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NEW ALLELE
C O N T R I B U T I O N S T O T H I S S E C T I O N WI L L NO T U N D E R G O P EE R RE V I EW , B U T W I L L B E R E V I E W E D B Y T H E ED I T O R
*
HLA-A*2313 is closest to
A*2301 but is likely to
stimulate T cells when
mismatcheda
P. A. Horn, M. Verboom & R. Blasczyk
Institute for Transfusion Medicine, Hanover
Medical School, Carl-Neuberg-Str. 1, 30625
Hannover, Germany
Key words: A*2313; HLA-A; sequencing-based
typing
We here describe
the identification The novel allele
of the novel allele HLA-A*2313 differs
from A*2301 by the
HLA-A*2313,
amino acid
which has been
exchange
detected in a
potential German His70Gln.
bone marrow
donor. The complete typing results
were A*2313, *3301; B*0702, *1402;
DRB1*010201, *150101.
Cycle sequencing of exons 2 and 3
was performed following haplotypespecific amplification essentially as
previously described (1) using an
Applied Biosystems 3730 sequencer,
resulting in the detection of the new
allele.
The new allele is identical to
A*2301 except for a non-synonymous
nucleotide exchange at position 209 in
exon 2 (T!C) changing codon 70
from CAC to CAG (Figure 1).
Because this triplet is frequently
found at codon 70 in a number of
alleles (e.g., most A*03, A*11, A*29,
A*34, A*66 and A*68 alleles), the
new A*23 allele probably originated
by gene conversion.
The data have been submitted to
the IMGT/HLA database (2), and
the name A*2313 has been officially
assigned by the WHO Nomenclature
Committee in August 2005. The
nucleotide sequence is available in the
EMBL, GenBank and DDBJ
Nucleotide Sequence Databases
under the accession number
AM072344.
The resulting amino acid exchange
His!Gln at position 70 of the
mature polypeptide (Figure 2)
affects a position that is part of the
alpha helix and that participates in
A*2301
A*2302
A*2303
A*2304
A*2305
A*2306
A*2307N
A*2308N
A*2309
A*2310
A*2311N
A*2312
A*2313
60
TGG
-------------------------
GAC
-------------------------
GAG
--------------C----------
GAG
-------------------------
ACA
-------------------------
65
GGG
--------------C----------
AAA
----------------T
---------
*
the formation of pockets A, B and C
of the peptide-binding groove. The
rather high Risler amino acid substitution score (3) of 68 indicates that
these two amino acids are relatively
dissimilar. Accordingly, it can be
expected that the peptide-binding
properties are altered. If so, a possible
mismatch with any other known
A*23 variant may stimulate alloreactive T cells and thus impair the
clinical outcome of stem cell
transplantation.
Correspondence
Prof Dr Rainer Blasczyk
Institute for Transfusion Medicine
Hanover Medical School
Carl-Neuberg-Str. 1
30625 Hannover
Germany
Tel.: þ49 511 532 6700
Fax: þ49 511 532 2079
e-mail: [email protected]
GTG
--------------A----------
AAG
-------------------------
GCC
-------------------------
70
CAC
------------------------G
TCA
-------------------------
CAG
-------------------------
ACT
-------------------------
GAC
--------------------C----
75
CGA
-------------------------
Figure 1 Nucleotide alignments of codon 60-75 of the new allele in comparison to the consensus of
all other A*23 alleles.
a
The name A*2313 has been officially
assigned by the WHO Nomenclature
Committee in August 2005. This follows
the agreed policy that, subject to the
conditions stated in the most recent
Nomenclature Report (4), names will be
assigned to new sequences as they are
identified. Lists of such new names will be
published in the following WHO
Nomenclature Report.
166
A*2301
A*2302
A*2303
A*2304
A*2305
A*2306
A*2309
A*2310
A*2312
A*2313
60
Trp
-------------------
Asp
-------------------
Glu
----------Gln
-------
Glu
-------------------
Thr
-------------------
65
Gly
----------Arg
-------
Lys
----------Asn
-------
Val
----------Met
-------
Lys
-------------------
Ala
-------------------
70
His
----------------Gln
Ser
-------------------
Gln
-------------------
Thr
-------------------
Asp
--------------His
---
75
Arg
-------------------
Figure 2 Deduced amino acid sequence from the sequences as shown in A.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
Received 01 November 2005; accepted 09
November 2005
doi: 10.1111/j.1399-0039.2005.00541.x
Acknowledgments
The authors thank Kerstin Müller and
Anja Hundt for excellent technical assistance. This study was supported in part
by grant KF0483301KLF3 (BMWA).
References
1. Kotsch K, Wehling J, Kohler S, Blasczyk
R. Sequencing of HLA class I genes
based on the conserved diversity of the
noncoding regions: sequencing-based
typing of the HLA-A gene. Tissue
Antigens 1997: 50: 178–91.
2. Robinson J, Waller MJ, Parham P
et al. IMGT/HLA and IMGT/MHC:
sequence databases for the study
of the major histocompatibility
complex. Nucleic Acids Res 2003: 31: 311–4.
3. Risler JL, Delorme MO, Delacroix H,
Henaut A. Amino acid substitutions in
structurally related proteins. A pattern
recognition approach. Determination of a
new and efficient scoring matrix. J Mol
Biol 1988: 204: 1019–29.
4. Marsh SG, Albert ED, Bodmer WF et al.
Nomenclature for factors of the HLA
system, 2004. Tissue Antigens 2005: 65:
301–69.
Identification of a novel
HLA-A*02 allele, A*0287, in
the cord blood of a Korean
baby
H.-S. Yun*, J.-Y. Baek*, S.-S. Hur, O.-J. Kwon &
K. Kwack
Pochon CHA University, Seongnam 463-836, Korea
Key words: HLA-A*0287; Korean; sequence-based
typing (SBT)
The HLA-A2 antigen is the most
common HLA-A antigen among
major ethnic groups, and HLA-A*02
is one of the most frequent and
polymorphic alleles at this locus.
*These authors contributed equally to
this work.
A novel HLAWe reported the
A*02 allele,
identification of a
A*0287, was
new HLA-A*02
detected in the
allele, A*0287,
cord blood of a
with a codon
Korean baby,
change at position
during HLA
77 in exon 2 in the
genotyping of
cord blood of a
cord blood
Korean baby.
donated to the
cord blood
bank. The nucleotide sequence of
HLA-A*0287 differs in exon 2 from
that of the A*0281 allele by one
nucleotide (a G to A substitution at
codon 77) (Figure 1). Donor’s HLA
types were HLA-A*020601, *0287;
HLA-B*150101, *270502; HLACw*010201, *040101; HLADRB1*010101, *0406; HLADRB4*010301; HLA-DQA1*0101,
*030101; HLA-DQB1*030201,
*050101; HLA-DPA1*010301,
*020202; HLA-DPB1*020102, *0501.
The new HLA-A*02 allele,
A*0287, was identified by the
sequence-based typing method.
Exons 2 and 3 of the HLA-A gene
were sequenced with a BigDyeTM
Terminator v3.1 Cycle Sequencing
Ready Reaction Kit (Applied
Biosystems, Foster City, CA, USA)
on an ABI PRISMÒ 3100 Genetic
Analyzer or ABI PRISMÒ 3730XL
DNA Analyzer (Applied Biosystems).
Accession numbers for HLA*0287,
DQ139310 (exon 2) and DQ139311
(exon 3) were assigned after deposition of the sequence in to the
GenBank, and an official name,
HLA-A0287, was assigned by the
WHO nomenclature committee in
August 2005.
The novel HLA-A*0287 generated
a single-nucleotide substitution in
exon 2, resulting in a change in the
amino acid sequence from serine
(AGC) to asparagine (AAC) in the
HLA-A*0281 allele.
Most polymorphisms of HLA class
I antigens are present in exons 2 and
3, which encode the a1 and a2
domains of HLA-A molecules,
involved in both peptide-binding and
HLA-restricted recognition by the
T-cell receptor. Residue 77 is located
on the longer curved a-helix in the
helical region of a1 and may make
contact with both peptide and TCR
(1). Compared with the HLA-A*0281
allele, the introduction of a bulky
amino acid, such as asparagine,
instead of serine, into the peptidebinding groove may affect HLA
functions.
In conclusion, a novel HLAA*0287 allele, detected in the cord
blood of a Korean baby, will help us
understand the HLA function and
the immune response.
A*0281
A*0287
1
5
10
15
20
25
GC TCT CAC TCC ATG AGG TAT TTC TTC ACA TCC GTG TCC CGG CCC GGC CGC GGG GAG CCC CGC TTC ATC GCA GTG
-- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
A*0281
A*0287
30
35
40
45
50
GGC TAC GTG GAC GAG ACG CAG TTC GTG CGG TTC GAC AGC GAC GCC GCG AGC CAG AGG ATG GAG CCG CGG GCG CCG
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
A*0281
A*0287
55
60
65
70
75
TGG ATA GAG CAG GAG GGT CCG GAG TAT TGG GAC GGG GAG ACA CGG AAA GTG AAG GCC CAC TCA CAG ACT GAC CGA
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
A*0281
A*0287
80
85
90
95
100
GAG AGC CTG CGG ATC GCG CTC CGC TAC TAC AAC CAG AGC GAG GCC G GT TCT CAC ACC GTC CAG AGG ATG TAT GGC
--- -A- --- --- --- --- --- --- --- --- --- --- --- --- --- - -- --- --- --- --- --- --- --- --- ---
A*0281
A*0287
105
110
115
120
125
TGC GAC GTG GGG TCG GAC TGG CGC TTC CTC CGC GGG TAC CAC CAG TAC GCC TAC GAC GGC AAG GAT TAC ATC GCC
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
A*0281
A*0287
130
135
140
145
150
CTG AAA GAG GAC CTG CGC TCT TGG ACC GCG GCG GAC ATG GCA GCT CAG ACC ACC AAG CAC AAG TGG GAG GCG GCC
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
A*0281
A*0287
155
160
165
170
175
CAT GTG GCG GAG CAG TTG AGA GCC TAC CTG GAG GGC ACG TGC GTG GAG TGG CTC CGC AGA TAC CTG GAG AAC GGG
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
A*0281
A*0287
180
AAG GAG ACG CTG CAG CGC ACG G
--- --- --- --- --- --- --- -
Figure 1 Alignment of nucleotide sequence of exons 2 and 3 of HLA-A*0281 and HLA-A*0287
allele. Dashes (-) indicate identity with the HLA-B*0281 sequence, and pipe (|) indicates the exon/
intron. Changed amino acid is indicated to a circle.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
167
NEW ALLELE Alerts
Acknowledgments
This study was supported by grants from
the Korea Science and Engineering
Foundation (R01-2002-000-00267-0) and
from the Ministry of Health and Welfare
(0405-BC02-0604-0004).
Correspondence
KyuBum Kwack or Oh-Joong Kwon
Pochon CHA University
Seongnam 463-836
Korea
Tel: 82 31 725 8376
Fax: 82 31 725 8350
e-mail: [email protected]
Received 10 November 2005; accepted 15
November 2005
doi: 10.1111/j.1399-0039.2005.00544.x
Reference
1. Bjorkman PI, Saper MA, Samraoui B,
Bennett WS, Strominger JL, Wiley DC.
Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987:
329: 506–12.
molecular typing. We further A new HLA-A allele,
investigated the HLA-A*3308, has been
sample by poly- identified in Chinese
individual by
merase chain
sequence-based typreactionsequence-based ing. This allele shows
five nucleotides
typing (PCRdifference with HLASBT).
A*330301 in exon 2 at
The new
codon 56(GGG!GGT),
A*3308 allele
codon 62(CGG!GGG),
showed unexcodon
63(AAC!GAG)
pected reaction
and
codon
patterns in routine PCR-SSO 66(AAT!AAA),
typing (Tepnel resulting in three
amino acids change
Lifecodes,
Stamford, CT, from Arg to Gly(R62G),
Asn to Glu(N63E) and
USA).
Asn to Lys(N66K).
According to
the data analysis, it can not assign to the final result.
A*330301
A*3308
Identification of a new
allele, HLA-A*3308, in the
Chinese population†
While we override the probe #1109(50 ACCGGAACACACGG.AATGT-30 )
from negative to positive, the assignment for A*02XX,33XX were
obtained. The SSO testing results
suggested that it might be something
new in A*33 within probe #1109
region; therefore, we further investigate the sample by using SBT method
after cloning.
To confirm the presence of a novel
allele in the sample, the entire coding
region of HLA-A gene was amplified
with PCR from genomic DNA.
Amplified PCR product was directly
ligated into pCR4-TOPO vector and
cloned by using TOPO cloning
sequencing kit (Invitrogen, Carlsbad,
CA, USA). Many recombinant colonies of the sample on Luria-Bertani
plates were selected at random, and
plasmid DNA was extracted from
74 G C T C C C A C T C C A T G A G G T A T T T C A C C A C A T 103
------------------------------
A*330301 104 C C G T G T C C C G G C C C G G C C G C G G G G A G C C C C 133
A*3308
------------------------------
A*330301 134 G C T T C A T C G C C G T G G G C T A C G T G G A C G A C A 163
L.-X. Yan, F.-M. Zhu, Q.-F. Lv, W. Zhang & J. He
HLA Typing Laboratory, Blood Center of Zhejiang
Province, Hangzhou, Zhejiang Province 310006,
China
Key words: HLA-A*33; new allele; SBT
A*3308
------------------------------
A*330301 164 C G C A G T T C G T G C G G T T C G A C A G C G A C G C C G 193
A*3308
------------------------------
A*330301 194 C G A G C C A G A G G A T G G A G C C G C G G G C G C C G T 223
More than 396 alleles of HLA-A have
been officially recongnized to date (1, 2).
In this report, we present a novel allele
which has been detected by unusual
probe reaction patterns during routine
A*3308
------------------------------
A*330301 224 G G A T A G A G C A G G A G G G G C C G G A G T A T T G G G 253
A*3308
----------------T-------------
A*330301 254 A C C G G A A C A C A C G G A A T G T G A A G G C C C A C T 284
†The name A*3308 has been officially
assigned by the WHO Nomenclature
Committee in June 2005. This follows
the agreed policy that, subject to the
conditions stated in the most recent
Nomenclature Report (2), names will
be assigned to new sequences as they
are identified. List of such new names
will be published in the following WHO
Nomenclature Report.
168
A*3308
--G--G-G--------A-------------
A*330301 284 C A C A G A T T G A C C G A G T G G A C C T G G G G A C C C 312
A*3308
------------------------------
A*330301 313 T G C G C G G C T A C T A C A A C C A G A G C G A G G C C G 343
A*3308
------------------------------
Figure 1 Alignment of the exon 2 of A*33 with the sequences of A*330301. Dashes indicated nucleotide
identity with the A*330301 sequence. Numbers above the sequence correspond to nucleotide position.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
each colony and used as template for
sequencing reaction. Both strands of
exons 2, 3 and 4 of HLA-A were
sequencing. The DNA sequence was
determined with a cycle sequencing
kit (BigDye terminator, Applied
Biosystems, Foster City, CA, USA)
and a genetic analyzer (ABI Prism
377, Applied Biosystems)
according to the manufacturers’
instructions (3). The sequencing
results showed HLA-A alleles of the
sample as A*0201 and the
variant allele. The variant allele has
been officially named A*3308 by the
WHO Nomenclature Committee. The
new A*3308 allele shows a sequence
identical to that of A*330301 in exons
3 and 4, but shows five nucleotides
mismatches with A*330301 in exon 2
(Figure 1) at nucleotide positions
240G!T, 256C!G, 259A!G,
261C!G and 270T!A, which result
in three amino acids change from Arg
to Gly(R62G), Asn to Glu(N63E)
and Asn to Lys(N66K). The sample
was typed as A*0201,*3308;
B*1511,5502; DRB1*1405,0803.
phenotypes in Chinese persons, including
a novel nonfunctional FUT1 allele.
Transfusion 2005: 45: 725–30.
A novel HLA-A*680104
varianta
G. A. Heymann, A. Lassahn, C. Schönemann &
A. Salama
Institute of Transfusion Medicine, Campus
Virchow-Klinikum, Charité – Universitätsmedizin
Berlin, Berlin, Germany
Key words: allele-specific amplification; DNA
sequencing; SSO; SSP
Correspondence
Prof. Li-Xing Yan
Director, Blood Center of Zhejiang Province
Wulin Road 345
Hangzhou, Zhejiang Province 310006
China
Tel: 86 571 85167801
Fax: 86 571 85065851
e-mail: [email protected]
We describe a novel human leukocyte
antigen A (HLA-A) allele, A*680104,
which was initially identified by lowresolution HLA-typing techniques in
an adult bone marrow transplant
recipient of Caucasian origin.
The variant
A novel, silent human
was recognized by low- leukocyte antigen A
allele, A*680104, with
resolution
a variation of codon
HLA-A typ181 (ACG!ACT)
ing of the
(conserved Thr) is
DNA sample
described.
using the
Dynal RELISSO HLA-A kit (batch no. PK622,
DynalTM, Oslo, Norway). This testing resulted in positive reactions at
positions 2, 3, 5, 9, 10, 11, 12, 14, 17,
19, 24, 26, 28, 30, 36, 38, 41, and 42
and did not match any allele
Received 16 July 2005; revised 28 July 2005,
12 August 2005, 7 September 2005; accepted
20 September 2005
a
doi: 10.1111/j.1399-0039.2005.00519.x
References
1. Schreuder GM, Hurley CK, Marsh SG
et al. The HLA Dictionary 2004: a summary of HLA-A, -B, -C, -DRB1/3/4/5
and -DQB1 alleles and their association
with serologically defined HLA-A, -B, -C,
-DR and -DQ antigens. Tissue Antigens
2005: 65: 1–55.
2. Marsh SG, Albert ED, Bodmer WF et al.
Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69.
3. Yan L, Zhu F, Xu X, Hong X, Lv Q.
Molecular basis for para-Bombay
The nucleotide sequence data presented in
this report have been submitted to the
EMBL nucleotide sequence database and
were assigned the accession number
AM062639. The name HLA-A*680104
has been officially assigned by the WHO
Nomenclature Committee in August 2005.
This follows the agreed policy that, subject
to the conditions stated in the most recent
Nomenclature Report (1), names will be
assigned to new sequences as they are
identified. Lists of such new names will be
published in the following WHO
Nomenclature Report.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
combination in the Dynal PATTERN
MATCH program database. Without
probe 41, a result (A*3201, 6808) was
found, but probe 41 does not reach
the codon 181. Finally, we cannot
blame any oligonucleotide of this
batch with a sequence in the user
manual for a detection failure. Next,
we performed HLA-A typing polymerase chain reaction sequence-specific primer SSP), including different
SSP kits from GenovisionTM, Vienna,
Austria [HLA-A low resolution (lot
no. N28), HLA-A*32 (lot no. N06),
HLA-A*68 (lot no. M67)]. When
comparing the sequences of primers
that resulted in amplification reactions
in concordance with the assumed
HLA-type A*3201,6801, the results
seemed to originate from primers not
affecting the nucleotide 618 of HLAA*6801. Serologic typing was performed using panels of antisera
(OneLambda by BMT, Merbusch,
Germany, Tray M144A, Lot 003). The
M144A tray gave positive reactions
with all antisera against A32 and A68.
Finally, we performed a sequencebased analysis of the novel HLA-A
variant in two different DNA probes
of the same patient using the
ProtransTM S4 HLA-A kit (lot no.
441E03, Protrans, Ketsch, Germany).
Briefly, the Protrans S4 kit allele-specific amplification of a region containing exons 2 and 3 resulted in
separate amplification products covering HLA-A*29/*32/*74 and HLAA*68/*69/*3401/*3405/*6602/*6603,
respectively. The remaining primers
and deoxyribonucleoside triphosphates were removed by ExoSAP-IT
(Amersham Biosciences, Cardiff, UK)
digestion. The amplification products
were used for the cycle-sequencing
reactions. For each sequencing reaction, 2 ml of the treated amplification
products were mixed with 2 ml BigDye
1.1 (Applied Biosystems, Warrington,
UK) and 6 ml sequencing primer and
incubated [(96˚ C, 1 min), 25 (96˚ C,
10 s; 50˚ C, 5 s; 60˚ C, 4 min), (4˚ C)]. The
following sequencing primers included
in the Protrans S4 HLA-A kit were
169
NEW ALLELE Alerts
Codon
>A*680101
>A*680102
>A*680103
>A*680104
>A*6814
>A*2906
>A*010103
>A*240302
.CAGCGC
.......
.......
.......
.......
.......
.......
.......
181
ACG
...
..C
..T
G..
G..
..T
..T
GACGCCCCC-.........-.---------.---------.---------.---------...C.....-.----------
Figure 1 Nucleotide alignment of the alleles of human leukocyte antigen A (HLA-A). Codon 181 is
labeled. A*680104 exhibits a sequence motif found in two other HLA-A alleles, but not A*68 group.
Some bases are indicated by dots. For A*2906, A*010103, and A*240302, see explanation in the text.
Six leading bases before codon 181 are given.
used: A-E2F (forward, intron 1), AE2R (reverse, intron 2), A-E3F (forward, intron 2), and A-E3R (reverse,
intron 3). By using this sequence-based
approach, clear identification of the
allele A*3201 and the new HLAA*680104 variant was possible. HLAA*680104, which is most closely
related to A*680101, exhibits a substitution of codon 181 (ACG!ACT).
This variation results in no amino acid
substitution. The sequence motif at
codon 181 is also found in A*010103
and A*240302 (Figure 1) (2). Besides
those alleles, A*680103 shows a silent
ACG!ACC exchange, and A*6814
and A*2906 (3) show an ACG!GCG
exchange, resulting in an amino acid
change Thr!Ala. In all other alleles of
the HLA-A, the amino acid at codon
181 is found to be a chemically neutral
threonine. Because A*680104 is structurally unaltered, finding a bone marrow donor may not be problematical.
Correspondence
Dr G. A. Heymann
Institute of Transfusion Medicine
Campus Virchow-Klinikum
Charité – Universitätsmedizin Berlin
Augustenburger Platz 1
13353 Berlin
Germany
Tel: 49 30 450 553089
Fax: 49 30 450 553988
e-mail: [email protected]
Received 8 September 2005; revised 20
September 2005; accepted 30 September 2005
doi: 10.1111/j.1399-0039.2005.00525.x
170
References
1. Marsh SGE, Albert ED, Bodmer WF
et al. Nomenclature for factors of the
HLA System, 2004. Tissue Antigens 2005:
65: 301–69.
2. Ellis J, Steiner N, Kosman C et al.
Seventeen more novel HLA-A locus
alleles. Tissue Antigens 2000: 55: 369–73.
3. Steiner NK, Rizzo M, Bradshaw D et al.
Seventeen novel HLA-A alleles. Tissue
Antigens 2003: 62: 256ff.
Identification of two new
HLA-A variants, HLAA*2911 and HLA-A*6827†
K. R. Alkharsah1, M. Dedicoat1, D. S. DeLuca2,
T. F. Schulz1 & R. Blasczyk2
1 Institute of Virology, Hannover Medical School,
Hannover, Germany
2 Institute of Transfusion Medicine, Hannover
Medical School, Hannover, Germany
Key words: HLA-A; new allele; South Africa; Zulu
†The names A*2911 and A*6827 have
been officially assigned by the WHO
nomenclature committee in December
2003 and in June 2004, respectively.
This follows the agreed policy that,
subject to the conditions stated in the
most recent nomenclature report (1),
names will be assigned to new
sequences as they are identified. List
of such new names will be published in
the following WHO Nomenclature
Report.
The HLA system comprises We report here the
the most poly- identification of the
morphic loci, new alleles HLAif not the most A*2911 and HLAA*6827 in two healthy,
at all, in the
human genome. unrelated Zulus
Improvements (Hlabisa, South
of new HLA- Africa).
sequencing
strategies contributed to the identification of new alleles and therefore to
the expansion of the HLA diversity.
About 340 HLA-A alleles have been
reported until October 2004 according to the WHO Nomenclature
Committee for Factors of the HLA
System. Here we present the identification of two new variants, HLAA*2911 and A*6827 (1), in two
unrelated female Zulus in Hlabisa,
South Africa.
The two new alleles were identified
during HLA-A typing in a group
of healthy mothers attending the
vaccination health clinics in Hlabisa,
South Africa. Genomic DNA was
extracted from saliva samples of the
two females by QIAamp DNA blood
mini kit (QIAGEN, Hilden, Germany).
Exon 2 and 3 of HLA-A were PCRamplified locus specifically and
sequenced in forward and reverse
directions (2). Sequencing analysis
was performed with the 3730 Genetic
Analyzer (Applied Biosystem,
Foster city, CA, USA), and the data
were analysed by the SEQPILOT program version 2.0 (Protrans, Ketsch,
Germany).
The sequence of HLA-A*2911
was confirmed by hemizygous
sequencing. This new variant when
compared to its closely related allele,
HLA-A*290201, was found to be
characterized by a non-synonymous
nucleotide exchange (A!C) at
nucleotide position 151 of exon 3.
This resulted in the amino acid
exchange Gln!Pro at position 141
of the mature polypeptide. This
position, located in the H1 helix of
the a2-domain, is probably not
directly involved in peptide binding.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
(A)
A*01010101
A*29010101
A*29010102N
A*290201
A*290202
A*290203
A*2903
A*2904
A*2905
A*2906
A*2907
A*2908N
A*2909
A*2910
A*2911
A*2912
A*2913
A*2914
135
GCG
-----------------------------------
GCG
-----------------------------------
GAC
-----------------------------------
ATG
-----------------------------------
5
ATG
---------------------------------------------------------------
AGG
---------------------------------------------------------------
TAT
---------------------------------------------------------------
TTC
---------------------------------------------------------------
GCA
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
--G
140
GCT
-----------------------------------
CAG
---------------------------C-------
ATC
-----------------------------------
ACC
-----------------------------------
TTC
-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-
10
TTC
--C
--T
--C
--C
--T
--T
--C
--T
--T
--T
--C
--T
--C
--T
--T
--C
--C
--C
--T
--T
--C
--T
--T
--T
--C
--T
--T
--C
--C
--C
--C
TCC
---------------------------------------------------------------
GTG
------A---------------------------A-----A-------------------A--
TCC
---------------------------------------------------------------
AAG
C-C-C-C-C-C-C-C-C-C-C---C-C-C-C-C--
145
CGC
-----------------------------------
AAG
-----------------------------------
TGG
-----------------------------------
GAG
-----------------------------------
GCG
-----------------------------------
150
GTC
-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-
CGG
---------------------------------------------------------------
15
CCC
---------------------------------------------------------------
GGC
---------------------------------------------------------------
CGC
---------------------------------------------------------------
GGG
---------------------------------------------------------------
GAG
---------------------------------------------------------------
20
CCC
---------------------------------------------------------------
(B)
A*01010101
A*680101
A*680102
A*680103
A*6802
A*680301
A*680302
A*6804
A*6805
A*6806
A*6807
A*6808
A*6809
A*6810
A*6811N
A*6812
A*6813
A*6814
A*6815
A*6816
A*6817
A*6818N
A*6819
A*6820
A*6821
A*6822
A*6823
A*6824
A*6825
A*6826
A*6827
A*6828
Figure 1 Alignment of nucleotide sequence displayed in codons of the HLA-A*29 and A*68 showing the
location of the new mutation. (A) Alignment of HLA-A*29 alleles. (B) Alignment of HLA-A*68 alleles.
Yet, since proline is a helix breaker
(3, 4), the structure of the neighbouring pocket F may be altered,
which might result in a change
of the peptide-binding
characteristics of the new
variant compared to the A*2902
molecule.
The mutation occurred in a region
which is highly conserved in all classical and non-classical HLA class I
alleles (Figure 1A). As no donor
allele could be identified, it is unlikely
that this mutation was generated by
gene conversion or other recombination events. Instead it is more likely
that a point mutation has occurred.
We found this allele in further seven
individuals of the same group, which
indicate that it is not uncommon in
Zulu population.
The sequence of the HLA-A*6827
variant is similar to A*6802 with a
non-synonymous nucleotide
exchange in exon 2 at position 33
(A!G), leading to amino acid replacement of methionine by valine
(Met!Val) (Figure 1B) at position
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
12 in the mature protein. This variation was further verified by the
separation of the two haplotypes
(A*6827 and A*680101) by sequencespecific sequencing primers after
repeated locus-specific PCR amplification. These sequencing primer
(A497S 50 -AGGTTCTCACACCAT
CCAGAT-30 for A*680101 and
A498S 50 -GGTTCTCACACCATCC
AGAG-30 for the A*6827) were
located between intron 2 position 240
and exon 3 position 19 because exon
2 was found not to have heterozygous positions. Amino acid position
12 is found on the beta sheet of the
peptide-binding pocket of the HLAA class I molecule. Its side chain is
facing away from the binding groove
and in the direction of b2-microglobulin. Since methionine and valine
are hydrophobic molecules, we do
not expect this mutation to have an
effect on the peptide binding of
HLA-A*6827 compared to A*6802.
Similar to A*2911, the A*6827 seems
to have arisen by a point mutation.
Correspondence
Rainer Blasczyk
Institute of Transfusion Medicine
Hannover Medical School
Carl-Neuberg-Str.1
30625 Hannover, Germany
Tel: þ49 511 532 6700
Fax: þ49 511 532 2079
e-mail: [email protected]
Received 13 August 2005; revised 18 August
2005; re-revised 26 August 2005; accepted 1
September 2005
doi: 10.1111/j.1399-0039.2005.00505.x
References
1. Marsh SG, Albert ED, Bodmer WF et al.
Nomenclature for factor of the HLA
system, 2002. Tissue Antigens 2002: 60:
407–64.
2. Kotsch K, Wehling J, Kohler S, Blasczyk R.
Sequencing of HLA class I genes based
on the conserved diversity on the noncoding regions: sequencing based typing
of the HLA-A gene. Tissue Antigens 1997:
50: 178–91.
3. Ballesteros JA, Weinstein H. Analysis
and refinement of criteria for predicting
171
NEW ALLELE Alerts
the structure and relative orientations of
transmembranal helical domains. Biophys
J 1992: 62: 107–9.
4. Barlow DJ, Thornton JM. Helix geometry in proteins. J Mol Biol 1988: 201:
601–19.
A novel identification of
HLA-B*07 allele, HLAB*0743, in the Korean
population
Y.-Y. Cha1*, J.-Y. Baek1*, Y.-S. Heo2, K. Kwack1 &
O.-J. Kwon3
1 Pochon CHA University, Seongnam 463-836,
Korea
2 CrystalGenomics Inc., Seoul, Korea
3 BioSenoom Institute of Bioscience &
Biotechnology, Seoul, Korea
Key words: HLA-B*07; HLA-B*0743; Korean;
sequence-based typing
To date, at least
The novel HLA40 HLA-B*07
alleles have been B*0743 allele was
identified from
identified (1).
cord blood of a
Here, we report
Korean baby.
the new allele
HLA-B*0743,
which was detected during routine
HLA genotyping of cord blood units
donated to the Cord Blood Bank.
During the routine HLA typing,
the sequence-based typing method
was employed. Thus, exons 2 and 3
of the HLA-B gene were sequenced
directly using the BigDyeTM
Terminator v3.1 Cycle Sequencing
Ready Reaction kit (Applied
Biosystems, Foster City, CA, USA)
and an ABI PRISMÒ 3100 Genetic
Analyzer or ABI PRISMÒ 3730XL
Genetic Analyzer (Applied
Biosystems). The sequence of the
HLA-B gene was checked in the
IMGT/HLA Sequence Database and
was identified as a novel HLA-B
*These authors contributed equally to
this work.
172
allele. To confirm the presence of a
new HLA-B allele, we sequenced the
exon 3 again using the allele-specific
sequence primers. The complete
HLA genotype of the donor was
HLA-A*020101, 240201; HLAB*0743, 150101; HLA-Cw*030301,
070201; HLA-DRB1*010101,
090102; HLA-DRB4*010301; HLADQA1*0101, 0302; HLADQB1*030302, 050101; HLADPA1*010301, Blank; HLADPB1*020102, 0402. We submitted
the nucleotide sequence of the HLAB gene to GeneBank, and it has been
assigned the accession numbers
DQ088145 (exon 2) and DQ088146
(exon 3). The name HLA-B*0743
was officially assigned by the WHO
nomenclature committee in June 2005.
The nucleotide sequence of HLAB*0743 is identical to that of HLAB*0731 except for a single nucleotide
change from G to T at position 539 in
exon 3 (Figure 1). This change results in
a non-synonymous amino acid change
from arginine to leucine at residue 156.
Residue 156 is located on the a2helix of the HLA class I molecule and
is involved in peptide binding by
constructing the D and E pockets of
the peptide-binding groove (2). While
a leucine instead of arginine at residue 156 of the B*0743 allele would
not change the peptide preference of
this allele from that of other B*07
alleles, it may elevate its peptidebinding affinity, because if the residues at P3, P5 and P7 of the bound
peptide are bulky hydrophobic residues, Leu156 of B*0743 would be
able to interact with these residues by
van der Waals contact. These interactions would enhance the peptidebinding affinity of the B*0743 allele
compared with that of other B*07
alleles bearing an arginine at residue
156. This tighter binding of peptides
to B*0743 might also influence the
recognition of the peptide–HLA
complex by the T-cell receptor and
thus may affect the immune response.
In conclusion, we identified a novel
HLA-B*07 allele from cord blood
from a Korean baby. Further studies
will be needed to determine whether
this novel allele could affect the success of transplantation.
B*070201
95
100
105
110
GG TCT CAC ACC CTC CAG AGC ATG TAC GGC TGC GAC GTG GGG CCG GAC GGG CGC CTC CTC
B*0731
-- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*0743
-- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
115
120
125
130
B*070201 CGC GGG CAT GAC CAG TAC GCC TAC GAC GGC AAG GAT TAC ATC GCC CTG AAC GAG GAC CTG
B*0731
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*0743
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
135
140
145
150
B*070201 CGC TCC TGG ACC GCC GCG GAC ACG GCG GCT CAG ATC ACC CAG CGC AAG TGG GAG GCG GCC
B*0731
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*0743
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
155
160
165
170
B*070201 CGT GAG GCG GAG CAG CGG AGA GCC TAC CTG GAG GGC GAG TGC GTG GAG TGG CTC CGC AGA
B*0731
--- --- --- --- --- --- --- --- --- --- --- --- AC- --- --- --- --- --- --- ---
B*0743
--- --- --- --- --- -T- --- --- --- --- --- --- AC- --- --- --- --- --- --- ---
175
180
B*070201 TAC CTG GAG AAC GGG AAG GAC AAG CTG GAG CGC GCT G
B*0731
--- --- --- --- --- --- --- --- --- --- --- --- -
B*0743
--- --- --- --- --- --- --- --- --- --- --- --- -
Figure 1 Comparison of the exon 3 nucleotide sequences of HLA-B*070201, HLA-B*0731 and
HLA-B*0743. Dashes (–) indicate identity with the HLA-B*070201 sequence. The change in the
nucleotide sequence is indicated in bold.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
Correspondence
KyuBum Kwack
Pochan CHA University, Seongnam
463-836, Korea
Tel: 82 31 725 8376
Fax: 82 31 725 8350
e-mail: [email protected]
Received 8 October 2005; revised 12 and 31
October 2005; accepted 8 November 2005
doi: 10.1111/j.1399-0039.2005.00535.x
Acknowledgments
This study was supported by grants from
the Korea Science and Engineering
Foundation (R01-2002-000-00267-0) and
from the Ministry of Health and Welfare
(0405-BC02-0604–0004).
References
1. Marsh SG, Albert ED, Bodmer WF et al.
Nomenclature for factors of the HLA
system, 2004. Tissue Antigens 2005: 65:
301–69.
2. Bjorkman PJ, Saper MA, Samraoui B,
Bennett WS, Strominger JL, Wiley DC.
Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987:
329: 506–12.
Characterization of the
novel allele HLA-B*3710y
S. Frison, E. Longhi, A. Espadas de Arias,
E. Andreini, F. Poli & M. Scalamogna
Dipartimento Trasfusionale e di Riferimento per i
Trapianti di Organi e Tessuti – Unità Operativa per
il Prelievo e la Conservazione di Organi e Tessuti,
†The allele B*3710 has EMBL nucleotide sequence Database Accession
Numbers
HWS10003117
and
AJ969934. The name B*3710 has been
officially assigned by the WHO
Nomenclature Committee in June
2005. This follows the agreed policy
that, subject to the conditions stated in
the most recent Nomenclature Report
(1), names will be assigned to new
sequences as they are identified. Lists of
such new names will be published in the
following WHO Nomenclature Report.
Ospedale Maggiore Policlinico, Mangiagalli, Regina
Elena – Fondazione IRCCS di Natura Pubblica,
Milan, Italy
Key words: direct sequencing; HLA-B*3710; PCR-SSO
HLA B*3710
differs from the In this report, we
most homolo- describe the identification of the new
gous allele
B*3701 by four allele HLA-B*3710,
nucleotide sub- which was found in a
Caucasian individual
stitutions in
exon 2 at posi- on the waiting list for
a kidney transplant in
tions 300
the North Italy
(G>A), 308
Transplant program
(G>C), 312
(C>G) and 313 organization.
(T>C). These
mutations result in three codon
changes: at codons 77 and 81 where
an aspartic acid is substituted by an
asparagine and an alanine by
a leucine, respectively, and at codon
79 without an amino acid
replacement.
Allelic polymorphism of HLA-B
locus is mainly located in exons 2 and
3 which encode for a1 and a2
domains within the antigenic peptidebinding site.
We report herein the characterization of a new HLA-B*37, officially
named HLA-B*3710 (1). This new
variant was identified in a male patient
(ID 108616) on the waiting list for a
kidney transplant in the North Italy
Transplant program organization.
This patient was typed with
serology for HLA-A,B as
A1,32;B37,40 (GTI, Waukesha, WI,
USA) and with polymerase chain
reaction-reverse sequence-specific
oligonucleotide (PCR-reverse SSO)
typing using a commercially
available kit (Lambda Array Beads
Multi-Analyte SystemÒ –
LABMAS, RSSO1B, One Lambda,
Canoga Park, CA, USA) (2) for
HLA-A,B,DRB1 as
A*01,*32;DRB1*11,*13. With the
interpretation software, no typing
was deducible at B locus because of
an anomalous hybridization
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
pattern. With PCR-sequence-specific primers (SSP) (Olerup SSP,
Saltsjoebaden, Sweden) (3), B locus
typing was B*40, X (w4, w6).
HLA-B sequence-based typing
was then performed with generic
primers which amplify both the
alleles from exon 1 to exon 5 (Atria
Genetics, San Francisco, CA, USA)
(4). Exons 2, 3 and 4 were
sequenced in both directions, and
the obtained heterozygous sequence
revealed the presence of a new allele
in combination with HLA-B*4001,
as the nucleotide sequence did not
match with any known allele
combination.
In order to separate the two alleles,
exons 2 and 3 were amplified with
group-specific primers provided by
Atria Genetics and sequenced.
The HLA-B new variant, officially
named HLA-B*3710, differs from
B*3701 by four nucleotide substitutions in exon 2 at positions 300
(G>A), 308 (G>C), 312 (C>G) and
313(T>C) (Figure 1). These mutations
result in three codon changes: at codon
77 (GAC>AAC) where an aspartic
acid is substituted by an asparagine, at
codon 81 (CTG>GCG) where an alanine is substituted by a leucine and at
codon 79 (CGG>CGC) with no
amino acid substitution.
The mutations are located in the
a1 helix of the peptide-binding groove (5).
These results explain the ambiguous hybridization pattern found
while performing HLA-B low resolution with reverse SSO. In fact, the
two probes (4, CTG CGC ACC
GCG CTC CGC TAC TAC and 26,
CAG ACT TAC CGA GAG
AAC þ ACC GCG CTC CGC TAC
TAC; Lot 006) that gave a positive
result even though not complementary to B37 and the probe specific for
B37 (83, CAG ACT TAC CGA
GAG GAC CTG; Lot 006) that
failed to hybridize are complementary to the DNA region spanning
from codon 72 to codon 85, where
the four mutations are located.
173
NEW ALLELE Alerts
1
GC TCC CAC TCC ATG AGG TAT TTC CAC ACC TCC GTG TCC CGG
B*3710
-- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*3710
2. Kellar KL & Iannone MA. Multiplex
microsphere-based flow cytometric
assays. Exp Hematol 2002: 30: 1227–37.
3. Olerup O & Zetterquist H. HLA-DR typing by PCR amplification with sequence
specific primers (PCR-SSP) in 2 hours: an
alternative to serological DR typing in
clinical practice including donor–recipient
matching in cadaveric transplantation.
Tissue Antigens 1992: 39: 225–35.
4. Pozzi S, Longo A, Ferrara GB. HLA-B
locus sequencing-based typing. Tissue
Antigens 1999: 53: 275–81.
5. Bjorkman PJ, Saper MA, Samraoui B
et al. Structure of the human
class I histocompatibility antigen,
HLA-A2. Nature 1987: 329: 506–12.
14
B*3701
-- Ser Tyr Ser Met Arg Tyr Phe Tyr Thr Ser Val Ser Arg
Exon 2
15
28
B*3701
CCC GGC CGC GGG GAG CCC CGC TTC ATC TCA GTG GGC TAC GTG
B*3710
--- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*3710
Pro Gly Arg Gly Glu Pro Arg Phe Ile Ser Val Gly Tyr Val
Exon 2
29
42
B*3701
GAC GAC ACC CAG TTC GTG AGG TTC GAC AGC GAC GCC GCG AGT
B*3710
--- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*3710
Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala Ala Ser
Exon 2
43
56
B*3701
CCG AGG ACG GAG CCC CGG GCG CCG TGG ATA GAG CAG GAG GGG
B*3710
--- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*3710
Identification of HLAB*4905 in a Caucasian
familya
Pro Arg Thr Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly
Exon 2
57
70
B*3701
CCG GAG TAT TGG GAC CGG GAG ACA CAG ATC TCC AAG ACC AAC
B*3710
--- --- --- --- --- --- --- --- --- --- --- --- --- ---
B*3710
E. Andreini1, E. Longhi1, S. Frison1, M. Bontempelli2,
L. Castellani2 & F. Poli1
1 Dipartimento Trasfusionale e di Riferimento per i
Trapianti di Organi e Tessuti – Unità Operativa
Complessa Immunologia dei Trapianti di Organi e
Tessuti, Ospedale Maggiore Policlinico,
Mangiagalli, Regina Elena – Fondazione IRCCS di
Natura Pubblica, Milan, Italy
2 U.O. Servizio di Immunoematologia e Medicina
Trasfusionale, Laboratorio di Immunologia e
Immunogenetica, Azienda Ospedaliera OO.RR.,
Bergamo, Italy
Pro Glu Tyr Trp Asp Arg Glu Thr Gln Ile Ser Lys Thr Asn
Exon 2
71
84
B*3701
ACA CAG ACT TAC CGA GAG GAC CTG CGG ACC CTG CTC CGC TAC
B*3710
--- --- --- --- --- --- A-- --- --C --- GC- --- --- ---
B*3710
Thr Gln Thr Tyr Arg Glu Asn Leu Arg Thr Ala Leu Arg Tyr
Exon 2
85
91
B*3701
TAC AAC CAG AGC GAG GCC G
B*3710
--- --- --- --- --- --- -
B*3710
Key words: DNA direct sequencing, group-specific
amplification, HLA-B*49
Tyr Asn Gln Ser Glu Ala Exon 2
Figure 1 The exon 2 nucleotide sequence for HLA-B*3710 is compared to that of B*3701. Dashes
indicate identity between the two alleles. In the protein sequence, aminoacidic substitutions are
reported in bold and the silent mutation in italic.
Correspondence
Francesca Poli
Dipartimento Trasfusionale e di Riferimento
per i Trapianti di Organi e Tessuti – Unità
Operativa per il Prelievo e la Conservazione
di Organi e Tessuti
Ospedale Maggiore Policlinico, Mangiagalli
Regina Elena – Fondazione IRCCS di
Natura Pubblica
Via Francesco Sforza 35
20122 Milan, Italy
Tel: 39 02 55034239
Fax: 39 02 55012573
e-mail: [email protected]
174
Received 30 August 2005; revised 20
September 2005; accepted 23 September 2005
This communication reports one previously undescribed HLA-B allele, officially named HLA-B*4905 (1), detected
in a Caucasian family from a North
Italian region, namely in a patient
doi: 10.1111/j.1399-0039.2005.00520.x
Acknowledgments
a
This study was supported by a grant from
‘Ricerca Corrente’ Ospedale Maggiore
Policlinico di Milano IRCCS, project
number 580/01, 2004.
References
1. Marsh SGE, Albert ED, Bodmer WF
et al. Nomenclature for factors of the
HLA system, 2004. Tissue Antigens 2005:
65: 301–69.
The name B*4905 has been officially
assigned by the WHO Nomenclature
Committee in September 2005. This
follows the agreed policy that, subject
to the conditions stated in the most
recent Nomenclature Report (1), names
will be assigned to new sequences as
they are identified. Lists of such new
names will be published in the
following WHO Nomenclature Report.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
(ID122142) and
A novel allele,
in his father
HLA-B*4905, is
(ID122139).
described.
The two
individualswere HLA-B*4905 shows a
Mendelian inheriserologically
typed as HLA- tance in a Caucasian
A23,24;B44,49; family. This variant
has four nucleotide
Cw7
(ID122142) and substitutions in
exon 3. These
HLAmutations result in
A23,25;one silent substitution
B49,51;Cw1,7
at codon 129
(ID122139)
(CCC!CCG) and in
(One Lambda
two aminoacid
Inc, Canoga
changes at codons
Park, CA,
138 (AAC!GAC)
USA). Low
and
140 (TTA!TCC),
resolution
Asn to Asp and
from
molecular typLeu to Ser,
from
ing was carried
respectively.
out with PCRSSO using a
commercially available kit (Lipa HLAA,B, Innogenetics N.V., Murex Biotech
Limited, Dartford, UK), but no typing
was deducible because of an anomalous
hybridization pattern. PCR-SSP did
not resolve the ambiguity (Olerup
SSPTM HLA-B low resolution/M26,
Saltsjoebaden, Sweden). In fact, one
couple of primers even not specific for
B*4901 amplified DNA from both the
subjects, while the other primer pair
specific for B*4901 did not amplify.
HLA-B sequence-based typing was
then performed with locus-specific
primers which amplify both the
alleles from exon 1 to exon 5 (Atria
Genetics, San Francisco, CA, USA)
(2). Exons 2, 3, and 4 were sequenced
in both directions and analyzed with
Assign SBTTM Version 3.2.7 software
(Conexio Genomics, Applecross,
Western Australia); the obtained
heterozygous sequence revealed the
presence of a new allele in combination with HLA-B*40020101, as the
nucleotide sequence did not match
with any known allele combination.
With the aim at separating the two
alleles, the sample was amplified with
group-specific primers (AlleleSEQR
HLA-B GSA SBT Kit, Atria Genetics).
nt 344
A*4901
A*4905
384
gg tct cac act tgg cag agg atg tat ggc tgc gac ctg ggg
-- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3
385
A*4901
A*4905
426
ccc gac ggg cgc ctc ctc cgc ggg tat aac cag tta gcc tac
--g --- --- --- --- --- --- --- --- g-- --- -cc --- --Exon 3
427
A*4901
A*4905
468
gac ggc aag gat tac atc gcc ctg aac gag gac ctg agc tcc
--- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3
469
A*4901
A*4905
510
tgg acc gcg gcg gac acc gcg gct cag atc acc cag cgc aag
--- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3
511
A*4901
A*4905
552
tgg gag gcg gcc cgt gag gcg gag cag ctg aga gcc tac ctg
--- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3
553
A*4901
A*4905
594
gag ggc ctg tgc gtg gag tgg ctc cgc aga tac ctg gag aac
--- --- --- --- --- --- --- --- --- --- --- --- --- --Exon 3
595
A*4901
A*4905
619
ggg aag gag acg ctg cag cgc gcg g
--- --- --- --- --- --- --- --- Exon 3
Figure 1 The exon 3 nucleotide sequence for HLA-B*4905 is compared with nucleotide sequence for
B*4901. Dashes indicate identity with B*4905. The allele B*4905 has EMBL Nucleotide Sequence
Database Accession Numbers HWS10003268 – AM076839.
The HLA-B new variant differs
from B*4901 by four nucleotide substitutions in exon 3: at codon 129
(CCC!CCG), at codon 138
(AAC!GAC), and at codon 140
(TTA!TCC). These variations result
in a silent mutation and two aminoacidic changes from Asn to Asp
and from Leu to Ser, respectively.
The nucleotide sequence of exon 3
of HLA-B*4905 is shown in Figure 1
aligned with that of HLA-B*4901.
The identification of this new allele,
HLA-B*4905, inherited in a Mendelian
fashion, contributes to the heterogeneity of HLA-B*49 that, although quite
frequent in the Caucasian population
(3), was monomorphic till 2001 when
the variant B*4902 was detected by our
group (4).
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
Acknowledgment
This study was supported by a grant from
‘Ricerca Corrente’ Ospedale Maggiore
Policlinico di Milano IRCCS, project n.
580/01, 2005.
Correspondence
Francesca Poli
Dipartimento Trasfusionale e di Riferimento
per i Trapianti di Organi e Tessuti – Unità
Operativa Complessa Immunologia dei
Trapianti di Organi e Tessuti
Ospedale Maggiore Policlinico
Mangiagalli
Regina Elena – Fondazione IRCCS di
Natura Pubblica
Via Francesco Sforza 35
20122 Milan
Italy
Tel.: þ39 02 55034239
Fax: þ39 02 55012573
e-mail: [email protected]
175
NEW ALLELE Alerts
Received 08 November 2005;
accepted 15 November 2005
doi: 10.1111/j.1399-0039.2005.00543.x
References
1. Marsh SGE, Albert ED, Bodmer WF
et al. Nomenclature for factors of the
HLA system, 2004. Tissue Antigens 2005:
65: 301–69.
2. Pozzi S, Longo A, Ferrara GB. HLA-B
locus sequencing-based typing. Tissue
Antigens 1999: 53: 275–81.
3. Gjertson DW, Geer L, Lee S et al.
Population studies. In: Gjertson DW,
Terasaki PI, eds. HLA, 1998. Lenexa,
Kansas: American Society for
Histocompatibility and Immunogenetics,
1998: 103.
4. Poli F, Longhi E, Frison S et al.
Identification of a novel HLA-B allele –
HLA-B*4902. Tissue Antigens 2001: 57:
173–4.
HLA-B*1586 is a novel,
hybrid HLA-B15/B22 allele
with unique serology and
haplotypic association
Y. Yang1, M. T. Aubrey2, G. Zhang1, Y. Ji1 & B. M.
Freed2
1 Immunogenetics Laboratory, Shanghai Blood
Center, Shanghai, 200051, China
2 ClinImmune Laboratories, University of
Colorado Health Sciences Center, Aurora, CO
80010, USA
Key words: gene conversion; HLA-B; SBT
HLA-B*15 is
HLA-B*1586 is a novel
the most
allele with hybrid
polymorphic
sequence and
of all HLA
serology.
groups having over 100 members (1, 2).
Numerous HLA-B alleles are presumptive hybrids of more common
alleles (3, 4). By nature, hybrid
alleles present challenges to our
classification schemes, and this is
reflected in their intermediate serology. In this communication, we
report on a new hybrid HLA-B*15
allele first characterized by our
group in a Chinese family. While
176
officially designated HLA-B*1586
(the name B*1586 has been officially assigned by the WHO
Nomenclature Committee in May
2004), this novel allele displays
serological reactivity overlapping
the HLA-B15 and HLA-B22,B59
groups and an HLA-C linkage
more consistent with HLA-B59
than seen with HLA-B62.
Unusual serological reactivity
(Figure 1), along with anomalous
SSO patterns, suggested the possible
presence of a new allele in a Chinese
leukemia patient. Additional samples were available on the sibling
and mother, which allowed SSP
characterization. SSP results were
also anomalous and did not match
any known allele. Heterozygous
sequencing (Applied Biosystems,
Foster City, CA, USA) identified a
novel polymorphism in the 5-prime
area of exon 3. The novel allele was
then separated by HaploPrep technology (Genovision, West Chester,
PA, USA) using probes B317T and
B355C (targeting the T and C at
positions 317 and 355, respectively).
Haploprep separations followed
by hemizygous sequencing in the
mother and sibling of the
B22/59
Sera
Score
B17/15/35
Sera
Score
9E
B54,
55
6D
9F
B54
6F
B55
B7
9D
B22
B67,
±B7
B8
B59
8F
B54,B55
B16,67
B59
B59
8
1
8
1
1
1
B63
8
8C
10C
10B
10A
11A
12D
7C
B17
B48,±44,
60
B51,53,18,13,46,78,
5102
B18,51,53,78
B53
B46
B57
B35
B35,±75
B35,1508,1511,62,75
B1508,
1511
B15,1508
8
B35,1508,
1511
6
8
1
1
1
2
7E
7F
12C
9B
B55,56
B7,27,42,67,
73,81,2708
7D
9A
±B56
B21
12B
Bw4
12A
Bw6
8
8
B53,38,48,57,
B22/15/35
Sera
Score
patient confirmed the B*1586
sequence.
B*1586 is a classic hybrid allele;
positions 74–362 of the nucleotide
sequence match B*15010101 while
the remaining nucleotides from 363
to the end of exon 4 match B*5401,
B*5502, B*5901 and others. As a
consequence, B*1586 has a unique
CACCCTCCAG AGGATGTATG
motif beginning at position 351.
The hybrid nature is reflected by
the unique serology of the
patient’s sibling depicted in
Figure 1. While there is clear reactivity with B22, B62, and B59, only
a fraction of the sera representing
those specificities was reactive.
Identical hybrid serological patterns
were observed in the patient and
mother.
The availability of the patient’s
sibling and mother allowed us to
confirm haplotypes. B*1586 was
inherited in this family as
A*1101-B*1586-Cw*0102DRB1*0901-DQB1*0303.
Reference to the IMGT/HLA
database (2) revealed that at least
three separate groups have
observed HLA-B*1586 (5). In all
cases, the allele was observed in the
B63
62, ±72,76
1
1
B35,B62,1508,1511,
70,75,77
8
1
B13
B62,
75,76
1
9C
B22
B17
B70,75
1
Figure 1 Serological pattern of an Asian serological tray (One Lambda of Canoga Park, CA)
observed in the sibling of the Chinese leukemia patient. The sibling has a molecular B-locus type
of B*1586, B*5801.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
Chinese population and in
combination with A11 and DR9.
Cw*0102 was reported as present
from the Australian Red Cross Blood
Service as well as our group. Identical
apparent linkages from three separate groups suggest that HLAB*1586 has a conserved haplotype
association as observed in other rare
alleles (4).
Correspondence
Michael T. Aubrey
ClinImmune Laboratoratories
University of Colorado
Health Sciences Center
Aurora
CO 80010
USA
Received 12 September 2005; revised 06
October 2005 and 12 October 2005;
accepted 13 October 2005
doi: 10.1111/j.1399-0039.2004.00531.x
References
1. Marsh SG, Albert ED, Bodmer WF et al.
Nomenclature for factors of the HLA
system, 2004. Tissue Antigens 2005: 65:
301–69.
2. Robinson J, Waller MJ, Parham P
et al. IMGT/HLA and IMGT/MHC:
sequence databases for the study of
the major histocompatibility
complex. Nucleic Acids Res 2003: 31:
311–4.
3. Marcos CY, Fernández-Vina MA,
Lázaro AM, Nulf CJ, Raimondi EH,
Stastny P. Novel HLA-B35 subtypes:
putative gene conversion events with
donor sequences from alleles common
in native Americans (HLA-B*4002 or
B*4801). Hum Immunol 1997: 53:
148–55.
4. Lebedeva TV, Huang A, Ohashi M
et al. The recombinant HLA-B*5518
allele supports the evidence of
conserved haplotype association of
rare alleles. Tissue Antigens 2005: 66:
156–9.
5. Yan L-X, Zhu FM, Lv Q-F, Zhang W.
Identification of a new HLA-DRB1
allele, HLA-DRB1*1212, and
confirmation of HLA-B*1586.
Tissue Antigens 2005: 65:
582–3.
Identification of two novel
HLA-C alleles, HLA-Cw*1217
and HLA-Cw*030404, in
Singapore Chinese†
S. W. Pang1, S. H. Chan1,2 & E. C. Ren2,3
1 WHO Collaborating Center for Research and
Training in Immunology, Yong Loo Lin School of
Medicine, National University of Singapore,
Singapore
2 Department of Microbiology, Yong Loo Lin
School of Medicine, National University of
Singapore, Singapore
3 Genome Institute of Singapore, Singapore
Key words: HLA polymorphisms; Singapore
Chinese; Cw*030404 allele; Cw*1217 allele
The major histocompatibility complex
(MHC) is the most polymorphic
region of our genome. Human leukocyte antigen-C (HLA-C) genes are
located in the class I region of MHC.
According to the latest version of the
IMGT/HLA Sequence Database
(release 2.11.0, October 2005) (2),
there are 2184 HLA alleles identified,
of which 210 are HLA-C alleles.
Accurate DNA sequencing for typing
and individual profiling is now commonplace and with
high-throughput typing methods,
sequence-based typing (SBT) is
easily performed in most laboratories,
resulting in numerous new alleles
†The nucleotide sequences reported in
this paper have been deposited in
GenBank,
Accession
Numbers
DQ206990
and
DQ206991
for
Cw*1217
and
DQ200948
and
DQ200949 for Cw*030404. The names
Cw*1217 and Cw*030404 were officially
assigned
by
the
WHO
Nomenclature
Committee
in
September 2005. This follows the
agreed policy that subject to the
conditions stated in the most recent
Nomenclature Report (1), names will
be assigned to new sequences as they
are identified. Lists of such new names
will be published in the following WHO
Nomenclature Report.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
being discovIn this article, we
ered each
report two novel
year. In this
human leukocyte
article, the
antigen-C (HLA-C)
sequences of
alleles, HLAtwo novel
HLA-C alleles Cw*030404 and HLACw*1217. Both are
are reported.
In perform- characterized by a
single nucleotide
ing population studies of change in exon 2
when compared to
HLA polytheir most homolomorphisms,
gous Cw alleles, but
we identified
only the nucleotide
two novel
change in Cw*1217
HLA-C
resulted in an amino
alleles, a
acid change.
Cw*12 varCompared to
iant and a
Cw*0304 var- Cw*120201, Cw*1217
had a single nucleoiant in two
different indi- tide substitution C>G
at nucleotide 295 of
viduals of
exon 2, resulting in a
Singapore
coding change, as
Chinese desArg75 is changed to
cent.
Gly75. Cw*030404 had
Cw*1217 was
a synonymous nucleodiscovered in
tide substitution of
a Singapore
Chinese male, A>G at nucleotide 264
compared to
designated
Cw*030401 and the
CM924, and
Cw*030404 in amino acid remains as
Thr64.
a Singapore
Chinese male,
designated CM795. Their HLA profiles are as follows: CM924 – HLAA*0207, 1101; HLA-B*1502, 2704;
HLA-Cw*0801, 1217; HLADRB1*1101, 1202; CM795 – HLAA* 1101, 1102; HLA-B*1301, 1527;
HLA-Cw*030404, 0401; HLADRB1*0406, 1602.
Novel nucleotide polymorphisms
were discovered during HLA-C
sequence-based typing (SBT) of
the samples, implying possible new
alleles. To verify the new alleles, a
nested polymerase chain reaction
(PCR) was performed. Specific
primers were then designed to
amplify the novel alleles in
isolation from their counterpart
HLA-C alleles. The full-length
177
NEW ALLELE Alerts
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
80
GCTCCCA
-------------------------
90
CTCCATGAAG
--------G--------G--------G--------G-
100
TATTTCTTCA
-------A--------A--------A--------A--
110
CATCCGTGTC
-CG-T-----CG-T-----CG-------CG-------
120
CCGGCCTGGC
------C--------C--------C--------C---
130
CGCGGAGAGC
-----G--------G----------------------
140
CCCGCTTCAT
---A--------A------------------------
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
150
CTCAGTGGGC
-G--------G--------G--------G--------
160
TACGTGGACG
-------------------------------------
170
ACACGCAGTT
-------------------------------------
180
CGTGCGGTTC
-------------------------------------
190
GACAGCGACG
-------------------------------------
200
CCGCGAGTCC
-------------------------------------
210
GAGAGGGGAG
------------------A--------A---------
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
220
CCGCGGGCGC
-------------------------------------
230
CGTGGGTGGA
-------------------------------------
240
GCAGGAGGGG
-------------------------------------
250
CCGGAGTATT
-------------------------------------
260
GGGACCGGGA
-------------------------------------
270
GACACAGAAG
------------G------------------------
280
TACAAGCGCC
-------------------------------------
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
290
AGGCACAGAC
--------------------------G--------G-
300
TGACCGAGTG
-------------------------------G-----
310
AGCCTGCGGA
-------------------------------------
320
ACCTGCGCGG
-------------------------------------
330
CTACTACAAC
-------------------------------------
340
CAGAGCGAGG
-------------------------------------
350
CCG|GGTCTCA
---|---------|---------|---------|-------
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
360
CACCCTCCAG
--T-A------T-A-----------------------
370
TGGATGTGTG
A------A-A------A-A------ACA------AC-
380
GCTGCGACCT
--------G--------G-------------------
390
GGGGCCCGAC
-------------------------------------
400
GGGCGCCTCC
-------------------------------------
410
TCCGCGGGTA
-------------------------------------
420
TGACCAGTAC
--------------------------C--------C-
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
430
GCCTACGACG
-------------------------------------
440
GCAAGGATTA
-------------------------------------
450
CATCGCCCTG
-------------------------------------
460
AACGAGGACC
--------T--------T-------------------
470
TGCGCTCCTG
-------------------------------------
480
GACCGCCGCG
------------------------T--------T---
490
GACACCGCGG
-----G--------G--------G--------G----
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
500
CTCAGATCAC
-------------------------------------
510
CCAGCGCAAG
-------------------------------------
520
TGGGAGGCGG
-------------------------------------
530
CCCGTGAGGC
-------------------------------------
540
GGAGCAGCGG
--------T--------T-------T--------T--
550
AGAGCCTACC
-------------------------------------
560
TGGAGGGCAC
--------CT
--------CT
-------------------
Cw*010201
Cw*030401
Cw*030404
Cw*120201
Cw*1217
570
GTGCGTGGAG
-------------------------------------
580
TGGCTCCGCA
-------------------------------------
590
GATACCTGGA
--------A--------A-------------------
600
GAACGGGAAG
---T--------T------------------------
610
GAGACGCTGC
-------------------------------------
620
AGCGCGCGG
---------------------------------
Figure 1 Nucleotide sequences of exons 2 and 3 of HLA-Cw*030404 and HLA-Cw*1217 compared with the sequences of similar HLA alleles. Dashes (–)
indicate the homology with the sequence of Cw*010201. Numbering is from the beginning of the coding region.
178
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
HLA-C gene was amplified using
primers described by Cox et al. (3),
followed by allele-specific
amplification of the novel alleles.
Exons 2 and 3 of the PCR products
were then sequenced in both
forward and reverse directions
using primers described by Cereb et al.
(4) and Dormoy et al. (5) to confirm
the novel polymorphisms.
In CM924, SBT revealed a novel
allele that is similar to Cw*120201
within exons 2 and 3, except for a
single nucleotide substitution in
nucleotide 295 of exon 2 (Fig. 1).
The C>G substitution resulted in an
amino acid change from Arg75 to
Gly75 and the allele has been officially
named Cw*1217. Such a C>G substitution remains unique amongst all the
known HLA-C alleles to date, with the
exception of Cw*0412.
In CM795, SBT revealed a novel
allele, Cw*030404, which is similar to
Cw*030401within exons 2 and 3,
except for a single nucleotide substitution (A>G) in nucleotide 264 of
exon 2 (Fig. 1), but there is no
resulting amino acid change and the
amino acid remains as Thr64. This is
a unique polymorphism amongst all
the known HLA-C alleles.
databases for the study of the major histocompatibility complex. Nucleic Acids
Res 2003: 31: 311–4.
3. Cox ST, McWhinnie AJ, Robinson J et al.
Cloning and sequencing full-length HLA-B
and -C genes. Tissue Antigens 2003: 61: 20–48.
4. Cereb N, Kong Y, Lee S, Maye P,
Yang SY. Nucleotide sequences of MHC
class I introns 1, 2 and 3 in humans and
intron 2 in nonhuman primates. Tissue
Antigens 1996: 47: 498–511.
5. Dormoy A, Froelich N, Leisenbach R,
Weschler B, Cazenave J-P, Tongio M-M.
Mono-allelic amplification of exons 2–4
using allele group-specific primers for
sequence-based typing (SBT) of the
HLA-A, B and -C genes: Preparation
and validation of ready-to-use pre-SBT
mini-kits. Tissue Antigens 2003: 62:
201–16.
Correspondence
Ee Chee Ren
Genome Institute of Singapore
Genome #02-01
60 Biopolis Street
Singapore 138672
Tel: þ65 68743316
Fax: þ65 67775720
e-mail: [email protected]
To date, at least
The novel HLAfive HLADRB1*09 alleles DRB1*0904 allele
was identified
have been identified (1). In this from cord blood of
two unrelated
report, we
Korean babies.
describe a novel
allele, DRB1*
0904, identified from two
unrelated Korean babies whose
cord blood was donated to the cord
blood bank and detected during
routine HLA typing by highresolution sequence-based typing
(SBT).
Received 11 October 2005; accepted 24
October 2005
doi: 10.1111/j.1399-0039.2004.00533.x
References
1. Marsh SGE, Albert ED, Bodmer WF et al.
Nomenclature for factors of the HLA
System, 2004. Tissue Antigens 2005: 65:
301–69 [Int J Immunogenet 2005: 32:
107–159; Hum Immunol 2005: 66: 571–636].
2. Robinson J, Waller MJ, Parham P et al.
IMGT/HLA and IMGT/MHC: sequence
Identification of a novel
HLA-DRB1*09 allele,
DRB1*0904, in the Korean
population, by sequencebased typing
C.-H. Song*, J.-Y. Baek*, Y.-S. Heo, K. Kwack &
O.-J. Kwon
Pochon CHA University, Seongnam 463-836, Korea
Key words: HLA-DRB1*09; HLA-DRB1*0904; sequencebased typing (SBT)
*These authors contributed equally to
this work.
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
For SBT, exon 2 of DRB1 from
Korean cord blood units was amplified by polymerase chain reaction for
sequence-based HLA-DRB1 typing.
Sequencing was performed with a
BigDyeTM Terminator v3.1 Cycle
Sequencing Ready Reaction Kit on
an ABI PRISMÒ 3730XL Genetic
Analyzer (Applied Biosystems,
Foster City, CA, USA).
We identified a novel DRB1
allele in two unrelated individuals.
SBT of these individuals was
performed with other class I and II
loci. Notably, the individuals had
common alleles in the HLA class II
group, with the genotypes
DRB4*010301, DQA1*0302,
DQB1*030302, DPA1*020201, and
DPB1*1901, and the novel HLADRB1*09 allele, DRB1*0904. The
nucleotide sequence of DRB1*0904
was submitted to GenBank and
assigned the accession number
AY772533 (exon 2). The name
DRB1*0904 was officially assigned
by the WHO Nomenclature
Committee.
As shown in Figure 1, allele
DRB1*0904 differed from
DRB1*090102 at one nucleotide
position in codon 67 (TTC!CTC),
resulting in an amino acid change
from phenylalanine to leucine at
residue 67 in exon 2 of the mature
protein, as deduced from the nucleotide sequence.
Residue 67 is located in the b1helix of the HLA class II molecule
and forms the E pocket of the
peptide-binding groove involved
in peptide binding (2). This implies
that the change at residue 67
affects the peptide preference of the
DRB1*0904 allele (3). Leu67 of
DRB1*0904 can interact with the
labeled leucine of the bound
peptide via van der Waals contact.
However, Phe67 of other DRB1*09
alleles cannot accommodate a
peptide with leucine at this position
due to an inevitable collision
between the leucine in the bound
peptide and Phe67. Therefore, other
179
NEW ALLELE Alerts
A
DRB1*090102
DRB1*090103
DRB1*0902
DRB1*0903
DRB1*0904
DRB1*0905
5
CA
------
CGT
-----------
TTC
-----------
TTG
-----------
AAG
-----------
10
CAG
-----------
GAT
-----------
AAG
-----------
TTT
-----------
CAG
-----------
15
TGT
-----------
CAT
-----------
TTC
-----------
TTC
-----------
AAC
-----------
20
GGG
-----------
ACG
--C
---------
GAG
-----------
CGG
-----------
GTG
-----------
25
CGG
-----------
TAT
-----------
CTG
-----------
CAC
-----------
AGA
-----------
DRB1*090102
DRB1*090103
DRB1*0902
DRB1*0903
DRB1*0904
DRB1*0905
30
GGC
-----------
ATC
-----------
TAT
-----------
AAC
-----------
CAA
-----------
35
GAG
-----------
GAG
-----------
AAC
-----------
GTG
-----------
CGC
-----------
40
TTC
-----------
GAC
-----------
ACC
-----------
GAC
-----------
GTG
-----------
45
GGG
-----------
GAG
-----------
TAC
-----------
CGG
-----------
GCG
-----------
50
GTG
-----------
ACG
-----------
GAG
-----------
CTG
-----------
GGG
-----------
DRB1*090102
DRB1*090103
DRB1*0902
DRB1*0903
DRB1*0904
DRB1*0905
55
CGG
-----------
CCT
-----------
GTC
---A-------
GCC
----T
-------
GAG
-----------
60
TCC
---A-------
TGG
-----------
AAC
-----------
AGC
-----------
CAG
-----------
65
AAG
-----------
GAC
-----------
TTC
------C----
CTG
-----------
GAG
------A
-----
70
CGG
----GAC
-----
AGG
-----------
CGG
------C
-----
GCC
-----------
GAG
-----------
75
GTG
-----------
GAC
-----------
ACC
-----------
GTG
--------TAC
TGC
-----------
DRB1*090102
DRB1*090103
DRB1*0902
DRB1*0903
DRB1*0904
DRB1*0905
80
AGA
-----------
CAC
-----------
AAC
-----------
TAC
-----------
GGG
-----------
85
GTT
-----------
GGT
-----------
GAG
-----------
AGC
-----------
TTC
-----------
90
ACA
-----------
GTG
-----------
CAG
-----------
AGG
-----------
CGA
-----------
95
G
-
B
DRB1*090102
DRB1*090103
DRB1*0902
DRB1*0903
DRB1*0904
DRB1*0905
10
GDTQPRFLKQ
*****----*****----*****----*****----*****-----
20
DKFECHFFNC
----------------------------------------------
30
TERVRYLHRG
----------------------------------------------
40
IYNQEENURF
----------------------------------------------
50
DSDVGEYRAV
----------------------------------------------
60
TELGRPVAES
---------------D--Y
----------------------------
70
WNSQKDFLER
---------------------------D
------L------------
80
RRAEVDTVCR
-------------------------------------------Y--
90
HNYGVGESFT
----------------------------------------------
100
VQRRVHPEVT
----******
----******
----******
----******
----******
Figure 1 The exon 2 nucleotide and amino acid sequences of HLA-DRB1*0904, compared with the
sequences of all HLA-DRB1*09 alleles. Dashes (-) indicate identity with the HLA-DRB1*090102 sequence
and asterisks (*) indicate unsequenced regions. The numbers above the sequence refer to codon position.
DRB1*09 alleles would prefer peptides with smaller residues at this
position.
In conclusion, we identified a
novel HLA-DRB1*09 allele,
DRB1*0904, that was found in
two unrelated Korean babies.
The new allele contains an aminoacid change in the antigenbinding site of the HLA protein,
which may alter its antigen-binding
properties.
Correspondence
KyuBum Kwack
Pochon CHA University
Seongnam 463-836
Korea
Tel: 82 31 725 8376
Fax: 82 31 725 8350
e-mail: [email protected]
Received 21st October 2005; Revised 7th
November 2005; Accepted 8th November 2005
doi: 10.1111/j.1399-0039.2005.00537.x
Acknowledgments
This study was supported by grants from
the Korea Science and Engineering
Foundation (R01-2002-000-00267-0) and
180
from the Ministry of Health and Welfare
(0405-BC02-0604-0004).
References
1. Marsh SG, Albert ED, Bodmer WF et al.
Nomenclature for factors of the HLA system, 2004. Tissue Antigens 2005: 65: 301–69.
2. Brwon JH, Jardetzky TS, Gorga JC et al.
Three-dimensional structure of the human
class II histocompatibility antigen HLADR1. Nature 1993: 364: 33–9.
3. Stern LJ, Brown JH, Jardetzky TS et al.
Crystal structure of the human class II
MHC protein HLA-DR1 complexed with
an influenza virus peptide. Nature 1994:
368: 215–21.
Identification of a novel
HLA-DRB1*11 allele:
DRB1*1152
G. Ozzella1, P. I. Monaco1, A. Tessitore2, A. Piazza1,
D. Piancatelli3 & D. Adorno1
1 Organ Transplant and Immunocitology, C.N.R.
Institute, Rome, Italy
2 Department of Experimental Medicine,
University of L’Aquila, Italy
3 Organ Transplant and Immunocitology, C.N.R.
Institute, Section of L’Aquila, Italy
Key words: HLA-DRB1; new allele; polymorphism
transplantation; polymorphism
The novel
New HLA-DRB1*1152
HLAallele differs from
DRB1*11
DRB1*1117 by a single
allele was
nucleotide substitudetected in
tion at codon 60
three mem(T!C).
bers of a
Moroccan
Berber family. It was suspected from
DRB1 genotyping discrepancies.
Low-resolution PCR-SSP typing of
the father’s DRB1 locus (DRB1,
Olerup SSP, Genovision, Vienna,
Austria) (1) revealed an ambiguity
because it was not possible to discriminate between rare DRB1*11
homozygosity (*1113/1117/1122,
*1123/1125) and DRB1*11,
DRB1*14 heterozygosity (*1101/
1104/1106/1112/1115/1124/1127–
1129/1132/1133/1135/1137–1139/
1143, *1408/1434).
DRB1 typing of the patient and his
matched brother were specific for
DRB1*14 (inherited from the
mother) and DRB1*1113/1117/1122
(inherited from the father). This
seemed to exclude the father from
being heterozygous for DRB1*11.
DRB1 typing of the sister was
specific for DRB1*14 inherited from
the mother and DRB1*11 inherited
from the father (*1101/1104/1106/
1112/1115/1124/1127–1129/1132/
1133/1135/1137–1139/1143 alleles).
This result excluded the possibility
that the father was homozygous for
DRB1*11.
The family was then studied by
high-resolution PCR-SSP typing. The
father’s DRB1*11 typing permitted
to assign DRB1*1104 allele. It was
not possible to define the second
allele because of an ambiguity
between DRB1*1117 and DRB1*14:
both showed one false positive reaction. However, it proved possible to
assign the DRB1*1117 allele to the
patient and his matched brother
without any mismatch. In fact, the
false positive reaction seen in the
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
NEW ALLELE Alerts
DRB1*1117
DRB1*1152
DRB1*1117
DRB1*1152
DRB1*1117
DRB1*1152
DRB1*1117
DRB1*1152
DRB1*1117
DRB1*1152
DRB1*1117
DRB1*1152
DRB1*1117
DRB1*1152
DRB1*1117
DRB1*1152
5
10
15
20
25
CA CGT TTC TTG GAG TAC TCT ACG TCT GAG TGT CAT TTC TTC AAT GGG ACG GAG CGG GTG CGG TTC CTG GAC AGA
-- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --P
*
R
-
F
-
L
-
E
-
Y
-
S
-
T
-
S
-
E
-
C
-
H
-
F
-
F
-
N
-
G
-
T
-
E
-
R
-
V
-
R
-
F
-
L
-
D
-
R
-
30
35
40
45
50
TAC TTC CAT AAC CAG GAG GAG TTC GTG CGC TTC GAC AGC GAC GTG GGG GAG TAC CGG GCG GTG ACG GAG CTG GGG
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --Y
-
F
-
H
-
N
-
Q
-
E
-
E
-
F
-
V
-
R
-
F
-
D
-
S
-
D
-
V
-
G
-
E
-
Y
-
R
-
A
-
V
-
T
-
E
-
L
-
G
-
55
65
70
75
60
CGG CCT GAT GAG GAG TAC TGG AAC AGC CAG AAG GAC CTC CTG GAG CGG AGG CGG GCC GAG GTG GAC ACC TAT TGC
--- --- --- --- --- C-- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --R
-
P
-
D
-
E
-
E
-
Y
H
W
-
N
-
S
-
Q
-
K
-
D
-
L
-
L
-
E
-
R
-
R
-
R
-
A
-
E
-
V
-
D
-
T
-
Y
-
C
-
80
85
90
95
AGA CAC AAC TAC GGG GTT GTG GAG AGC TTC ACA GTG CAG CGG CGA G
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- R
-
H
-
N
-
Y
-
G
-
V
-
V
-
E
-
S
-
F
-
T
-
V
-
Q
-
R
-
R
-
V
*
Figure 1 Exon 2 nucleotide and predicted amino acid sequences alignment of DRB1*1117 and the new DRB1*1152. Dashes indicate identity with
DRB1*1117; stars denote undefined amino acid.
father could now be used for the
assignment of the DRB1*1401 allele
inherited from the mother. The typing of the sister’s DRB1 alleles permitted to assign unambiguously
DRB1*1401 inherited from mother,
with DRB1*1104 inherited from
father. All these results supported the
hypothesis of a novel DRB1*11 allele
in the father, the patient and his
matched brother. To confirm this,
DRB1 sequence-based typing was
carried out after a preliminary PCRSSP screening of the major DRB1
allele groups using DRBAMP-52 and
DRAMP-B primers (twelfth IHW)
(2). PCR products were sequenced in
both directions by Big Dye
Terminator chemistry. The complete
sequence of exon 2 of the DRB1 gene
was amplified using primers within
intron 1 and intron 2 (3), and the
PCR product was cloned using the
pGEM-T Easy Vector System II
Cloning Kit (Promega, Madison, WI,
USA) to distinguish the alleles. DNA
inserts from the cloned allele were
amplified and sequenced. The new
sequence obtained differs from
DRB1*1117 by single nucleotide substitution at codon 60 (TAC!CAC),
changing the amino acid from tyrosine (Y) to histidine (H).
The nucleotide sequence was submitted to GenBank in March 2004
and given the accession number
AY574194. The name DRB1*1152
was officially assigned by the WHO
Nomenclature Committee for
Factors of the HLA System in May
2004 (4) (Fig. 1). This follows the
agreed policy that, subject to the
conditions stated in the most recent
Nomenclature Report, names are to
be assigned to new sequences as they
are identified.
Comparison of DRB1*1152 with
all DRB1*14 sequences on the
IMGT/HLA database showed that
the triplet CAC at codon 60 is shared
with some DRB1*14 alleles such as
DRB1*1408 and DRB1*1434, as
suggested by low-resolution DR
genotyping. The novel DRB1*1152
allele segregated together with
DQB1*0503; this DR11-DQ5
phenotype was in contrast to already
known HLA linkages.
The alteration in the amino acid
sequence encoded by DRB1*1152
changes the amino acid produced by
codon 60 from a neutral tyrosine to a
positively charged histidine. This
mutation is located on the a-helix
portion of the b1 chain of DRB1 that
contributes to form the side of the
Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard
peptide-binding groove (5). Histidine
often contributes to the hydrogen
bond network formed between the
peptide and a1/b1 a helical domains
(6). This tyrosine-to-histidine substitution might interfere with binding
energy, contributing to stabilize or
destabilize peptide–HLA II interactions and thus might affect antigen
presentation.
Correspondence
Giuseppina Ozzella
Istituto C.N.R. peri Trapianti D’Organo e
l’lmmunocitologia, P. le dell’Umanesimo
10, 00144 Rome, Italy
e-mail: [email protected]
Received 3 October 2005; revised 17 October
2005; accepted 2 November 2005
doi: 10.1111/j.1399-0039.2005.00534.x
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Ó 2006 The Authors
Journal compilation 67 (166–182) Ó 2006 Blackwell Munksgaard