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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
1
2
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
RESULTS
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.
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.
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 B*0801DRB1*0301, B*1302-DRB1*0701 and B*4402-DRB1*0401,
respectively; two samples from the Korea Marrow Donor
Program (B*3701-DRB1*1001); two from a Mongolian
population study (B*5801- DRB1*0301); and two samples
a Zambian HIV-1 transmission study (B*4201-DRB1*0302).
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).
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.htm
International Immunogenetics Project (IMGT) HLA Sequence
Database: http://www.ebi.ac.uk/cgi-bin/imgt/hla/get_all.cgi
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.
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 MICA*028- was typed as
heterozygote for MICA*00201/020 and
*00901. We obtained the same result by
SBT. The cell line TISI was reported as
MICA*017 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 MICA*019
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 MICA*049 or
*00901, and listed as *00901 / *049 in the
IHWG and IMGT-HLA databases. Our
SBT method typed this cell line as
homozygous for MICA*00901 (and STR
allele A6). The only difference between
MICA*00901 and *049 is at the
nucleotide position 109 in exon 3 and
LUY has G at this position (not C).
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
B*4403-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. MICA*00801 (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.
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.
REFERENCES
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(2000) Further characterization of MHC haplotypes demonstrates
conservation telomeric of HLA-A: update of the 4AOH and 10IHW cell
panels. Eur J Immunogenet 27:397-426
Dawkins R, Leelayuwat C, Gaudieri S et al (1999) Genomics of the MHC:
haplotypes, duplication, retroviruses and disease. Immunol Rev 167:275304
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 9:93-110
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 5:371-4
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 62:1-20