Polymorphism of bovine MHC class II genes.
Joint report of the Fifth International Bovine Lymphocyte Antigen
(BoLA) Workshop,Interlaken, Switzerland, 1 August 1992.
C.J.
Davies, I. Joosten, L. Andersson, M.A. Arriens, D. Bernoco, G.
Byrns, B. Bissumbhar, M.J.T. van Eijk, B. Kristensen, H.A. Lewin,
S. Mikko, A.L.G. Morgan, N.E. Muggli-Cockett, Ph.R. Nilsson, R.A.
Oliver, C.A. Park, J.J. van der Poel, M. Polli, R.L. Spooner,
& J.A. Stewart*
* See Appendix of 5th BoLA workshop, class
I report for addresses of workshop participants.
Correspondence/proofs: Dr. C.J. Davies
Department of Microbiology
Immunology and Parasitology
Schurman Hall
College of Veterinary Medicine
Cornell University
Ithaca
New York 14853-6401
U.S.A.
Telephone: (1) 607-253-3734
Fax: (1) 607-253-3384
Reprints:
Dr. J.J. van der Poel
Department of Animal Breeding
Wageningen Agricultural University
P.O. Box 338
6700 AH Wageningen
The Netherlands.
Short title: Fifth International BoLA Workshop - class II genes
Summary
Polymorphism of the bovine DRB, DQA, DQB, DYA, DOB and DIB genes
was investigated using restriction fragment length polymorphism
(RFLP) analysis, isoelectric focusing (IEF), class II serology
and polymerase chain reaction (PCR) based typing techniques. The
simultaneous application of multiple typing techniques and the
characterization of multiple genes resulted in a greatly enhanced
picture of the bovine class II regions. Thirty-eight class IIa
(DR-DQ) and 5 class IIb (DYA-DOB-DIB) haplotypes were defined.
It was found that IEF types were associated with DRB3 polymorphism
defined by DRB3 PCR-RFLP and DRB3 microsatellite PCR. Serologically
defined polymorphism was associated with distinct molecular/IEF
motifs and, therefore, DR and DQ specificities could be tentatively
distinguished. Although the DR and DQ genes are tightly linked,
neither DR nor DQ typing defined all of the class IIa region polymorphism.
Furthermore, even the most powerful DRB3 typing technique, DRB3
PCR-RFLP, failed to detect all expressed DRB3 polymorphism. All
detected DRB3 polymorphism could, however, be distinguished with
a combination of two molecular techniques: DRB3 PCR-RFLP and DRB3
microsatellite PCR. RFLP typing with transmembrane probes detected
significantly less polymorphism than typing with cDNA or exon
probes. However, the transmembrane probes were useful because
they were locus specific. The presence of only 5 of 12 possible
class IIb haplotypes was unexpected and indicates that the DYA,
DOB and DIB genes are tightly linked.
Keywords:
Bovine Lymphocyte Antigens, BoLA, Major Histocompatibility
Complex, MHC, class II, bovine, cattle, serology, isoelectric
focusing, restriction fragment length polymorphism, polymerase
chain reaction, DRB, DQA, DQB, DOB, DIB, DYA.
Introduction
The bovine lymphocyte antigen (BoLA) class II genes are located
in two regions that are separated by a recombination frequency
of approximately 17% (Andersson
et al., 1988; Stone & Muggli-Cockett,
1993; van Eijk et al., 1993).
The two regions are henceforth referred to as the class IIa and
class IIb regions. The class IIa region is tightly linked to the
class I region and contains the bovine DR and DQ genes (Andersson
et al., 1986a, b).
Both genomic and cDNA clones of bovine DR and DQ genes have been
isolated and homology with the human genes has been established
(Muggli-Cockett &
Stone, 1988, 1989;
van der Poel et al., 1990;
Groenen et al., 1990; Burke
et al., 1991; Xu et al., 1991,
1993). Allelic series of DRB,
DQA and DQB genes have been defined by sequencing the highly polymorphic
exon 2 following PCR amplification (Sigurdardóttir
et al., 1991a, 1992,
van der Poel et al., 1992).
The class IIb region contains the DOB, DYA, DYB and DIB genes,
and probably also the DNA gene (Andersson
et al., 1988; Stone & Muggli-Cockett,
1993). The bovine DOB and DNA genes have not been cloned but
are probably homologous to their human counterparts. DYA, DYB
and DIB are apparently novel bovine genes. The bovine DYA and
DIB genes have been cloned (van
der Poel et al., 1990; Stone
& Muggli-Cockett, 1990) and sequence polymorphism in DYA
has been identified (van Eijk
et al., 1992a).
A number of methods are now available for studying class II polymorphism
of cattle. In this workshop restriction fragment length polymorphism
(RFLP) analysis, isoelectric focusing (IEF), class II serology
and polymerase chain reaction (PCR) based typing techniques were
utilized to characterize the polymorphism of class IIa and class
IIb region genes and gene products. The simultaneous application
of multiple typing techniques and the detailed characterization
of groups of tightly linked genes has resulted in a greatly enhanced
picture of the two bovine class II regions and the bovine MHC
as a whole.
The Fifth International BoLA Workshop involved the characterization
of both class I and class II polymorphism. In an accompanying
paper results for the class I region were presented (Davies
et al., 1994). Here results pertaining to the class II genes
are presented and discussed.
Materials
and Methods
General
organization
The workshop was based on the distribution of 62 blood samples
(cells) from 60 selected animals (2 duplicates). Details about
the organization of the workshop are given in an accompanying
paper (Davies et al., 1994).
Class
II serology
Three laboratories, BER, EDS and WGN (see appendix of Davies
et al., 1994 for laboratory codes) performed serological class
II typing. A total of 151 antisera derived from 84 unique alloantisera
originating from BER ( Arriens
et al., 1991), BMD (Davies
et al., 1992a), EDS ( Williams
et al., 1991), ITH (Davies
& Antczak, 1991) and WGN (Nilsson
et al., 1994) were used. The difference between the total
number of antisera and the number of unique antisera is due to
the use of antisera: in more than one laboratory, from multiple
bleeds, at multiple dilutions, and/or following distinct absorptions.
BER and EDS used the standard complement-mediated lymphocyte microcytotoxicity
assay (Terasaki et al., 1978)
with B and T lymphocyte enriched cell populations (Vaiman
et al., 1983). WGN used the two colour fluorescence test (van
Rood et al., 1976) with unfractionated peripheral blood mononuclear
cells (Nilsson et al., 1994).
Serum reactions and local specificity assignments were reported
to WGN. BER reported results for 59 animals, EDS for 52 animals
and WGN for 48 animals.
Class
II isoelectric focusing (IEF)
EDS and UTN contributed class II IEF data. Typing was performed
using the workshop protocol described in the class I workshop
report (Davies et al., 1994).
Two monoclonal antibodies (mAb) were used for immunoprecipitation
of class II molecules: IL-A21 raised against bovine class II molecules
(a gift of A.J. Teale, ILRAD, Nairobi), and TH14B raised by immunization
with lymphoid cells from multiple species (Davis
et al., 1987). The laboratories submitted original autoradiographs,
annotated pictures and allele assignments. Interpretation of gels
was based on visual pattern recognition.
Restriction
Fragment Length Polymorphism (RFLP)
Five laboratories, DAV, LUT, MLI, URB and WGN, performed RFLP
typing using previously published methods (Andersson
et al., 1986a; Andersson
& Rask, 1988; Muggli-Cockett
& Stone, 1988; Teutsch
et al., 1990). The probes used in each laboratory are specified
in Table 1. DAV, URB and WGN used the restriction enzymes TaqI
and PvuII with all probes employed. MLI used TaqI
and PvuII for DQA and DQB typing but only used TaqI
for DRB. LUT used TaqI and BglII with the DRB-TM
probe, TaqI and PvuII with the DQB-TM probe, TaqI
with the DIB-TM probe and PvuII with the human DOB cDNA
probe.
RFLP patterns were ascribed by the laboratories and reported to
WGN using a predefined format. The nomenclature for DRB, DQA and
DQB RFLP patterns detected with cDNA or first and second domain
(exon 2 and exon 3) probes was based on the nomenclature of
Sigurdardóttir et al. (1988). One important modification
was that DQA and DQB patterns were ascribed independently. When
two previously defined DQ haplotypes had identical DQA or DQB
patterns the name of the lower numbered haplotype was retained
for the DQA or DQB pattern. Nomenclature for DRB-TM (Muggli-Cockett
& Stone, 1991) and DQB-TM RFLP patterns has not been established.
The nomenclature used for the DYA TaqI RFLP detected with
the human DQA cDNA probe corresponds to that described by Andersson
et al. (1988). DYA alleles 1 and 2 have 1.4 and 1.9 kb TaqI
fragments, respectively. The nomenclature used for the DOB PvuII
RFLP was proposed by Andersson
and Rask (1988). DOB alleles 1 and 2 have 9.5 and 4.0 kb
PvuII fragments, respectively. The DIB TaqI RFLP
was described previously (Muggli-Cockett
& Stone, 1991), however, the alleles were not named. DIB
alleles 1, 2 and 3 have 5.9, 3.9 and 1.6 kb TaqI fragments,
respectively.
DRB3
and DYA PCR-RFLP
DRB3 and DYA PCR-RFLP typing were carried out by URB using previously
described methods (van Eijk
et al., 1992a, 1992c).
DRB3 typing involved a two stage heminested PCR amplification
of the second exon, restriction enzyme digestion of aliquots of
PCR product with RsaI, HaeIII and BstYI,
and separation of restriction fragments on 6% polyacrylamide minigels
followed by staining with ethidium bromide. The DRB3 PCR-RFLP
nomenclature proposed by van
Eijk et al. (1992c) was used. DYA typing involved amplification
of exon 2 using two primers one of which contained a mismatch
that introduced a HindIII restriction site in the allele
with a G rather than an A at position 219 (numbering of van
der Poel et al., 1990), digestion with HindIII, and
separation of fragments on 6% polyacrylamide minigels. The DYA
PCR-RFLP nomenclature is concordant with the DYA RFLP nomenclature
(see results). Allele 1 has 219G resulting in restriction fragments
of 151 and 23 bp while allele 2 has 219A resulting in an uncut
PCR product of 174 bp.
DRB3
microsatellite (DRB3-MS) PCR amplification
Typing of the microsatellite located in the second intron of the
DRB3 gene was performed by UPS using previously described methods
(Ellegren et al., 1993).
Following PCR amplification of the microsatellite the length polymorphism
was resolved using an ALF automated sequencing/fragment analysis
system (Pharmacia LKB, Uppsala, Sweden). Alleles are defined on
the basis of the length of the amplified fragments. COL also performed
DRB3 microsatellite typing on 44 of the workshop samples. The
COL primers generated PCR products 55 bp shorter than those generated
with the UPS primers and failed to amplify one allele which had
sequence heterogeneity in the region of the COL forward primer.
The COL results served in a comparative role to the more complete
UPS results presented in this report.
Data
analysis
Allele assignments, specificity assignments and serum reactions
were reported to WGN using a predefined data format. DAV and WGN
performed cluster analysis on the serological data and ascribed
workshop specificity assignments to the cells. Comparison of typing
results obtained using other methods was performed by WGN using
the Cytofile computer programs (Davies,
1988). Data from different laboratories were compared and
consensus assignments were established. The consensus assignments
were entered in a computer database and haplotypes were established
on the basis of segregation and statistical correlations.
Class IIa haplotypes (DH) have been numbered to facilitate discussion
of the data. However, the DH numbers are not to be construed as
official haplotype names. The DH numbering scheme is based on
splits of the DRB3 PCR-RFLP patterns. Haplotypes that only differed
for DRB RFLP patterns and, consequently, are expected to express
identical DR and DQ products, have not been given separate numbers.
Results
Class
IIa region
Serology. Cluster analysis revealed 5 well defined specificities
that corresponded to local specificities and were defined by more
than 3 antisera originating from at least 2 laboratories. These
specificities were accepted as workshop D region specificities
(Dw) and henceforth shall be known as: Dw1, Dw2, Dw3, Dw4 and
Dw8. The sera used to define these specificities are listed in
Table 2. Also shown are the local
specificities ascribed to these reagents. Eight additional specificities
that were associated with particular BoLA class Iia haplotypes
were identified. These specificities were defined by 1 to 4 antisera
originating from a single laboratory. Many of the antisera used
to define these specificities were used in duplicate or triplicate
and in more than one laboratory. The workshop cluster names (Dc)
and local names for these specificities are as follows: Dc5 =
BMD-Dx5 and WGN-Ds5; Dc6 = EDS-ESD4 and WGN-Ds6; Dc7 = WGN-Ds7;
Dc9 = BMD-Dx9 and WGN-Ds9; Dc11 = WGN-Ds11; Dc12 = WGN-Ds12; Dc13
= BER-BeIII and WGN-Ds13; and Dc20 = BER-BeVI. Although these
specificities are not well enough defined to be given official
workshop recognition they have been ascribed to the animals (Table
3) and included in the haplotype definitions because they
provide valuable information about the complexity of the class
Iia region. Specificities Dw8, Dc5, Dc7 and Dc9 had tails that
showed weak and/or inconsistent reactivity patterns (Table
3). Because it is probable that the sera used to define these
specificities were not monospecific, these specificities have
only been assigned to haplotypes when it was clear that the primary
specificity included that haplotype (see below).
IEF.
In previous IEF studies, DRB polymorphism was defined using a
polyclonal anti-class II antiserum (Joosten
et al., 1989, 1990; Williams
et al., 1991; Watkins et
al., 1989). With the mAbs IL-A21 and TH14B, the same pairs
of bands were detected. However, whereas TH14B appeared to recognize
solely the DR molecules, IL-A21 showed some additional activity,
pointing to cross-reactivity with DQ products. The workshop analysis
was restricted to the definition of DRB polymorphism. A consensus
workshop nomenclature was established. IEF patterns were named
by indicating the locus followed by a F to indicate that polymorphism
was defined by IEF, i.e. DRBF for DRB patterns. The DRBF names
for the previously defined EDF and UDF types are shown in Table
4. Twelve DRBF types were well enough defined in both laboratories
to be given DRBF names. Although EDS did not differentiate DRBF10
and DRBF11, the UDF11/UDF20 split was accepted because it was
supported by serology, DRB3 PCR-RFLP and DRB3-MS PCR (Table
3). UTN defined 4 DRB IEF types in addition to the 12 DRBF
types accepted by the workshop. The 4 additional types need confirmation
and are referred to here using the local UDF nomenclature (Table
4).
Figure
1 is a schematic drawing of the DRBF and unconfirmed UDF types.
The difference between DRBF3 and DRBF4 is minimal. Although these
patterns can be distinguished when present on a single gel, it
is exceedingly difficult to tell which pattern is represented
when only one is present. Consequently, it is worth noting that
the DRBF4 top band is a doublet following precipitation with either
IL-A21 or TH14B.
RFLP
of DRB genes. Table 5 shows
the 46 DRB RFLP patterns defined to date using cDNA or exon 3
probes. Thirty DRB patterns, including 12 new patterns, were represented
in the workshop panel (Tables 3
and 5). DRB patterns are complex,
consisting of 2 to 7 TaqI fragments. The complex patterns indicate
that cattle have multiple DRB genes. DRB patterns can not be grouped
on the basis of the presence of particular fragments. Consequently,
the DRB nomenclature is based on the DQ alleles with which DRB
patterns were first associated (Sigurdardóttir
et al., 1988). New patterns identified in the workshop have
been named accordingly (Table 5).
Four of the new DRB patterns (DRB*1F, DRB*3F, DRB*3I and DRB*5D)
were confirmed by segregation. In addition, one pattern, DRB*3D,
was found in a Holstein Friesian class II homozygote (WK5-62)
and 2 British Friesian heterozygotes (WK5-07 & WK5-33) carrying
a w40(A14,A8)-DH11A haplotype, identical to one of the haplotypes
carried by WK5-62 (Table 3). The
7 remaining patterns (DRB*1E, DRB*1G, DRB*3E, DRB*3H, DRB*5C,
DRB*8C and DRB*14), 6 of which were associated with unique haplotypes
but were found in only a single animal or 2 genotypically identical
animals, need to be confirmed.
Two different DRB*3C patterns have been defined in previous studies
(Joosten et al., 1990; Bernoco
et al., 1991). The pattern described by Joosten
et al. (1990) has been renamed DRB*3G. Animal WK5-55 is a
member of the same Brown Swiss family used for the definition
of the DRB*3C pattern in the Fourth BoLA Workshop (Bernoco
et al., 1991). However, the fragments ascribed to WK5-55 were
significantly different from the fragments assigned to the DRB*3C
haplotype in the Fourth BoLA Workshop (Table
5). Obviously the fragments that make up the DRB*3C pattern
need clarification.
Eleven DRB2-TM patterns were found in the workshop animals (Table
6). Each pattern consisted of one TaqI and one BglII
fragment, demonstrating the locus-specific nature of the probe.
The DRB2-TM patterns were associated with specific class IIa haplotypes
(Table 3 and below).
RFLP of DQA genes. The 31 DQA patterns defined thus far using
cDNA or exon 2 and exon 3 probes are shown in Table
7. Twenty-four DQA patterns, 5 of which were new, were present
in the workshop animals (Tables 3
and 7). Two of the new patterns,
DQA*1D and DQA*3C, were present in 6 and 5 animals, respectively,
and were confirmed by segregation (Table
3). The other new patterns, DQA*1E, DQA*7F and DQA*11F, were
associated with unique haplotypes but were present in one animal
or two genotypically identical animals and, therefore, require
confirmation. Table 7 indicates
the fragments that hybridize selectively with the W1 exon 2 and
exon 3 probes. However, there are two distinct groups of DQA alleles
and exon 2 probes of the DQA5 type have a different pattern of
reactivity than exon 2 probes of the W1 type (Sigurdardóttir
et al., 1991b). Nevertheless, since the majority of fragments
carry exon 3 sequences, the overall patterns defined with probes
of the two types are virtually identical (Table
7). The sizes of several high molecular weight fragments were
adjusted on the basis of the BRL 1 kb ladder which includes several
high molecular weight markers. The new size estimates are given
in Table 7.
Animal WK5-56 was assigned DQA*1D despite the fact that her dam
WK4-17 was assigned DQA*1B (Bernoco
et al., 1991). As DQA*1B and DQA*1D were both represented
in the workshop animals and were associated with distinct DRB
types it would appear that DQA*1D is the correct assignment.
RFLP
of DQB genes. Table 8 gives
the 26 DQB RFLP patterns defined to date using cDNA or exon 2
and exon 3 probes. Nineteen patterns, including 1 new pattern,
were represented in the workshop panel (Tables
3 and 8). The new pattern,
DQB*3C, was associated with two haplotypes: an A32-DH22A haplotype
found in 3 British Friesians (WK5-08, WK5-34 & WK5-36) and two
Danish Black Pieds (WK5-26 & WK5-27), a dam-daughter pair,
and an A22(w49)-DH22G haplotype found in one Brown Swiss cow (WK5-60).
Table 8 includes information on
the probes that hybridize with each fragment. Although the DQbeta-1
cDNA probe apparently hybridized with all DQB fragments, hybridization
with fragments only containing exon 2 were sometimes weak.
Table
9 shows the 12 DQB-TM patterns found in the workshop animals.
With the exception of the DQB-TM*213 pattern which has 2 PvuII
fragments, each pattern is comprised of 1 TaqI and 1 PvuII
fragment. This indicates that the DQB1-TM probe behaves in a locus-specific
manner. With the notable exception of haplotypes DH1A, DH18A and
DH31A, which all have the DQA*5,DQB*5 RFLP type, each class Iia
haplotype was associated with a single DQB-TM RFLP pattern (Table
3 and below). Nine animals with haplotypes DH1A, DH18A or
DH31A were typed. No results were reported for WK5-05, a DQA*5,DQB*5
homozygote, and five DQA*5,DQB*5 heterozygotes were assigned only
a single DQB-TM RFLP type. The results for the remaining 3 animals
were inconsistent. These results imply that the DQB1-TM probe
hybridizes poorly with the DQB genes carried by DH1A, DH18A and
DH31A.
DRB3
PCR-RFLP. Nineteen of the 30 previously defined DRB3-PCR RFLP
patterns were represented in the workshop panel (Table
3; van Eijk et al., 1992b).
In addition, one new pattern was defined. The new pattern, pattern
31, is comprised of: RsaI pattern i (50, 54 &
180 bp fragments); BstYI pattern b (284 bp fragment);
and HaeIII pattern f (4, 48, 65 & 167 bp fragments).
Pattern 31 was gametically associated with an A19(A6)-DH31A haplotype
inherited by 4 offspring of Klaus, a Danish Black Pied bull (Table
3).
DRB3-MS
PCR. DRB3-MS alleles of 14 different lengths have been described
previously (Ellegren et al.,
1993). Eleven of the previously described alleles and 6 new
alleles (157, 165, 167, 173, 175 and 191 bp) were found in the
workshop animals (Table 3). Alleles
157, 165, 167 and 175 were found in two or more animals and were
in gametic association with specific class IIa haplotypes. Alleles
173 and 191 were each present in a single class IIa heterozygous
animal carrying a unique haplotype in combination with a well
defined haplotype. Animal WK5-45 was assigned DRB3-MS*179 when,
on the basis of segregation data for the DH22B haplotype in other
Danish Black Pied cattle, DRB3-MS*175 was expected. It is not
known if the discrepancy is due to a typing error or a mutation.
Class
IIa region haplotypes. On the basis of segregation data, statistically
significant associations and information from other studies (
Sigurdardóttir et al., 1988; Joosten
et al., 1990; Bernoco et
al., 1991; Davies et al.,
1992a; van Eijk et al.,
1992c; Ellegren et al.,
1993), the class IIa haplotypes carried by 59 of the 60 workshop
animals were determined (Table 3).
The data submitted for animal WK5-47 was inconsistent with any
known haplotype. Consequently, the haplotypes carried by this
animal could not be deduced. Table 10 lists the 38 clearly defined
class IIa haplotypes found in the workshop animals. There were
4 pairs of haplotypes (DH7A, DH11A, DH18A and DH21A; each pair
having a single DH number) that only differed for DRB RFLP patterns.
Since the haplotypes in these pairs had identical DRB IEF and
DRB3 PCR-RFLP patterns, and DRB3 is evidently the only actively
transcribed DRB gene (Burke et
al., 1991), the two haplotypes are believed to express identical
DR and DQ alleles. Consequently, there were apparently a maximum
of 34 class IIa haplotypes expressing distinct products. Haplotypes
with different DRB IEF types or class II serotypes must express
different class II alleles. Furthermore, a DRB3 PCR-RFLP difference
indicates distinct class II products unless all of the substitutions
are silent substitutions. The DH9A, DH9B and DH11A, DH11B haplotypes
have similar RFLP patterns and were not distinguished by DRB3
PCR-RFLP, IEF or serology. It is, therefore, possible that the
haplotypes making up these pairs express identical products. There
were two other pairs of haplotypes (DH10B, DH10C and DH12A, DH12B)
that were not distinguished by a product-based or exon-specific
typing method, however, the haplotypes constituting these pairs
have very different DQA and DQB RFLP patterns and, undoubtedly,
express different DQ alleles.
Table
11 shows the 30 class IIa haplotypes for which the serological
specificities could be determined with reasonable confidence sorted
by class II serotype. Sorting the haplotypes in this manner suggested
that the serological specificities were associated with distinct
molecular/IEF motifs. Associations with molecular/IEF motifs made
it possible to tentatively determine the locus with which most
of the class II specificities were associated (Table
11). It was not possible to determine the locus association
of the Dw2 specificity because all known class IIa haplotypes
expressing the determinants defined by this specificity are closely
related and appear to express identical DR and DQ products. However,
all of the other Dw specificities appear to be DQ specificities.
Furthermore, the serological specificities associated with DR
products appear to be related to individual alleles as defined
by DRB3 PCR-RFLP and IEF, whereas the specificities associated
with DQ products appear to be broad allospecificities. The significance
of this observation will be discussed below. Table
11 also shows that the DRB2-TM and DQB-TM RFLP patterns frequently
define groups of closely related haplotypes. Of particular interest
is the group of haplotypes expressing the Dw3 specificity and
carrying the DQB-TM*45 pattern. Included in this group were haplotypes
DH15A, DH15B, DH20A, DH24A and DH27A, which have a single set
of DQ genes, and haplotypes DH23A and DH28A, which have two sets
of DQ genes (Andersson &
Rask, 1988). The similarity between the DQ genes in this group
of haplotypes is also manifest in the DQB RFLP patterns, all of
the fragments of the DQB*1 pattern are present in the DQB*7A pattern
(Table 8; Sigurdardóttir
et al., 1988; see also Sigurdardóttir
et al., 1992).
Class
I - class IIa haplotypes. The class IIa region is tightly
linked to the class I region and, therefore, the definition of
class I - class IIa haplotypes was straightforward. With the exception
of two crossbred animals, each carrying two unique class I - class
IIa haplotypes, and one animal for which class Iia haplotypes
could not be determined, it was possible to assign class I - class
Iia haplotypes to the animals with a considerable degree of confidence
(Table 3). Table
12 summarizes the number of animals of each breed carrying
each of the 48 class I - class IIa haplotypes defined in this
workshop. Haplotypes were established on the basis of:
- segregation
in dam-calf pairs
- occurrence
opposite a segregating haplotype
- presence
in class IIa homozygotes
- statistically
significant associations, and/or
- occurrence
opposite a haplotype established on the basis of a statistically
significant association.
Only
haplotypes present in class IIa homozygotes or animals for which
segregation data was available should be considered confirmed.
Class
IIb region
Two DYA, 2 DOB and 3 DIB alleles have been identified in previous
studies (Andersson et al.,
1988; Andersson & Rask, 1988; Muggli-Cockett
& Stone, 1991; Stone &
Muggli-Cockett, 1993; van
Eijk et al., 1992a) and were found in the workshop animals.
The fragment sizes for the alleles were given in the materials
and methods section. Both RFLP and PCR-RFLP results for DYA were
available for 34 animals. The DYA RFLP and PCR-RFLP results were
completely correlated indicating a tight gametic association between
the independent polymorphisms detected by the two methods. Fifty-four
animals were typed for DYA, DOB and DIB. DYA-DOB-DIB haplotypes
could be unequivocally determined for: 15 animals homozygous at
all 3 loci, 12 animals homozygous at 2 of the 3 loci, and an additional
11 animals for whom segregation data was available. Only 5 of
the possible 12 DYA-DOB-DIB haplotypes were found in these 38
animals (Table 13). The class
IIb haplotypes for the remaining 16 animals could be unambiguously
determined if it was assumed that only the 5 haplotypes were present.
The data show that there were only 4 common class IIb haplotypes,
and probably only a single rare haplotype, in this diverse population
which included animals of 9 different breeds.
Previous reports have indicated that the class IIb region is separated
from the rest of the BoLA complex by a high recombination frequency
(Andersson et al., 1988;
Stone & Muggli-Cockett, 1993;
van Eijk et al., 1993).
Although the workshop included only 1 informative half sibling
family, 1 of the 7 offspring of Klaus, a Danish Black Pied bull,
was a recombinant. Four offspring inherited an A19(A6)-DH31A-DYA*1-DOB*1-DIB*2
haplotype, 2 offspring inherited a w36(A20)-DH8A-DYA*2-DOB*1-DIB*1
haplotype and the last offspring inherited a recombinant w36(A20)-DH8A-DYA*1-DOB*1-DIB*2
haplotype.
Discussion
The results of this workshop demonstrate the power of the simultaneous
application of multiple typing techniques to a common group of
animals. The use of multiple techniques made it possible to characterize
BoLA haplotypes in considerable detail and helped to elucidate
the relationships between the haplotypes (Tables
10 and 11). The data suggest
that both mutation and recombination have been involved in the
evolution of BoLA class IIa haplotypes. Recombination between
the DR and DQ genes would be the likely explanation for haplotypes:
DH3B, with DRB genes closely related to DH3A and DQ genes closely
related to DH12B; and DH22F, with DRB genes closely related to
DH22C, DH22E and DH22B and DQ genes closely related to DH9A, DH11A
and DH11B (Table 11). It is noteworthy
that a DRB*11A,DQA*3A,DQB*4A haplotype that could be the predecessor
of DH22F has been identified in Angus cattle (Davies
et al., 1992a). DH7A is an intriguing haplotype because it
has been found in every cattle population that has been investigated
and, therefore, appears to be old, yet closely related haplotypes
expressing distinct class II products have not been identified.
It is possible that this haplotype has been conserved because
it diverged rapidly and, therefore, due to its unique sequence
could not recombine with other haplotypes, i.e. this may be an
example of a conserved polymorphic genetic block (Rayssiguier
et al., 1989; Radman, 1991).
Both DR and DQ typing are required to define all of the polymorphism
encoded in the class IIa region. Furthermore, multiple techniques
must be used, or multiple loci must be typed, to define all of
the expressed polymorphism at any given locus. For example DRB3
PCR-RFLP, which detects sequence variation in exon 2 which encodes
the antigen presentation groove and is the most powerful locus
specific technique available, misses a significant amount of expressed
DRB3 polymorphism detected by IEF (Table
10). It is noteworthy that the combination of two DNA based
techniques, DRB3 PCR-RFLP and DRB3-MS PCR, apparently detected
all of the expressed DRB3 polymorphism. Moreover, in the three
instances where haplotypes had similar DRB IEF and DRB3 PCR-RFLP
types but different DRB3-MS alleles, significant DQ and/or DR
RFLP differences were present (Table
10). It is also noteworthy that in all but one instance a
given class IIa haplotype, even if present in multiple breeds,
was associated with a single DRB3-MS allele. The one exception,
DH22B with DRB3-MS*179, was only found in one animal, WK5-45,
and was not verified by retyping. The results from the workshop
together with the results presented by Ellegren
et al. (1993) indicate that the DRB3-MS is highly stable and,
therefore, is a useful marker for DRB3 polymorphism.
DRB RFLP patterns defined using cDNA or exon probes are complex
and the blots are difficult to interpret. Twelve new DRB RFLP
patterns were defined on the basis of the workshop results. However,
7 of the new patterns were only found in 1 or 2 animals and need
confirmation. Actually additional segregation data for all of
the DRB RFLP types would be useful because some fragments probably
represent class IIb region genes (Andersson
et al., 1988). In many instances there were multiple DRB RFLP
allelic patterns associated with identical DRB3 PCR-RFLP patterns
and IEF types (Table 10). Therefore,
if DRB3 is the only functional bovine DRB gene, as the study by
Burke et al. (1991) suggests,
much of the DRB RFLP polymorphism is likely to be functionally
irrelevant.
DQA and DQB RFLP patterns are simpler to interpret than DRB patterns.
Furthermore, because some or all of the haplotypes with duplicated
DQ genes express both sets of genes (Bissumbhar
et al., 1994), DQ RFLP patterns probably reflect expressed
polymorphism reasonably well. The number of DQA and DQB genes
present in different haplotypes can be deduced from the number
of fragments detected (Tables 7
and 8). Class IIa haplotypes with
DQA*1,DQB*1, DQA*2,DQB*2, DQA*3,DQB*3, DQA*4, DQB*4 and DQA*14,DQB*14
have single DQA and DQB genes; haplotypes with DQA*13,DQB*13 have
2 DQA genes and 1 DQB gene; and the remaining haplotypes have
duplicated DQA and DQB genes (Andersson
& Rask, 1988; Sigurdardóttir
et al., 1991b).
For each DRB IEF allele two bands were observed (Figure
1). In an earlier collaborative effort involving UTN and EDS,
11 DRB IEF alleles were described (Joosten
et al., 1989). In this workshop a consensus was reached on
12 DRB IEF alleles, which were renamed DRBF1-12. Alleles not confirmed
by more than one laboratory will continue to be referred to using
the local, EDF or UDF, nomenclature. Seven of the newly accepted
DRBF types, DRBF1, 3, 8, 9, 10, 11 and 12 were associated with
single DRB3 PCR-RFLP types. DRBF11 was associated with two different
DRB3-MS alleles; however, it is likely that haplotypes with identical
DRB3 PCR-RFLP and DRB IEF types express identical DRB3 alleles.
DRBF2, 4, 5, 6 and 7 were associated with more than one DRB3 PCR-RFLP
type and, therefore, these DRB IEF types do not define unique
DRB3 alleles.
Determination of the serological specificities associated with
each class Iia haplotype was complicated by:
- the
small population size
-
missing data
- lack
of segregation data
-
tails associated with some sera,
- confounding
of specificities present on both haplotypes, and
- lack
of information on locus specificity.
Nevertheless,
because of the wealth of information available on the haplotypes
carried by the animals, it was possible to tentatively identify
the locus specificity of the 5 workshop (Dw) and 8 provisional
(Dc) class II specificities (Table
11). It is interesting that all of the Dw specificities, the
better defined specificities, appear to be DQ specificities. The
DQ specificities seem to be supertypic specificities while the
DR specificities appear to define individual alleles (Table
11). This presumably reflects the distinct DQA and DQB subtypes
defined by RFLP and sequence analysis (Sigurdardóttir
et al., 1991b; Sigurdardóttir
et al., 1992), as well as the restriction of most expressed
DR polymorphism to the first domain (exon 2) of DRB3. The DQA
and/or DQB proteins may have both first and second domain epitopes.
Furthermore, some haplotypes evidently express two distinct DQ
products (Bissumbhar et al.,
1994).
It is exceedingly useful to define serological specificities on
the basis of the alleles with which they react. The relationships
between specificities are also important. Although in the workshop
Dw4 behaved as a Dw1 subtype, Dw4 also encompasses the DQA*6,DQB*6
haplotypes (Bernoco et al., 1991;
Davies et al., 1992a) which
have DQA genes related to those found in the DQA*5,DQB*5 haplotype
(Sigurdardóttir
et al., 1991b) but do not express Dw1. It is essential that
the associations between specificities and DR or DQ alleles tentatively
identified here be confirmed in segregation studies. Furthermore,
as many of the antisera had complex reactivity patterns, absorption
studies need to be conducted. Since a few associations were probably
masked in the small workshop population and all known haplotypes
have not been tested, the inventory of alleles encompassed by
the specificities should be considered tentative and incomplete.
Forty-eight class I - class IIa haplotypes were defined in the
workshop animals. In the populations that have been studied only
a limited number of class I - class IIa combinations have been
found. This is a reflection of the tight linkage that exists between
the class I (BoLA-A) and class IIa regions. The fact that in the
small workshop population 7 class I - class IIa haplotypes were
found in more than one breed supports previous findings suggesting
that the BoLA class I and class IIa regions exhibit strong linkage
disequilibrium. Haplotypes occurring in multiple breeds are candidates
for ancestral haplotypes. The A11-DH24A, w36(A20)-DH8A and w40(A14,A8)-DH11A/DH11B
haplotypes are particularly strong candidates because these haplotypes
occur in a large number of breeds and are often found at a high
frequency (Bernoco et al., 1991;
Davies et al., 1992a).
It was found that the DYA, DOB and DIB genes are tightly linked
and are part of a conserved MHC block (Table
13). This was an unexpected finding and is intriguing as the
class II genes in this region are not particularly polymorphic.
Although DYA and DIB are complete, intact class II genes (
van der Poel et al., 1990; Stone
& Muggli-Cockett, 1990) there is no evidence for their
expression (Stone & Muggli-Cockett,
1993) and their function is unknown. The MHC peptide transporter
(TAP1 and TAP2) and proteasome (LMP2 and LMP7) genes are located
just centromeric to DOB in the human MHC (Trowsdale
et al., 1991). Consequently, the bovine TAP1 and LMP7 genes
identified by Davies et al.
(1992b) are probably located in the class IIb region. Since TAP
polymorphism can effect class I mediated antigen presentation
(Powis et al., 1992), better
characterization of the class IIb region of cattle is clearly
needed.
The Fifth BoLA Workshop has greatly enhanced our understanding
of the bovine class IIa and class IIb regions. A new laboratory
workshop has not been undertaken at this time. However, the development
of a sequence based class II nomenclature has been set as an objective
for the period leading up to the 1994 Animal Genetics meeting.
Anyone wishing to contribute sequence data should contact Dr.
Noelle Muggli-Cockett, LUT.
Acknowledgments
The chairman, C.J. Davies, was supported by: the Department of
Animal Breeding, Wageningen Agricultural University; the Organization
for Economic Co-operation and Development, Project on Biological
Resource Management; and the Helminthic Diseases Laboratory, United
States Department of Agriculture.
