H E R E 55-15

GENETICAL STUDIES ON THE

Rh BLOOD GROUP SYSTEM
By AAGE HEIKEN and MARIANNE RASMUSON
STATE INSTITUTE FOR BLOOD GROUP SEROLOGY, STOCKHOLM 60, AND
INSTITUTE OF GENETICS, UNIVERSITY OF UPPSALA, SWEDEN
(Received March 25th, 1966)

INTRODUCTION

S EROLOGICAL research has, since the first Rh blood groups were

discovered by LANDSTEINER and WIENER(1940),revealed a system of
the most fascinating antigen-antibody complexity with important med-
ical applications. The nomenclature used to communicate the serolog-
ical findings have been based almost entirely on the two classic hypoth-
eses concerning the genetics of the system, one of which has been sug-
gested by WIENER(cf. WIENERand WEXLER,1963),the other by FISHER
and RACE (cf. RACEand $ANGER, 1962). In the present investigation, the
nomenclature of FISHER and RACEwill be used.
According to WIENER’S hypothesis, the Rh antigens are produced by
a series of multiple alleles of one gene; while, in the FISHER-RACE con-
cept, the Rh antigens are the product of three closely linked genes, ar-
ranged in the linear sequence D , C and E. The antigens c and e are
antithetic to C and E, respectively; c is produced by c , a n allele of C ,
and e is produced by e, a n allele of E. The occurrence of a d antigen
was presumed, but this antigen has never been demonstrated.
As appears from this basic scheme, a n Rh gene complex could be
assembled in eight different ways. Considering the fact that there are
three orders of frequency with which these Rh gene complexes occur in
the English population (frequent: DCe, dce and D c E ; infrequent: Dce,
dcE, dCe and DCE; very infrequent, and not observed at the time of the
presentation of the hypothesis: d C E ) , FISHER-RACE suggested that the
infrequent combinations might be maintained by occasional crossing-
over from the three frequent complexes. The rise of dCE require a cross-
ing-over involving at least one of the infrequent complexes d c E , dCe or
DCE. The reason for the given sequence of genes was that the frequency
ratios of the gene complex dcE to DcEldce, which represents a crossing-
over between D and E , are considerably larger than the ratios of dCe to
 RH BLOOD GROUP SYSTEM 193

UCeldce (crossing-over between C and U ) and of DCE to DCelDcE

(crossing-over between C and E). It has hitherto been impossible to
obtain the large number of pedigrees, which seem necessary to prove
the close linkage hypothesis directly, and all studies have not con-
clusively supported the crossing-over theory. Recently, however, STEIN-
BERG (1965) has reported a case, which might be the first recognized
example of a crossing-over within the system.
Subsequent research has disclosed additional alleles of the Rh loci:
D" (STRATTON, 1946), C" (CALLENDERand RACE, 1946), C" (RACEet al.,
1948), C" (STRATTON and RENTON,1954), E" (GREENWALTand SANGER,
1955) , E" (CEPPELLINI et al., 1950), es (SANGER et al., 1960) and ei (LAY-
RISSE et al., 1961). However, with the exception of D" and C", all these
alleles are unusual or rare in Caucasian populations. Specific antibodies
demonstrating the antigens D", C", E" and ei have not been found. They
distinguish themselves by giving some but not all the reactions expected
of the ordinary antigens. It seems now most probable that C" is identical
with the C gene occurring in the complex D (C) ( e ) to be mentioned below
(the brackets representing the weakness of the corresponding antigens).
A series of antibodies demonstrating so-called compound antigens
have been isolated: anti-ce (ROSENFIELDe f al., 1953), anti-ces (DENA-
TALE et a/., 1955), anti-Ce (ROSENFIELDand HABER,1958), anti-DC
(ALLEN and TIPPETT,1958), anti-CE (TIPPETTet al., 1961) and anti-cE
(KEITH et al., 1965). It has also been established that it is possible to
subdivide the antigens D (UNGER and WIENER,1959 a, b, c; UNGER
et al., 1959; SACKS et al., 1959; TIPPETT and SANGER, 1962; SUSSMAN and
WIENER,1964; UNGER,1964), E (Vos and KIRK,1962) and e (SHAPIRO,
1960, 1964). Mainly based on these observations, it has been questioned
whether the Rh complex might be of cistron character (cf. HELMBOLD,
1959; HACKEL,1961; YOKOYAMAet al., 1961; BOORMAN and LINCOLN,
1962; RACEand SANGER, 1962) and, if this should be the case, whether
more than one cistron is involved. An attempt of a cis-trans test has
been performed by RACEand SANGER (1962) on the basis of which they
concluded that C and E may be in one cistron. Whether or not D is
present in the same cistron could not be determined.
Some unusual, but theoretically very important Rh types have been
encountered in which some or all the expected antigens are lacking or
are giving weak reactions. The first of these types was described by
RACEet al. (1950). The propositus was assumed to be homozygous for a
rare gene complex which was given the symbol D- -, as it produced no
detectable C-c or E-e antigens. Successively, thirteen new instances have
13 - Heredifas 55
194 AAGE HEIKEN AND MARIANNE RASMUSON

been reported (a detailed list is given by RACE and SANGER, 1962). Even
in the heterozygous state the D-- gene complex has been met with
(HENNINGSEN, 1957; BROMANand HEIKEN, 1962; LAWLERand MAR-
SHALL, 1962; HEIKENand GILES, 1965, 1966 a ) . A minute deletion in the
Rh chromosome was first considered to be responsible for this complex;
but since it was observed that there was a high consanguinity rate
among the parents of individuals homozygous for the D- - complex, and
also that the sibs of such propositi were more often D- -ID- - than ex-
pected, RACE and SANGER(1960, 1962) suggested that this fact might
indicate the involvement of complementary mutational genes in the
control of the type.
Five members of a large family, described by GUNSONand DONOHUE
(1957), were found to be homozygous for the gene complex DCw-.
Further serological investigation showed that the Cw antigen produced
by this complex is notably weaker than that produced by DCwe (TIP-
PETT et al., 1962) ; it seems therefore appropriate to denote this complex
by D (Cw)-.The same is true for the Dc- gene complex described by TATE
e f al., (1960), which produces a weaker c antigen than usually corre-
sponds to the production of one c gene: it could suitably be called D ( c )-.
Two gene complexes with depressed antigenic expression have been
described in connection with the detection of the compound antigens
DC and CE, uiz., d ( C )( e ) (ALLEN and TIPPETT, 1.c.; see also LEVINE
et al., 1961) and d ( C )(E) (TIPPETT et al., 1961). In an A4mericanNegro
family, ROSENFIELDet al. (1960) discovered another complex of this
category, D ( C ) ( e ) . It has been shown that this complex is not confined
to Negroes but also occurs among Caucasians (BROMAN et al., 1963). A
gene complex, characterized by producing weak c and e antigens, was
detected by METAXASand METAXAS-BUHLER (1961). Owing to the pair-
ing chromosomes, it could not be established whether this complex was
D ( c )( e ) or D"(c)( e ) ;however, for reasons of frequency, they considered
the former possibility as the most probable. This has later been sup-
ported by comparative studies including two unrelated families (BRO-
MAN et a/., 1964; HEIKENand GILES, 1965).
The - - - (or d- -) gene complex, in the heterozygous state, has been
suggested to be responsible in three independent instances of apparent
contrary homozygosity between mother and child reported by HEN-
NINGSEN (1958), PROKOP and SCHNEIDER (1960), and BROMANand
HEIEEN (1.c.). An individual with no detectable Rh antigens was en-
countered by Vos e f al. (1961). It was suggested that this person was
homozygous for the complex - - -. Recently, however, a similar example
 RH BLOOD GROUP SYSTEM 195

has been described by LEVINEet al. (1964); but, in this case, quite
another genetic mechanism seems to be responsible for the lack of
antigens. The parents of the propositus, who apparently was - - -I-- -,
belonged to the CCDee and CcDee phenotype, respectively; but all the
antigens present in their erythrocytes showed reduced activity. The
same was true for the child of propositus, who was CcDee, and many of
the other members of the family. Since it has been established that the
Rh and LW genes segregate independently of each other (LEVINEet al.,
1963; SWANSON and MATSON, 1964), LEVINEet al. (1964) considered it
to be more than a coincidence that the two only known carriers of Rhnull
are LW-negative. They postulated that both systems require the same
precursor substance to produce their normal phenotypic expression and
that in the presence of a basic X'r gene in the homozygous state, suf-
ficient precursor substance is available to be acted on by the Rh and
LW genes to produce complete expression of their respective antigens.
The family members showing reduced Rh antigenic reactivity were sug-
gested to be heterozygous for a mutant precursor gene, Xor, while the
propositus was suggested to be homozygous for this gene, i.e., XorXor.
If such a concept of basic precursor substance for both the LW and
Rh systems and the outlined interrelationship of their genes is correct,
it is possible to postulate that Rhnunmay arise in two ways. The first
way is that in which both LW and Rh genes are normal but the X'r
gene is absent. The second possibility would arise when two X'r genes
are present, but the Rh genes have mutated in such a way as to produce
no detectable Rh antigens. Since the second possibility does not affect
the LW genes, these would act on the normal amount of precursor sub-
stance. As the co-existence of two unrelated genes, both exeedingly rare,
would be unlikely, a n Rh,n of this type would most probably be LW-
positive.
Recently, a scheme of biosynthetic pathways with a sequence of Rh
genes acting on precursor substance has been suggested by BOETTCHER
(1964), who was not aware of the study of LEVINEet al. (1964). How-
ever, his hypothesis concerning the origin of the deviating Rh types
could be extended to incorporate the LW system quite well. He proposed
that there are three adjacent loci, D, C and E, each controlling the speci-
ficity of a single enzyme. The enzyme specified by the genes on one
chromosome are spatially arranged adjacently in the erythropoietic tis-
sue and act so that the precursor substance is passed subsequently to the
D, C and E enzymes. Each enzyme adds a new specificity to the sub-
stance, but the C enzyme is dependent on the action of the D enzyme,
196 A A G E HEIKEN AND M A R I A N N E RASMUSON

and the k: enzyme is dependent on the action of the C enzyme. Thus, if

a mutation occurs somewhere along the pathway, a block will be the
result and the rest of the antigenic structure will not be expressed in the
phenotype.
As it is known (cf., e.g. HARTMAN and SUSKIND, 1965) that the reduc-
tion in enzymic activity effected by a mutation may be complete, i.e., it
results in complete absence of the enzyme, or it may be partial, i.e., it
results in the formation of altered enzyme that has some limited degree
of activity, the theory of BOETTCHER (l.c.) might explain even the gene
complexes producing antigens with weak expression. This concept also
offers an explanation of the enhanced D-antigen reactions observed iD
individuals possessing the gene complexes D- -, D(Cw)-,D ( c ) - and
D ( C ) ( e ) (arrows indicate the production of weaker antigens than brack-
-1 1
ets only).
The basic assumption of BOETTCHER(l.c.) was that the Rh blood
group substances are carbohydrate-polypeptide complexes such as the
ABH substances, and that these substances therefore must be at least
two steps removed from the genes. So far, however, very little is known
about the chemical nature of the Rh antigenic substances ( c f . PROKOP
and UHLENBRUCK, 1963).
Indications that recessive suppressor genes may influence the action
of the Rh complexes have been obtained by DUNSFORD and TIPPETT
(quoted by RACEand SANGER, 1962) and GILES and BEVAN(1964); and,
recently, a mutation influencing the antigenic expression of the DcE
complex has been found by HEIKENand GILES (1966 b).
The present study was undertaken to investigate the frequencies of
the Rh gene complexes, including the deviating forms D ( C )( c ) , d ( c )( e ) ,
D- - and - - -, in the Swedish population and to examine their mode of
inheritance.

Material and methods

The material consisted of (1) a regionally divided sample of 8297
children, (2) 9468 mother-child combinations and (3) 101 families with
231 children. The children of the first sample were partly included in
the second sample. They were picked out at random in connection with
routine tests in paternity cases, and so were the mother-child combina-
tions of the second sample. The families voluntarily contacted the State
Institute for Blood Group Serology in Stockholm in order to participate
in the investigation. The regional classification of the first sample was
 RH BLOOD GROUP SYSTEM 197

made on the basis of the administrative division of the country into

counties, and the informations of place of residence at the time of the
investigation.
The serological tests were carried out during the years 1961-1965.
The blood specimens were tested with anti-D, anti-CCw, anti-E, anti-c
and anti-e by means of LOW'S papain technique. Specimens positive
with anti-CC" were tested with pure anti-c", but no pure anti-C was
available to test those specimens, which proved positive with anti-C"
and negative with anti-c. Such specimens, which were negative with
anti-D, but positive with anti-CC" and/or anti-E, were tested for the pre-
sence of D" by means of the indirect anti-human-globulin technique
with a strong incomplete anti-D. Only bloods which were positive with
anti-E were tested with anti-e. The individuals of the mother-child
sample were not tested with anti-C". All antisera used in the investiga-
tion were checked by known control cells before each series of test.

RESULTS
The regionally divided sample of 8297 children was tested for pheno-
typic homogeneity among regions. The 25 counties were aggregated into
ten groups, each consisting of two to five counties. The distribution OP
the five most common phenotypes is shown in Table 1. A f-test for
homogeneity among groups was performed for each of the five common
phenotypes and for the sums of the more uncommon types. I n no case
was heterogeneity indicated (Table 1). The Lappish intermixture in the
most northern counties (group X) has not been strong enough to in-
fluence these phenotypic frequencies. For instance, the ccdee pheno-
type, which is known to be of low frequency in the Lapps, has a fre-
quency of 15.2 "/. in group X as compared with 14.9 % in the total
material. The C" antigen, however, which was not included in the tests
of Table 1 , has a significantly higher frequency in group X, 6.03 "/.
against 3.99 % in the total material. In spite of this discrepancy it seems
justified to use the total sample for the estimation of the frequencies of
the Rh gene Complexes in Sweden.
Seven cases of Rh phenotypes with weak antigen reactions were ob-
served in this sample (see Table 10). The abnormal gene complexes re-
sponsible for these reactions will be dealt with below; for the present
purpose of estimating the frequencies of the normal gene complexes the
weakly reacting types have not been distinguished among the pheno-
types.
A maximum likelihood (ML) estimation of nine gene frequencies and
198 AAGE HEIKEN AND MARIANNE RASMUSON

TABLE 1. T h e regional distribution of 8297 Rh tested children

-
~

Phenotype
Region No.
Total
Counties.
CcDee CCDee 1 CcDEe ccDEe ccdee others
~

I M,L,K 369 202 154 137 170 59 1091

I1 N,O 320 173 147 135 139 54 968
I11 E,F,G,
H, 1 340 160 137 104 146 48 935
IV P , R 168 89 78 80 84 28 527
V S, T 182 90 81 70 88 32 543
VI C, D , U 226 107 89 96 94 40 652
VII A , B 735 402 316 231 311 103 2098
VIII w, X 212 100 101 78 89 32 612
IX Y,Z 142 80 78 49 47 27 423
X AC,BD 141 92 74 57 68 16 448
Total 2835 1495 1255 1037 1236 439 8297
% 34.16 18.01 15.12 12.50 14.89 5.29
Heteroge-
neity-xz 6.26 8.21 1 7.40 14.60 7.18 5.83
Probability 0.8-0.7 0.2-0.1 0.7-0.5 0.8-0.7

*The counties are designated according to the official Swedish motor register

TABLE 2. Rh gene complex frequencies and their standard deviations

estimated f r o m 8297 Rh tested Swedish children b y t h e m a x i m u m
likelihood method

Gene
complex
1 Frequency
Standard
deviation

DCe 0.40356 0.00387

D C We 0.01983 0.00110
dCe 0.00489 0.00085
dCWe 0.00034 0.00024
DCE 0.00082 0.00034
DcE 0.16701 0.00294
dcE 0.00295 0.00070
Dce 0.01855 0.00166
dce 0.38205 0.00399
 RH BLOOD GROUP SYSTEM 199

their standard errors was performed according to the method given by

FISHER(1946, 1947), and the result is summarized in Table 2. Two
Cwcdee phenotypes revealed the presence of the dCwe complex in the
sample, but dCE was not observed.
In Table 3 the observed phenotypic frequencies are shown together
with the expected phenotypic and genotypic frequencies. The agree-
ment between observed and expected distribution was tested. The re-
sulting xa-value of 12.564 has four degrees of freedom, since nine para-
meters are estimated from the sample with 13 classes. This corresponds
to a probability a little below the 5 % significance level.
The ML estimated gene frequencies were used for calculating the
expected phenotypic frequencies in the other samples included in the
present study. The 9468 mothers of the mother-child combinations con-
stitute a material which to a substantial degree is independent of the
regionally divided sample of children. Table 4 shows the agreement
between the observed phenotypic distribution in the mother sample and
that expected from the ML gene frequencies. The discrepancies are not
significant. Gene frequencies were also estimated from the mother
sample according to the method described by MOURANT (1954). These
estimates are well within the confidence intervals of the ML estimates.
The children in the mother-child combinations are partly identical
with those in the regional sample. Accordingly, their total phenotypic
distribution shows a satisfactory agreement with the expectations ob-
tained from the ML gene frequencies (~'=9.031;9 degrees of freedom;
P=O.5-0.3).
Furthermore, the wholly independent sample of the 202 parents in
the family material shows no significant discrepancies from expecta-
tions. Of more interest is the testing of the hypothesis of random mating
by a comparison between the observed and the expected distribution of
mating types. The sample includes 25 different mating types, as shown
in Table 7. No suspicious deviations are found, but because of the small
expected numbers the material in this form is not suited for a f-test.
Instead, three different tests have been performed with regard to the D,
C and E loci, respectively. These are shown in Table 8. For the D locus
the two reciprocal heterogenic mating types have been separated. Since
the Ee and E E types are so infrequent they had to be added before the
performance of the test. Thus analyzed no significant f-values were
obtained, and the hypothesis of random mating is supported by the
family sample.
A genetic hypothesis based on random combinations of the nine gene
TABLE 3. Observed Rh phenotype frequencies i n the regionally divided
sample and expected frequencies of phenotypes and genotypes

Phenotypes
Expected frequencies

Designa- Reaction with anti-

1 Observed frequencies Genotypes in

tion :C'"CW c D E c
/o
o/ genotypes

ccdee 1236 14.897 dceldce 14.5962 14.596

ccDee 123 1.482 Dceldce 1.4174 1.452
Dce!Dce 0.0344
ccdEe 18 0.217 deEldce 0.2254 0.225
ccdEE O* 0 dc E jde I:' 0.0009 0.001
ccDEE 256 3.085 Dc E l D c E 2.7892 2.888
DcEldcE 0.0985
ccDEe - ++++ 1037 12.499 DcEldce 12.7612 13.392
DeEIDce 0.6196
Dceldel:' 0.0110
Ccdee +-+-- 32 0.386 dCeldce 0.3736 0.374
CcDce +--t+- 2715 32.723 DCeldce 30.8360 32.351
DCelDce 1.4972
DceldCe 0.0181
CWcDee 120 1.446 DCWeIdce 1.5152 1.590
DCWelDce 0.0735
dCWel Dce 0.0013
CcdEc A - -1- - ++ O* 0 dCeldcE 0.0029 0.003
CcDEe + - + + + -t- 1200 14.463 DCelDcE 13.4797 13.947
DCeldcE 0.2381
dCelDcE 0.1633
DCEldce 0.0627
DCEIDce 0.0030
CcDEE -t-+++- 4* 0.048 DCEIDcE 0.0274 0.028
DCEldcE 0.0005
CwcDEe $+++++ 55 0.663 DCWeIDcE 0.6624 0.685
DCWeldcE 0.0117
dCWelDcE 0.0113
CWcdEe 0* 0 dCWe lde E 0.0002 0.000
CWcdee 2* 0.024 dCWe/dce 0.0260 0.026
CCdee O* 0 dCeldCe 0.0024 0.002
CC Dee 1341 16.163 DCelDCe 16.2861 16.681
DCeldCe 0.3947
CWCdec O* 0 dCWe1dCe 0.0003 0.000
dCWe!dCWe 0.0000
CWCDee 154 1.856 DCWelDCe 1.6005 l.(iN8
DCweldCe 0.0194
DCwelDCWt 0.0393
dC We 1D Ce 0.0274
dCWe/DCWe 0.0014
CCDEc 4* 0.048 D C E 1D Ce 0.0662 0.067
DCEIdCe 0.0008
CCDEE 0* 0 DCEIDCE 0.0001 0.000
CWCDEe O* 0 D CWe/D C E 0.0033 0.003
dCWelDCE 0.0001
Total 8297

*Uncommon phenotypes which h a v e been aggregated i n t h e X2-test

 RH BLOOD GROUP SYSTEM 201

TABLE 4. Rh type distribution among the 9468 mothers in the

mother-child sample

Expected number
according to
Observed
Phenotype
number
ML estimates
estimates
from sample

ccdee 1403 1381.970 1401.208

ccDee 123 137.458 122.015
ccdEe 31 21.342 29.867
ccdEE 2* 0.082* 0.159*
ccDEc 1242 1267.934 1262.841
ccDEE 291 273.414 274.110
Ccdce 34 37.836 34.238
CcDee 3236 3213.573 3218.799
CcDEe 1363 1385.383 1383.045
CcDEE 3* 2.639* 1.289*
CCdee 1* 0.259 * 0.209*
CCDee 1736 1739.156 1736.609
CCDEe 3* 6.656* 3.244*
Others O* 0.298 * 0.367*
Total 9468
*Uncommon phenotypes which have been
aggregated in the x2-tests:
9.934 5.268
x2 8.873 4.176
Degrees of 9 1
freedom 3
Probability

Gene complex frequencies estimated from sample:

DCe dCe DCE DcE dcE Dee dee
0.4236 0.0047 0.0004 0.1661 0.0041 0.0164 0.3847

complexes of Table 2 and their ML estimated frequencies can be tested

in those samples which extend over two successive generations, viz. the
mother-child combinations and the family sample. Within each pheno-
typic class of the mothers in the mother-child combinations an expected
distribution of children can be calculated from the expected genotypic
distribution within groups of equal phenotype (as given in Table 3) and
the population gene frequencies (assuming the fathers to be a random
sample of the population). I n the same way the expected genotypic
 TABLE 5. Phenotypic distribution of children within each of the nine largest phenotypic groups of mothers i n t h e
mother-child sample
~
-
~
-
-

Phenotypes of children
Mothers Num- ~
I.f
x=
phenotype her
1 ccdee zDee ccDEE ccDEe CCDee 1 CcDEe 1 CCDee Ccdee ccdEe :CDEE XDE, others
- -
ccdee 1402 0 552 25 238 575 3 5 4 2.471 5
E 535.63 26.01 234.15 593.59 1.15 7.33 4.15
ccDee 122 0 18 27 19 58 0.59 1.942 3
E 22.76 26.11 20.56 51.98
ccDEE 290 0 49 114 125 2 0.28 0.009 2
E 49.27 114.28 124.27 1.89
ccDEe 1242 0 230 28 124 325 264 261 6 3 1 10.269 6
E 226.07 22.69 105.54 352.37 263.07 266.67 3.09 1.94 0.56
CcDee 3236 0 612 70 255 1321 274 695 6 2 1
E 589.20 58.97 270.44 1333.26 276.32 693.50 8.42 4.55 1.33 0.01 6.076 7
CcDEc 1363 0 1 119 253 299 410 269 4 3 3 2
E 1.12 115.27 267.84 269.97 407.24 290.79 3.13 4.46 1.08 1.87 0.27 5.806 5
CCDee 1735 0 682 308 735 8 1
E 687.05 294.81 743.55 8.00 1.42 0.17 0.743 3
Ccdee 34 0 7 12 4 2 9
E 6.50 7.51 2.85 7.20 6.58 3.36 8.155 4
ccdEe 31 0 8 5 10 8
6.56 6.58 5.97 5.97 9.540* 4
~

Not included: Total 45.011 39

Rare pheno-
types 9 *P < 0.05
Cases of con-
trary homozy-
gocity 4
__
Total 9468

In the Xz-tests classes with expectations < 5 have been added until an expectation > 5 was obtained
O=observed number; E=expected number
 RH BLOOD GROUP SYSTEM 203

TABLE 6. Distribution o f D-positive ( D ) and D-negative ( d d ) children

among offspring f r o m D-positive and D-negative mothers in the
mother-child sample
Mothers X2-tests for goodness of fit with expectations
D dd Total derived from ML gene frequency estimates:

D mothers:

Children
dd ' 895 i
I
594 1489 D dd Total
Observed 7102 895 7997
Expected 7121.04 875.96 7997
x2 = 0.465; 1 d.f.; P = 0.5 - 0.3

dd mothers:
Children
D dd Total
Observed 877 594 1471
Expected 896.97 574.03 1471
x2 = 1.140; 1 d.f.; P = 0.3 - 0.2

distribution within mating types is used for obtaining the expected dis-
tribution of children in the family sample.
Deviations from the expected distribution may be of a qualitative or
a quantitative kind. Qualitative discrepancies may, if technical reliabil-
ity is assumed, be due to mutations, rare recombinations within the Rh
gene complex or to the occurrence of abnormal Rh complexes lacking
one or more antigenic expressions. Four such discrepancies, represented
by apparent contrary homozygosity between mother and child in the
C or E locus were found in the mother-child sample (Table l l ) , but
none were found in the family sample. The case histories of these ex-
ceptions have been reported by BROMAN and HEIKEN(1.c.) and HEIKEN
and GILES (1965),who attributed them to the presence of the abnormal
Rh complexes D- - or - - -. These four cases have been excluded from the
quantitative tests.
Quantitative discrepancies are revealed by significant f-values in
tests for goodness of fit. They may indicate that selective forces are
operating in the system, or that the assumption of random mating is
false. In the Rh system the most interesting question is the possible in-
fluence of mother-child incompatibility on viability. Since it is known
204 AAGE HEIKEN AND MARIANNE RASMUSON

Phenotype

I
~-
I 1
~~

ccdee ccDEe 'ccDEE

I 1
CcDee CcDEe CCDee
I ccDee
I- - Ccdee CW~Dec1CC"Dec
I

ccdeeO 5 6 13 2
E 2.15 3.95 9.54 ~ :.ll ~ 4.92

S o t observed mating types: E 10.57

Total 101
0 = observed number; E = expected number

TABLE 8. X2-tests for distribution of mating types as regards D, C , and

E loci t a k e n separately
I
Mating
JI 1:
Obs. Exp. Exp. I
I
Mating Obs. Exp.

dd x dd 5 2.342 cc x cc 14 10.704 47.753

D x dd 13 13.038 cc x c c 35 32.225 39.339
dd x 1) 15 13.038 cc x cc 5 12.127 4.051
D x D 68 52.582 Cc x Cc 32 24.254 8.102
c c x cc 11 18.255 1.669
cc x c:c 4 3.435 0.086
'x:

Probability
3.601; 3 d.f.

hl =mother; F=father
0.5-03
x2 = 10.892;
0.1 -0.05
5 t1.f.

I x2 = 0.297; 2 t1.f.
0.9-0.8
 RH BLOOD GROUP SYSTEM 205

that the D antigen is the most effective in cases of immunization of

mothers through pregnancies, the crucial distributions are those be-
tween D and dd children from dd mothers, or, when the phenotype of
the father is known, from the matings between dd mothers and D
fathers.
Tests for goodness of fit have been performed for the distribution of
the children within each of the nine largest phenotypic classes of
mothers in the mother-child sample (Table 5). Only in the smallest
phenotypic class, where all the expected frequencies are small, and x2
therefore is probably over-estimated, was a significant deviation found.
A comparison between observed and expected phenotypic frequencies
among the children in the family sample is shown in Table 9. The ex-
pected numbers are obtained by adding the expectations from each of
the 25 mating types. The Xe-value for this comparison is remarkably
low. Thus, neither the mother-child combinations nor the family mate-
rial reveals any quantitative discrepancies in the overall distribution
among the children.

TABLE 9. Observed and expected distribution of children in t h e family

sample
The expected numbers are obtained b y addition
of the expectations from each of the 25 mating types

Phenotypes of children

~ ccdee I C c D L p e e ~ C C D E F [ ~ ~ ccDee
E E I I ! I
CCwDee Ccdee CwcDEe others I Total

Obs.148 164 j40 ~ 3 1 130

Exp. 47.92 67.95 35.70 28.24 32.98
~6
6.97
1 4
4.15
1 4
2.49
13 1 1 10
2.785 1.01 0.805
(231
- ' d

11.12 7.09

x2 = 1.516; 6 d.f.; P = 0.99 - 0.95

,
Children from 15 families of type dd mother x D father:

Phenotypes of children

! i
1
D Total

1
~

I
Obs. 16 20 ~ 36
Exp. 17.17 18.83 x2 = 0.152; 1 d.f.;
P = 0.7 - 0.5
206 AAGE HEIEEN AND MARIANNE RASMUSON

TABLE 10. Observed instances of phenotypes w i t h w e a k antigen ex-

pression d u e t o t h e presence of t h e Rh gene complexes D(C)(e) and
d(c)(e)

Number of Phenotypes
Sample individuals _.
tested (C)cDE(e) ~ (C)cDee ~ C(c)DE(e) I C(cjDee
Total %o

Children
reg. div. 8297 G 0 1 0 7 0.84
Mother-child
combin.
mothers 9468 0.95
children 9468 0.84

Tests for the distribution on Rh-negative (dd) and Rh-positive (D)

children in the offspring from dd mothers are shown in Table 6, and
from d d X D matings in Table 9. No significant deviations are found,
but there is a non-significant deficit of 1.4 % D children among the
offspring from dd mothers. This deficit is not refound in the offspring
from D mothers, which is also shown for comparison in Table 6. It
corresponds to a selection coefficient of 0.05 for Rh-positive children
from Rh-negative mothers. The influence of mother-child incompat-
ibility on viability must therefore be considered as very slight in the
present material, a circumstance which can certainly be explained by
the great proportion of first pregnancies.
Among the exceptional types of Rh complexes, which were mentioned
in the introduction, both such with depressed and such lacking antigen
expressions were found in the present material. Table 10 summarizes
the observed instances with weak antigen expressions, which are attri-
buted to the presence of the D ( C )( e ) and d ( c )( e ) complexes. Individuals
carrying the complexes D - - or - - - in the heterozygous state will appear

TABLE 11. Observed instances of apparent contrary homozygocity

among t h e 9468 mother-child combinations

Phenotype Most probablm Phenotype Most probablc

of mother genotype of child genotype

ccdee dcel--- cc DEE DcEl---

CCDee DCelD-- ccDee dcelD--
ccDee dcelD-- CCDEE L)cEID--
ccDEE DcEID-- ccDee dcelD--
 RH BLOOD GROUP SYSTEM 207

TABLE 12. Estimated frequencies of four exceptional Rh complexes

Standard
Rh complex Frequency
deviation

D(C)( e ) 0.00067 0.00020

d(c)(e) 0.00012 0.00008
D-- 0.0005-0.0006
--_ 0.0001-0.0002

to have a homozygous phenotype, and the presence of these complexes

can only be recognized in cases of apparent contrary homozygosity be-
tween parent and offspring. The four instances found among the 9468
mother-child combinations are shown in Table 11. The first case sug-
gests the presence of the - - - complex, the following three the presence
of D--.
The frequencies of these exceptional Rh complexes in the Swedish
population, as represented by the present material, have been estimated
and are summarized in Table 12. For the two complexes with weak
antigen reactions frequencies and standard deviations were estimated
from the mother-child combinations by the iterative gene counting
method of SMITH(CEPPELLINI, SINISCALCO and SMITH, 1955; SMITH,
1957), starting with preliminary values obtained from the regional
sample. For D -- and - - - the same method lead to unacceptably high
estimates, probably because of a slight excess of homozygotes in the
mother-child combinations. Tentative frequencies for these complexes
were instead obtained from a comparison between observed and ex-
pected number of cases of apparent contrary homozygosity between
mother and child. A more detailed account of the estimation procedure
has been given elsewhere (RASMUSONand HEIKEN, 1966).

DISCUSSION
While the Rh complexes characterized by depressed antigenic expres-
sion hardly can give rise to misinterpretations in paternity cases, given
that several testsera and adequate serological techniques are used, the
occurrence of complexes lacking expression of one or more antigens
obviously causes a decrease in the genetic reliability of the system.
The observed cases of apparent contrary homozygosity between mother
and child indicate the possibility of corresponding cases in which a true
father might be falsely excluded. However, there are two ways which
208 AAGE HEIKEN AND MARIANNE RASMUSON

may be helpful in the attempts to avoid misinterpretations: ( I ) inves-

tigation of the parents (or family) of the alleged father, and (2) test of
the red cells with suitable incomplete anti-D sera in saline suspension;
but it ought to be stressed that more comprehensive family studies are
only possible in a very limited number of cases and that they may fail
to be informative. Although useful in many cases, the serological meth-
od mentioned cannot solely be considered to give conclusive evidence
for the presence or absence of the D -- complex in genetico-legal inves-
tigations.
All instances of apparent contrary homozygosity between mother and
child analyzed in Sweden are found to be due to Rh complexes which
lack both C-c and E-e antigenic expression. No complexes have been
reported which produce an E (or e) antigen but not a C (or c) antigen,
and n o complexes have been reported as producing a C (or c) antigen of
normal strength at the same time as they fail to produce an E (or e )
antigen. Thus, the risk of false exclusion of a true father seems to exist
only when both father and child are of homozygous C (or c ) and E
(or e ) phenotypes-given that weak reactions do not occur.
From Table 12 the added frequency of the D- - and - - - complexes is
found to be 0.0006-0.0008. A joint frequency, q, can also be obtained
from the total frequency of apparent contrary homozygosity in the
mother-child sample. The probability of contrary homozygosity in C is
2 p c . p c - q ; in E 2pE*pe'q, and the total probability is the sum of these

TABLE 13. Possible cases of apparent contrary homozygosity between

alleged man and child
q=frequency of Hh complexes lacking C-c and E-e antigenic expression.
Tentative value of q: 0.00068; upper limit of 99 ::confidence interval: 0.0013

Apparent Phenotype of Paternity index Securitv

contrary ~ ~~

rate
iomozygo alleged Tentative Upper in 76
sity in man value limit

C CC or C C W
0.0028 0.0052 99.7-99.5
cc
E I2 E ee
0.0048 0.0090 99.5-99.1
ce EE
C and E CCee ccEE
14.lg 0.0096 0.0181 99-98
ccEE CCee
CCEE ccee 640q 0.43G 0.484 56-52
ccee CCEIS 0.674 0.798 33-20
 RH BLOOD GROUP SYSTEM 209

minus the probability of contrary homozygosity in both C and E, which

is 2 ( p c e . p C ~ + p pce)
c ~ . * q. By equalizing this probability to the observed
frequency of 419648, the estimated value of q=0.00068 is obtained. An
upper limit of the confidence interval can be calculated from a formula
given by HALD (1952). The 99 % upper limit is q=0.0013. The numer-
ical values in Table 13 are calculated from these estimates.
The paternity index, I, defined as the statistical probability of an
alleged man being the father of a child (GURTLER, 1956), can be cal-
culated also for cases where apparent contrary homozygosity exists be-
tween this man and the child. In Table 13 the paternity index has been
calculated for the different possible cases of contrary homozygosity in
the Rh system. The calculations are based on the assumption that all
cases of apparent contrary homozygosity are due to the abnormal Rh
complexes D- - and - - -, and not to modificative lack of manifestation
or inhibiting effect of suppressor genes at other loci. The two last men-
tioned cases in the table are certainly very improbable, since the CCEE
phenotype has an expected frequency of only 7 - in the population.
Excluding these cases the genetic security rate of the Rh system is found
to be 99 % or higher.

SUMMARY
The frequencies of the Rh blood groups in Sweden are studied on a
regionally divided sample of 8297 unrelated children tested with anti-
CC", -C", -c, -D, -E, and -e. No regional heterogeneity was indicated
except for the C" antigen, which has a higher frequency in the northern
part of the country. A maximum likelihood estimation for nine Rh gene
complexes was performed on the total sample.
The estimated gene frequencies were used for tests of the genetic
model on a sample of 9468 mother-child combinations and a family
sample of 101 families with 231 children. These samples afforded no
significant indications of selective forces or assortative matings.
Phenotype with weak antigenic expressions due to the exceptional
complexes D ( C ) (e) and d ( c ) (e) were observed in the material with a
frequency of about 1760. Further, four cases of apparent contrary ho-
mozygosity between mother and child were found in the mother-child
sample. On the basis of earlier studies of case histories they were attri-
buted to the presence of the exceptional Rh complexes D- - and - - -, Le.,
complexes lacking C-c and E-e antigenic expression. The frequencies of
these abnormal Rh complexes have been estimated and are found to be
14 - Hereditas 55
210 AAGE HEIKEN AND MARIANNE RASMUSON

of the same magnitude as the DCE and dCwe complexes, or even rarer.
On account of the presence of the D- - and - - - complexes in the Swedish
population the risk for false exclusions in paternity cases and the genet-
ic security of the Rh system are discussed.

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 RH BLOOD GROUP SYSTEM 21 1

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212 AAGE HEIKEN AND MARIANNE RASMUSON

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