lIB11711110-
lmmunogenetics26: 216-219, 1987
genetics
© Springer-Verlag 1987
Identification of HLA-B44 subtypes associated with extended MHC
haplotypes
Margot S. Kruskall, Elizabeth E. Eynon, Zuheir Awdeh, Chester A. Alper, and Edmond J. Yunis
The Center for BloodResearch, Departmentsof Pediatrics, Pathology,and Medicine, Children's HospitalMedicalCenter, BethIsraelHospital, DanaFarber CancerInstituteand HarvardMedical School, Boston, Massachusetts,USA, and the AmericanRed Cross BloodServices- NortheastRegion,
Dedham, Massachusetts, USA
Abstract. The HLA class I antigen B44 is found in each of
two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of
HLA-B44 were identified. The basic variant was found in
all cell lines with the extended haplotype HLAB44, DR7, FC31, and the acidic variant in all cell lines with
the extended haplotype HLA-B44, DR4, SC30. The
occurrence of each antigen variant with a unique extended
haplotype explains previous observations concerning the
nonrandom association of B44 variants with DR antigens.
Introduction
Extended major histocompatibility complex (MHC)
haplotypes are certain allelic combinations of HLA-B,
HLA-DR, and the complement components BF, C2, C4A,
and C4B, which occur more frequently than expected from
their individual allele frequencies (Awdeh et al. 1983).
These fixed allele combinations constitute nearly 30 % of
all Caucasian 6p haplotypes (Alper et al. 1984). The increase in frequency of individual MHC alleles among
patients with diseases such as the 21-hydroxylase form of
congenital adrenal hyperplasia and insulin-dependent diabetes mellitus has been demonstrated to be attributable to
the increase in frequency of specific extended haplotypes
(Fleischnicket al. 1983, Raumet al. 1984). Studies tomore
precisely define the characteristics of these haplotypes are
important to the understanding of these and other diseases
where relationships to MHC alleles have been reported.
The extensive polymorphism of class I antigens of the
MHC was originally demonstrated through the use of serologic techniques. More recently, cell-mediated lympholy-
Address correspondenceto: MargotS. Kruskall, MD, BloodBank, Beth
Israel Hospital, 330 Brooldine Avenue, Boston, MA 02215, USA
sis (CML) typing and biochemical analysis using electrophoresis and isoelectric focusing (IEF) have revealed
even greater diversity of many class I antigens than previously established. For example, individuals with the serologically identified antigen HLA-B27 have been shown to
fall into multiple distinct antigen groups by CML typing
and one-dimensional gel electrophoresis (BreurVriesendorp et al. 1986, Choo et al. 1986, M61ders et al.
1983, Neefjes et al. 1986, Vasilov et al. 1983). Diversity
has also been established for a number of other class I antigens (Gaston et al. 1983, Gotch et al. 1985, Horai et al.
1982, Neefjes et al. 1986, van der Poel et al. 1983, Yang
et al. 1984). Recent studies have identified two subtypes of
HLA-B44, and it has been suggested that there may be linkage disequilibrium between individual B44 subgroups and
other HLA antigens, including DR4, DR7, and A2 (Hahn
et al. 1984, Kato et al. 1982, Tekolfet al. 1982, Yang et al.
1984). Previous work from our laboratory has identified
two extended haplotypes which include B44: B44,
DR7, FC31 (in FC31, BF=F, C2 =C; C4A = 3; C4B= 1),
with a frequency in the Caucasian population of 3.7%
and B44, DR4, SC30 (in SC30, BF= S, C2 = C; C4A = 3;
C4B=QO), with a frequency of 3.4% (Alper et al. 1984).
Because we suspected that a different and distinct B44 subgroup could be associated with each haplotype, further
characterization of the antigen in patients with such haptotypes was carried out using one-dimensional IEF.
Materials and methods
Cellselection. Serotypingfor HLA-A, -B, -C, and -DR was performed
by the HLA Laboratoryof the AmericanRed Cross using standard NIH
microcytotoxicitytechniques. Complementtypes for BF, C2, C4A, and
C4B were determinedusingpreviouslypublished methods(Marc'usand
Alper 1986). Peripheralbloodlymphocytesfrommembersof sevenfamilies withone or the other B44-containingextendedhaplotypewere transformed with Epstein-Barr virus supernatants to produce B-lymphoblastoid cell lines (B-LCLs) and frozen. Two control cell lines, each
HLA-B44 subtypes and extended MHC haplotypes
217
Results
homozygous for a B44-containing haplotype (complement data
unknown), were treated similarly.
Isoelectric focusing. After thawing, cells were grown in RPMI-1640
(GIBCO, Grand Island, New York) supplemented with 10% fetal calf
serum (KC Biological, Lenexa, Kansas), glutamine, and penicillin/streptomycin. When there were approximately 5-10 x 106 cells, the
cultures were resuspended in RPMI without methionine and subsequently
labeled with 100 gCi/ml of 35S-methionine (Amersham, Arlington
Heights, Illinois) overnight. Cells were then lysed using the detergent
Nonidet P-40 (NP-40). Supernatants with the radiolabeled antigens were
collected. Supernatants were precleared using a polyclonal rabbit antimouse antibody (Litton Bionetics, Charleston, South Carolina) to remove
HLA class II antigens by adding the antibody-supernatant mixture
sequentially to aliquots of inactivated Staphylococcus aureus cells until no
additional absorption occurred. Specific immunoprecipitation was
carried out using the monoclonal antibody 4E (Dana Farber Cancer Institute, Boston, Massachusetts) bound to S. aureus cells. This antibody
recognizes a common epitope on HLA-B, but not most A locus antigens
(Yang et al. 1984). The washed immunoprecipitates were treated with
neuraminidase (type VI, Sigma Chemical Company, St. Louis, Missouri)
for 1 h at 37 °C to remove sialic acid residues. After further washing, the
immunoprecipitates were resuspeuded in an IEF sample buffer containing
8 M urea, 5 % ampholine, pH 3.5-10, and 2 % NP-40, to a final volume
of between 30 and 50 gl. IEF gels were made with 8 M urea, 60:1.7
acrylamide: bis acrylamide, 1.7 % NP-40, and the following ampholines:
0.8 ml pH 3.5-10, 0.6 m l p H 5-7, and 0.6 ml pH 6-8, for a total volume
of 23.5 ml. One-millimeter thick gels were poured. When the gel solidified, samples were applied to the anode side of the gel using filter paper
strips (Whatman 4*3). The gel was run for 16 h at room temperature using
constant power and 600 V. After electrophoresis, the gel was fixed in
ethanol/acetic acid/water (35:10:55, v/v) to remove urea and ampholytes, and soaked in Autoflor (National Diagnostics, Somerville, New
Jersey). Autoradiographs were made following standard procedures.
Table 1 lists the MHC haplotypes of the two individuals
homozygous for B44-containing haplotypes (controls), as
well as members of each of seven families in which
B44-containing extended haplotypes were found, and the
relative location (acidic or basic) of the B44 band after isoelectric focusing. The IEF pattern obtained from the two
homozygous B44 control cells is shown in Figure I.
Although both celllines are serologically B44 positive, two
different band locations are seen: the band in lane 6, from
the B44 DR4 homozygous cell, is more acidic than the band
in lane 1, from the B44 DR7 homozygous cell. Sample IEF
patterns from members of two families with extended
haplotypes are also shown. The Bo family (lanes 4 and 5)
has the extended haplotype HLA-B44, DR7, FC31. JoBo
(lane 4) and E1Bo (lane 5) have identical, basic B44 bands
which match that of the basic (B44 DR7) control. JoBo's
second, slightly more acidic band is the antigen HLA-B7.
By contrast, the Pa family (lanes 2 and 3) has the extended
haplotype HLA-B44, DR4, SC30. E1Po (lane 2) has a band
identical with the acidic (B44 DR4) control. CaWi (lane 3)
is HLA-B44-positive (acidic variant) but without either extended haplotype; the other, more acidic band is HLA-B8.
In total, six individuals, from four different families, with
the extended haplotype HLA-B44, DR7, FC31 all had the
basic HLA-B44 variant on IEF, and six other individuals,
from three different families, with the extended haplotype
Table 1. B44-positive cell lines with extended haplotypes
Family Individual HLA haplotypes
Extended haplotype
B44, DR4, SC30
Control
De
Re
Pa
Ru
ER-4
JB-12
A2, B44, Cw5, DR4/A2, B44, Cw5, DR4
A2, B44, DR7/A2, B44, DR7
JoMu
PaDe
A2, B44, Cw5, DR4, SC30/A1, Bw57, DRw6,SC61
A2, B44, Cw5, DR4, SC30/A1, B8, DR3, SC01
X
X
GeRe
JeRe
A2, B44, Cw5, DR4, SC30/A1, B44, Cw6, DR2, SC31
A2, B44, Cw5, DR4, SC30/A2, B14, DR7, SC31
X
X
RaHi
E1Po
CaWi
A24, B44, DR4, SC30/A24, B44, DR1, SC31
A24, B44, DR4, SC30/A23, B49, DR7, SC61
A24, B44, DR1, SC31/A2, B8, DR3, SC01
X
X
JilRu
JoRu
RiRu
NaRu
MaRu
JimRu
Bo
E1Bo
JoBo
B44 BAND on IEF
B44, DR7, FC31
-
A
-
B
-
A
-
A
-
A
-
A
-
A
-
A
-
A
A29, B44, DR7, FC31/A26, B45, DR4, $1C21
A29, B44, DR7, FC31/A2, B35, Cw4, DR5, SC31
A2, B35, Cw4, DR5, SC31/A26, B45, DR4, S1C21
A2, B8, DR5, SC31/A26, B45, DR4, $1C21
A2, B8, DR5, SC31//t26, B45, DR4, $1 C21
A2, B8, DR5, SC31/A2, B35, Cw4, DR5, SC31
X
X
-
B
B
Neither
Neither
Neither
Neither
A3, B44, DR7, FC31/A1, B13, Cw6, DR7, SCO1
A3, B44, DRT, FC31/A1, B7, DR4, SCO1
X
X
B
B
Ti
ATi
A26, B44, DR7, FC31/A28, B51, DRw6, SC31
X
B
Rei
TRei
A2, B44, DR7, FC31/A29, B35, Cw4, DR4, SC31
X
B
218
Fig. 1. Autoradiograph of IEF gel class I HLA-B antigens obtained from
35S-methionine-labeled B-LCL extracts. Lane 1, JB-12 (basic variant
B44 homozygous control). Lane 2, Pa family: EIPo, A24,
[B44, DR4, SC30]/A23, B49, DR7, SC61. Lane 3, Pa family: CaWi,
A24, B44, DR1, SC31/A2, [B8, DR3, SC01]. Lane 4, Bo family: JoBo,
A3,[B44, DR7, FC31]/A1, B7, DR4, SCO1. Lane 5, Bo family: E1Bo,
A3,[B44, DR7, FC31]/A1, B13, Cw6, DR7, SC01. Lane 6, ER-4 (acidic
variant B44 homozygous control)
HLA-B44, DR4, SC30 all had the acidic HLA-B44 variant.
This difference is significant at P < 0.05 (using Fisher's
exact test for comparison of two proportions).
Cell lines from five other unrelated B44-positive individuals with either unknown or nonextended haplotypes,
and individuals from two families with nonextended haplotypes, were also studied by IEF. Nine examples of
B44-containing haplotypes were evaluable, since one individual and one family were homozygous B44-positive.
Four antigens were shown to be the acidic variant B44; five
were the basic variant. All five basic variant antigens occurred in individuals who also were DR7-positive; in three
of these individuals the (nonextended) haplotype was
known, and the B44-containing haplotype always included
DR7.
M.S. Kruskall et al.
MHC extended haplotype: the basic B44 antigen on the extended haplotype HLA-B44, DR7, FC31 in all individuals
of four families, and the acidic B44 antigen on the extended
haplotype HLA-B44, DR4, SC30 in all individuals of three
families. The antigen frequency of B44 in our population
of over 1700 Caucasian haplotypes is 15.3 %. Because the
two extended haplotypes which contain B44 occur with a
combined frequency of6.1%, nearly 40 % of the incidence
of B44 is in association with one or the other of the two
extended haplotypes. Thus, the existence of these two
common extend haplotypes is the most likely explanation
for the observation by Tekolf and others (1982) that each
of their two CML-defined B44 variants displayed linkage
disequilibrium with a DR antigen (their 44.1 with DR4,
and 44.2 with DR7).
In the present study, the basic B44 variant also appeared frequently in association with DR7 in individuals
with nonextended or unidentified haplotypes. However,
the small number of haplotypes studied makes drawing a
conclusion about any relationship difficult. In Tekolf's
study, extended haplotypes were not identified. It therefore remains unclear whether, after excluding the association contributed by extended haplotypes, there is any
residual linkage disequilibrium between the B44 basic
variant and DR7.
Thus, the presence of even more diverse alMic variability than appreciated through serologic techniques
makes the phenomenon of linkage disequilibrium more
readily demonstrable. The association of each B44 antigen
variant with a specific extended haplotype provides further
information about these highly conserved segments of the
MHC region of the human genome.
Acknowledgments. This work was supported by NIH grants HL 29583, AI
14157, AM 16392, HD 17461, CA 20531, CA 06516, and a grant from
the American Red Cross.
References
Discussion
The results of this study confirm the presence of two variants of B44, separable by IEF. The acidic variant has a pI
between that of HLA-B7 and -B8, and the basic variant is
cathodic to B8. These two antigens have also been identified by other groups using IEF, including Yang and coworkers (1984), who called the acidic antigen primary and
the basic antigen a variant; and Hahn and associates
(1984), who called the acidic antigen type I and the basic
variant type II. Furthermore, two corresponding variants
(44.1 and 44.2) are also detectable by CML typing (Tekolf
et al. 1982).
However, of particular interest in the present study is
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Received February 27, 1987; revised version received May 5, 1987