotid saliva. The complex pattern of bands Sourcebook in Forensic Serology, Zmmunologv, and Biochemistry could be divided into six regions, and the variation was ob- and 2, were observed. The best genetic explanation for the served in region 4. Two bands were involved; both could be data was an autosomal locus Sgd, with a pair of codominant present or absent. In addition, either one could be present in alleles Sgd' and SgcP. The fact that Sgd is autosomal shows the absence of the other one. The proteins were inherited, that it is separate and distinct from Gd, which is well estab- and the most viable genetic explanation postulated two loci lished as being X-linked. The Sgdl frequency was about 0.75 called Sal Iand Sal 11, each of which had a dominant and a in Caucasians, 0.66 in Japanese and 0.7 in Chinese, all living recessive allele. Sal I controlled the "fast" band, and its in Hawaii. alleles were designated Fand f; the alleles of Srrl II were cor- Genetic polymorphism in salivary (parotid) peroxidase respondingly designated S and s. The "null" condition (no (SAPX)was described in detail by Azen (1977). The isoen- bands) was attributed to the ffsgenotype. Azen (1978) said zymes were detected in concentrated parotid saliva samples that examination of the patterns in the photograph of Bala- by polyacrylamide gel electrophoresis in Tris-lactate buffers krishnan and Ashton (1974) suggested that the two bands at pH 2.4. The gels were then washed in alkaline buffers to might represent two of the Pr proteins. However, the bring the pH to the 8.0-8.4 range prior to detection of ac- genetic analysis, and particularly the f~ndingof the "null" tivity. Detection was canied out using a modification of the phenotype, would not fit with this explanation. procedure descnied by Uriel (1958) for Cp detection (sec- 47.3 Polymorphic Salivary Enzyme tion 45.5). It may be rded that Cp is often detected by taking advantage of its oidase activity. Azen (1977) found Systems two comparatively common and one rare SAPX pheno- The best studies of polymorphic enzymes in saliva is prob- types. SAPX l, the most common type, had a major fast ably amylase, and this system has been discussed elsewhere, ("F") band, a faster minor "f' band (probably a conver- as noted in section 47.1. In this section are dkcused salivary sion product of F), and several slower bands. SAPX 2, the acid phosphatases (Sap), &erases (Set or Set,), Glc-6-phos- next most common type, showed a major slow ("S") band, phate dehydrogenase (Sgd) and peroxidase (SAPX). and several assoc6te-d slower bands, the first of which ("s") The presence of an acid phosphatase activity in saliva was probably represents a conversion product of S. SAPX 3 was noted by Gi in 1936. It had a higher activity with hexose seen in one family. These phenotypes could be accounted diphosphate than with giycerophosphate, and the pH opti- for on the basis of three alleles, SAPX, SAPX and mum was 2 to 3 units higher with the former. Using poly- SAPX. One informative family in the study showed that acrylarnide gel electrophoresis, Tan and Ashton (1976a) there was an intimate relationship between SAPX and Pa. found five patterns of salivary acid phosphatase, and one This frnding implied that SAPX would also show a relation- deficient type. These patterns could be explained on the ship with Pr and Db as well, and this suspicion was con- basis of two separate genetic loci, called Sap-A and Sap-B. fmed. Extensive biochemical studies on the proteins and Sap-A had three alleles, sap-^^, sap-^*' and SpAO, genetic studies on the interrelationships of the determining while Sap-B had two alleles, Sap-BB and SapBO. Opera- genetic loci showed that there is a perfect correlation be- tion of these loci gave rise to the phenotypes AB (which was tween SAPX and Pa products. SAPX behaves as a reces- common), B, AA' ,A'B, AA'B, A and 0.The relationship sive, while SAPX and SAPX behave as complete domi- of these isozymes to other human acid phosphatases (section nants. The SAPX 2 and 3 products appear to be genetically 29.3) will eventually want some further explanation. controlled modifications of SAPX 1, rather than different In 1976, Tan found polymorphism among a pair of car- primary gene products. Pa 1 is always assochted with boxylesterase bands in saliva using polyaaylamide gel elec- SAPX 2, and Pa 2 is always awxiated with SAPX 3. The trophoresis and detection with a-naphthyl acetate and Fast Pa locus may be regarded, on the basis of these data, as a Blue RR salt. Of the two bands, a fast one and a slow one, kind of "modifier" locus, whose products modify the both or either could be present in a given person, giving the SAPX 1 type. When the Pa type is Pa 0, the SAPX type phenotypes F, FS and S. The locus controbg this poly- is 1. It is probable that Pa 1 or Pa 2 monomers complex morphism was called Set-I, and the observations could be with SAPX 1 through Widebonds to yield SAPX 2 or understood in terms of two alleles, Set-lF and Set-lS. SAPX 3. Set-lF had a frequency of about 0.61 among Caucasians and about 0.5 among Japanese, both in Hawaii. As with the 47.4 Typing Salivary Polymorphic case of Sap isoenzymes noted just above, the relationship of Proteins Set-1 isozymes to the other wboxylesterases of human Polyacrylamide gel electrophoretic methods for typing tissues and their genetic loci (section 31.3.6) will require ad- the salivary proteins and enzymes were collected and organ- ditional study. ized by Tan and Teng (1979) in a useful summary paper. Tan and Ashton (1976b) described a fairly well distrii- Typing some of the proteins in a reproduaile way is ob- uted polymorphism in salivary glucose-6-phosphate dehy- viously somewhat involved (Azen, 1978). More than one drogenase, denoted "Sgd" (it may be rdedthat the locus electrophoretic system must be employed to discern all the denoting the red cell Glc-6-PD is often denoted Gd--see in variants in some systems. Azen (1978) gave a thorough secton 33.1). Polyacrylamide gel electrophoresis was used to discussion of the most optimal typing methods for the separate the koemymes, and three phenotypes called 1,2-L various systems, based upon his considerable experience. Salivary Polymorphic Proteins

Friedman and Allushuski (1975) described a special electro- other populations from various sources. The probability of phoretic system for defining Pr-Db-Pa types. It appears excluding a falsely accused father using each of the systems, that the typing of the polymorphic isoenzymes, leaving aside and for the Pa, Pr, Db, Pm and Ph systems combined, was SAPX which is closely associated with Pa, may be more calculated from the Japanese population data. They noted straightforward. that the combination of bloods groups, isoenzyrnes, and Most workers have used parotid saliva for this work, at serum group systems currently employed for paternity least in the case of the nonenzymatic proteins. This material testing (which did not include HLA)had a combined PE of is collected in a special way in a little cup, quickly frozen, about 0.92. The five salivary systems gave a combined PE of and then lyophilized. Reconstitution of the lyophilizate for 0.305, and when combined with all the other marker typing results in a several-fold concentration of the proteins. systems, raised the overall CPE to 0.944. The value Many authors have noted that these proteins seem to be calculated for the combined PE (CPE) of the five salivary especially subject to proteolytic degradation, which changes systems is, however, almost certainly in error on the high the electrophoretic patterns and results in the inabiity to side. The Japanese workers assumed independent segrega- determine the type. Whole saliva is considered unsuitable tion for all the loci in doing the computation, and it is now for typing many of the proteins, apparently because of the clear, as Yu et al. (1980b) pointed out and as was discussed rapid proteolytic activity that occurs in the oral cavity. The above, that Pa, Pr, Db and Pm are all closely linked, and apparent sensitivity of the proteins to degmhtive change, that some allelic combinations at these loci show significant and the necessity to concentrate even fresh material in order linkage disequilibria. Accordingly, independence of the loci to diagnose the types in several of the systems, indicate that cannot be correctly assumed in computing the PE for any the nonenzymatic proteins may not be very suitable candi- combinations of these systems. dates for saliva stain identification work. There is no reason Pronk et al. (1979) briefly noted some of the problems why specialized laboratories could not employ some of the associated with changes in the Pb patterns in saliva between systems, which have simple and clear cut inheritance pat- the time of collection and that of actual analysis. These terns, in disputed parentage cases. Indeed, some of the alterations are of a proteolytic nature. systems are beginning to find their way into medicolegal Hayashi and Hayashi (1979) have descriied a polyacryla- work already (see immediately below). mide disc gel electrophoretic procedure for Set phenotyping, 47.5 Medicolegal Applications which was applicable to whole saliva and to saliva stains up to 6 weeks old. The conditions used to examine the stains With a single exception, to be noted below, the few papers were slightly different from those used for fresh material on medicolegal applications of the salivary polymorphic (which was frozen at once after collection, and thawed just proteins have been concerned with disputed parentage. prior to typing). The polymorphism was recommended for Ikemoto et al. (1979b) carried out extensive population paternity investigations and as an identification marker in studies on the Pa, Pr, Db, Pm, Ph and Amy, systems in salivary stains. Japanese. The data were compared with published data for Hair Polymorphic Proteins

SECTION 48. POLYMORPHIC PROTEINS IN HAIR

A number of genetic markers have been detected in hair, Dr. Baden and his collaborators at Harvard have recently which shares them in common with blood and other tissues. carried out extensive studies on the structural proteins of These markers have been discussed in previous sections de- hair. The data indicate that there are detectable differences voted to the various systems. in these proteins in hairs from different individuals, and that ABO grouping of hair was discussed in 19.10.7.1. ABO is some of these differences are simply inherited. The major the only blood group antigen marker system that has been structural proteins of hair (keratins) consist of fibrous pro- detected in hair, and the only marker that is present in the teins and the amorphous matrix proteins. The fibrous pro- hair shaft. There seems to be no doubt that the glycopro- teins consist of a number of different polypeptides, having teins which possess ABH antigens are present in the hair many CySSCy residues and MW in the range of 40,O- shaft. Yet, while some workers have reported entirely 58,000. These proteins can be separated and examined by reliable results in typing hairs, others have found that the disc polyaayhide gel electrophoresis if they are first type corresponding tgthat of the blood cannot always be reductively denatured in urea, and the S-carboxymethyl determined correctly. : derivatives prepared. Baden et al. (1975) reported a genetic A number of isoenyme markers which are expressed in variant in one of the peptides of the fibrous proteins using red cells as well as in tissues can be diagnosed in the cells of this technique. The variant type occurred in about 5% of the hair root sheath. These markers appear to be detennin- Caucasians. Hairs from any body site in variant persons able about as reliably in hair roots as they are in blood showed the variant pattern. Four families were studied, and (though, perhaps, many are determinable longer in dried indicated that the trait was autosomal and dominant. In- blood than in plucked hairs). Among these are PGMI types terestingly, the characteristic variant pattern could also be and subtypes (27.4.3), ESD (31.4.4), PGD (33.2.4) and detected in nails. GLO (34.5.2). PGD asidl,, these systems give reasonably The matrix proteins are a little more difficult to separate, good discrimination in most populations, and would yield but Lee et al. (1978) reported a procedure for doing so. In considerable individualizing. information if they were all about 300 people, two variant proteins were detected that used on the same sample. A root sheath is required, how- showed as extra bands in the usual pattern. The extra ever, in order to make typing possible. variant bands were called VI and V2. V, was inherited in one Hair is common physical eyidence, and techniques other motherchild pair. The inheritance of V2 was not studied. than morphological compariSons that might help to in- Another variation was seen in which one of the usual bands dividualize human hair would be most welcome in the foren- was significantly reduced. This pattern was reproducible in sic science laboratory. Many hairs encountered in case work people who exhibited it, and it appeared to have been inher- lack root sheaths, so the enzyme markers cannot be used. ited in one family. These data were regarded as preliminary, And, since everyone is not in agreement about the reliability suggesting further family and population studies as well as of ABO typing, morphological comparison of questioned searches for additional variation. While this work is at an and known hairs is usually the only remaining approach. early stage, the results do seem to offer hope that detectable While such comparisons have indisputable value, especially polymorphic protein systems may well be present in the in excluding common origin,' less subjective procedures structural proteins of hair. Further study will be needed would be desirable. before these systems can be exploited for medicolegal work. Referencesfor Unit VII

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Revillard.--~p~ J.P.. M. Robert. E. DuPont. H. Betuel. G. Rifle and J. raised by the Isf system of human immunoglobulins. Vox Sang. 14: ~rae~ei.1973. Inhibition df mixed lymphocyte culture by alloantibodies 458459 in renal transplantation and in prcgnaucy. Tnmspkmtation. Proc. 5: Ropartz, C., L. Rivat and P.Y. Ro~u.1962. Deux nouveaux facteurs 331-335 dans les systemes hereditaires de gamma-giobulina: Le Gm(e) a Rim, G., U. Maaa. R.M. Turi, P.G. Pich, C. Camaschclla. G. Saglio I'Inv(l), in: Proc. 9th Congr. Int. Soc. Blood Trausf., Mexico City, and L.F. Bernini. 1976. S~icanceof a new type of human fetal Bibliotheca Haematol., Fasc. 19, 455458 (1%4) hemoglobin canying a replacement isoleucincthnoninc at position 75 Ropartz, C., L. Rivat and P.Y. Rousseau. 1963. Le Gm(b) a ses prob- (E19) of the y chain. Hum. Genet. 32: 305-313 lemes. Vox Sang. 8: 7 17-723 Rieder, R.J., J.B. Clegg, H.J. Weiss, N.P. Christy and R. Rabinowitz. Ropartz. C., L. Rivat, P.Y. Rousseau, H.H. Fudenberg. R. Molter and C. 1976. Hemoglobin At-Rooseveh: adrmVPI-.chr.Biochim. Biophp. Salmon. 1966. Seven new human srmm factors presumably supported Acta 459: 501-504 by the gamma globulins. Vox Sang. 11: 99-102 Rirnon, A., Y. Shamash and B. Shapiro. 1966. The plasmin inhibitor of Ropartz. C., P.Y. Rousseau, L. Rivat and J. Lenoi. 1%1b. Etude g6ntti- human pksma. IV. Its action on plasmin, trypsin, chymotrypsin and que du facteur stri~ueInv frQuence dans cmaina populations. Rev. thrombin. J. Biol. Chem. U1:5102-5107 Fr. Etud. Clin. Biol. 6: 374-377 References for Unit VII

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