MICA Poster [M.Tevfik DORAK]

1
Re-examination and Completion of MICA Typings on
Conserved Extended Haplotypes Relationship with
Other Polymorphisms M Tevfik Dorak 1, Wenshuo
Shao 2, Helmut KG Machulla 3, Myoung Hee Park 4,
Elena Lobashevsky 2, Jianming Tang 2, Richard
Kaslow 2 1 School of Clinical Medical Sciences,
Newcastle University, U.K 2 Dept of
Epidemiology, University of Alabama at
Birmingham, USA 3 Interbranch HLA Lab, Martin
Luther University Medical School, Germany 4 Dept
of Lab Medicine, Seoul National University
College of Medicine, Korea
INTRODUCTION Despite having numerous published
reports on the MHC loci in conserved extended
haplotypes (CEH), also called ancestral
haplotypes, considerable amount of data are still
missing and there are also inconsistencies. We
aimed to unambiguously characterise 37 CEHs at a
number of MHC loci, including MICA, using
primarily IHWG reference cell lines in a single
study (Dorak et al, 2006). Here, we present the
MICA results.
RESULTS Table 1 summarises the complete results
on CEHs. We obtained the MICA types for CEHs
35.3, 35.4, 44.4, 50.1, 51.1, 62.2, 64.1 and 65.2
(represented by cell lines SPE/G, TISI, HOR,
EAV/AC (heterozygote), BM92, COL/E, CF996 and
HO301). The whole cell panel did not have any
example of MICA alleles 005, 013, and 014
similar to the results of other studies.
We confirmed that all CEHs with the same HLA-B
allele have the same MICA STR allele except HOR.
The cell line HOR is a heterozygous example of
CEH 44.4 but differs from other B4403-bearing
CEHs (STR allele A4 as opposed to A6). One
observation was that different alleles of
serologically indistinguishable specificities
(1401/1402 3501/3502 4001/4002 4402/4403)
were associated with different MICA STR alleles
in CEHs. MICA00801 (which includes STR A5.1 in
its allele definition) was present on a highly
divergent set of CEHs 7.1 7.2 8.1 13.1 37.1
44.1 47.1 60.1 60.2 and 60.3. This set did not
share any other allele at other loci.
Table 1. MICA typings in 37 conserved extended
haplotypes
A SNP at nt130 of exon 3 of the MICA gene has
functional importance in binding to NKG2D
receptor of NK cells. We investigated the
relationship of this polymorphism with HLA
haplotypes. With one exception (CEH 58.1), the
minor allele 130A was on the LTA 252A - NFKBIL1
-62A haplotype (CEH 18.1, 18.2, 18.3, 35.2, 35.3,
38.1, 51.1, 54.1, 57.1, 65.1 and 65.2). 10 out of
11 haplotypes that carried LTA allele 252G had
MICA 130G. This relationship may confound
associations with either loci.
MATERIAL AND METHODS We genotyped 101 IHWG
reference cell lines and nine additional
anonymous samples representing all 37
unambiguously characterized CEHs at MICA,
NFKBIL1, LTA, NCR3, AIF1, HSPA1A, HSPA1B, BF,
NOTCH4 and a SNP at HLA-DQA1 as well as MICA,
NOTCH4, HSPA1B and all five TNF STR polymorphisms
(Dorak et al, 2006). Complete typing results on
these samples are available on request as an
Excel file. CEH assignments were taken from the
latest update (Cattley et al, 2000) and reviews
(Dawkins et al, 1999 Yunis, 2003). Five CEHs
(35.1 58.2 59.1 61.1 62.4) usually included
in provisional lists of CEHs could not be
analysed because of insufficient characterisation
of them, in particular at the DNA level. Ten
additional HLA-B - DRB1 homozygous samples from
normal populations were also included in the
study. Three of these samples were from our
previous newborn study (origin South Wales,
U.K.) homozygous for B0801-DRB10301,
B1302-DRB10701 and B4402-DRB10401,
respectively two samples from the Korea Marrow
Donor Program (B3701-DRB11001) two from a
Mongolian population study (B5801- DRB10301)
and two samples a Zambian HIV-1 transmission
study (B4201-DRB10302). We only typed the cell
lines that were representatives of CEHs with
missing data or had inconsistent MICA results in
the literature using the modified SBT scheme
described by us (Shao et al, 2004).
CONCLUSION The overall CEH study 1) provided an
extensive catalogue of MHC polymorphisms in all
CEHs 2) unravelled interrelationships between
HLA and non-HLA haplotypical lineages 3)
resolved reported typing ambiguities, and 4)
described haplospecific markers for a number of
CEHs. The complete results on multiple MHC loci
on CEHs will be useful in design and
interpretation of HLA and disease association
studies. This study highlighted the need to
reexamine previously reported genotype data. This
is most urgent for the complotypes, the main
characteristics of CEHs, which are not routinely
typed by DNA-based methods.
Rees et al (2005) updated the MICA/B typings
that were inconsistent in the literature using a
new PCR-SSP scheme. The cell line EHM -the only
reference cell line for MICA028- was typed as
heterozygote for MICA00201/020 and 00901. We
obtained the same result by SBT. The cell line
TISI was reported as MICA017 elsewhere but our
result agreed with that of another study (016).
We confirmed the MICA type of the cell line HOR
as 004 (but not as 007). The cell line CF996
was typed as MICA019 by us disagreeing with some
other results. The MICA type of cell line HO301
was 011 but not 008. The cell line LUY has been
reported to be MICA049 or 00901, and listed as
00901 / 049 in the IHWG and IMGT-HLA databases.
Our SBT method typed this cell line as homozygous
for MICA00901 (and STR allele A6). The only
difference between MICA00901 and 049 is at the
nucleotide position 109 in exon 3 and LUY has G
at this position (not C).
REFERENCES Cattley SK, Williamson JF, Tay GK,
Martinez OP, Gaudieri S, Dawkins RL (2000)
Further characterization of MHC haplotypes
demonstrates conservation telomeric of HLA-A
update of the 4AOH and 10IHW cell panels. Eur J
Immunogenet 27397-426 Dawkins R, Leelayuwat C,
Gaudieri S et al (1999) Genomics of the MHC
haplotypes, duplication, retroviruses and
disease. Immunol Rev 167275-304 Dorak MT, Shao
W, Machulla HKG, Lobashevsky ES, Tang J, Park MH,
Kaslow, RA (2006) Conserved Extended Haplotypes
of the Major Histocompatibility Complex Further
Characterisation. Genes Immun (in press) Rees
MT, Downing J, Darke C (2005) A typing system for
the MHC class I chain related genes A and B using
PCR with sequence-specific primers. Genet Test
993-110 Shao W, Lobashevsky ES, Kaslow RA,
Dorak MT (2004) MICA intron 1 sequences of
conserved extended HLA haplotypes implications
for sequencing-based typing. Genes Immun 5371-4
Yunis EJ, Larsen CE, Fernandez-Vina M et al
(2003) Inheritable variable sizes of DNA
stretches in the human MHC conserved extended
haplotypes and their fragments or blocks. Tissue
Antigens 621-20
Several studies reported a variant form of the
MICA-STR allele R6 on different examples of CEH
65.1. We were unable to detect any difference
between the cell lines representing CEH 65.1 and
other cell lines carrying R6. The reason may be
that we separated amplification products on
polyacrylamide gels rather than with capillary
electrophoresis. We therefore could not confirm
the presence of a variant MICA-STR allele.
ONLINE DATABASES FOR CELL LINE DATA European
Collection of Cell Cultures Centre (ECACC)
http//www.ecacc.org.uk International
Histocompatibility Working Group (IHWG) Cell
Gene Bank http//www.ihwg.org/shared/cbankover.ht
m International Immunogenetics Project (IMGT)
HLA Sequence Database http//www.ebi.ac.uk/cgi-bi
n/imgt/hla/get_all.cgi