Identification of a novel allele, DRB1*1340, in two Brazilian individuals

Abstract

We identified a novel HLA-DRB1 allele, named DRB1*1340 by the WHO HLA Nomenclature Committee, in two Brazilian individuals. Typing by polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) showed a DRB1*13 allele with an unusual hybridization pattern. DNA sequencing of both strands and comparison of the sequence with previously described DRB1 alleles revealed that the most similar allele is DRB1*1301, from which DRB1*1340 differs by a single nucleotide (T-->A) in exon 2, at position 127, codon 47 (Phe-->Tyr). The sequence received accession number AJ237964 from the EMBL database.

Full text

Key words:
HLA-DRB1; DRB1*1340; Brazilian population
Acknowledgments:
Thanks are due to Hospital Napolea
˜o Laureano
for the management of samples and to CNPq,
FAPESP, UFPR and LICR for financial support.
Received 26 January, revised,
accpeted for publication 10 April 2000
Copyright cMunksgaard 2000
Tissue Antigens . ISSN 0001-2815
Tissue Antigens 2000: 56: 194–196
Printed in Denmark . All rights reserved
194
Brief communication
P.C. Maciag
Identification of a novel allele, DRB1*1340,
K.S. Junes
in two Brazilian individuals
L.L. Villa
M.L. Petzl-Erler
Abstract: We identified a novel HLA-DRB1 allele, named DRB1*1340 by
the WHO HLA Nomenclature Committee, in two Brazilian individuals. Typ-
ing by polymerase chain reaction using sequence-specific oligonucleotide
probes (PCR-SSOP) showed a DRB1*13 allele with an unusual hybridization
pattern. DNA sequencing of both strands and comparison of the sequence
with previously described DRB1 alleles revealed that the most similar allele
is DRB1*1301, from which DRB1*1340 differs by a single nucleotide
(T»A) in exon 2, at position 127, codon 47 (Phe»Tyr). The sequence re-
ceived accession number AJ237964 from the EMBL database.
HLA-DRB1 is the most polymorphic among the human class II
genes. More than 240 HLA-DRB1 alleles are known (1, 2). Some of
these have a wide geographic distribution, but most are restricted
to particular ethnic groups. DR13 is one of the most heterogeneous
of the DRB1 allele groups (2). Here we report a novel HLA-DRB1*13
allele identified by DNA-based methods in two white Brazilian indi-
viduals born in Salgado de Sa
˜oFe
´lix, Paraı
´ba State, and living in
Joa
˜o Pessoa, the main city of the same state, in northeastern Brazil.
DNA was obtained from cervical smears by a conventional
phenol-chloroform protocol. HLA-DQA1, DQB1 and DRB1 2nd ex-
ons were amplified by polymerase chain reaction (PCR) and the
alleles discriminated by sequence-specific oligonucleotide probes
(SSOP) (3, 4). Generic HLA-DRB typing of the two samples revealed
heterozygosity for alleles of the DR4 and DR52 groups. By group-
specific typing, allele DRB1*0402 was identified. The DRB1 allele
of the DR52 group was specifically amplified using primers
2DRBAMP-3 (5ø-CACGTTTCTTGGAGTACTCTAC-3øforward) and
The name DRB1*1340 has been officially assigned by the WHO Nomenclature
Committee in January 2000. This follows the agreed policy that, subject to the
conditions stated in the most recent Nomenclature Report (Bodmer et al. 1999),
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.
Authors’ affiliations:
P.C. Maciag
1,2
,
K.S. Junes
2
,
L.L. Villa
2
,
M.L. Petzl-Erler
1
1
Laboratory of Human
Molecular Genetics,
Genetics Department, UFPR,
Curitiba, Brazil,
2
Ludwig Institute for Cancer
Research, Sa
˜o Paulo
Branch, Sa
˜o Paulo, Brazil
Correspondence to:
M. Luiza Petzl-Erler
Departamento de Gene
´tica,
UFPR
Caixa Postal 19071
81531–990 Curitiba
Brazil
e-mail: perler/bio.ufpr.br

Maciag et al : Identification of DRB1*1340
Fig. 1. Comparison of the nucleotide
sequence of exon 2 of alleles
DRB1*0101, DRB1*1340, DRB1*1301,
DRB1*1308, DRB1*1332 and DRB1*1336.
A dash (-) indicates identity between the
sequences.
2DRBAMP-B (5ø-CCGCTGCACTGTGAAGCTCTC-3øreverse).
Identification of the allele was performed by hybridization with a
set of 52 probes (4). The hybridization results allowed to character-
ize the DR52 allele as DRB1*13, although the pattern obtained did
not match that of any previously described DR13 allele. The same
results were obtained amplifying the allele with primers 2DRBAMP-
3 and 2DR86AMP-VR (5ø-CTGCACTGTGAAGCTCTCCA-3ø).
Sequencing was performed for both samples to confirm the new
allele. Briefly, the 2nd exon of the DRB1*13 allele was amplified
with intronic primers DRB-GH46 (5ø-CCGGATCCTTCGTGTCCC
CACAGCACG-3ø) and CRX37 (5ø-GAATTCCCGCGCCGCGCTCA
CCT-3ø) (5). Amplification products were purified from a 1.5% agar-
ose gel in a DEAE-cellulose membrane and cloned in pUC18
195
Tissue Antigens 2000: 56: 194–196
plasmid. Competent Escherichia coli DH5awere submitted to elec-
trotransformation and then selected in LB agar with IPTG, X-gal
and ampicillin. Plasmids containing the DRB1*13 sequence were
identified by the DR52 group-specific PCR reaction. In 8 recombin-
ant plasmids, forward and reverse DNA sequencing reactions were
performed using the fluorescently labeled M13 universal and re-
verse primers (Genosys), respectively, by the dideoxy-mediated
chain termination method (6). Reaction products were submitted to
electrophoresis in an automated DNA sequencer (ALFexpress;
Pharmacia Biotech, Uppsala, Sweden). Nucleic acid sequence align-
ment is shown in Fig. 1.
Comparison of the sequences revealed that DRB1*1340 differs
from DRB1*1301 by a single T-to-A substitution at position 127 of

Maciag et al : Identification of DRB1*1340
exon 2, codon 47, that changes the encoded amino acid from Phe
(TTC) to Tyr (TAC). Alleles DRB1*1308, DRB1*1332, and
DRB1*1336 also differ from DRB1*1340 at a single codon in exon 2,
but nucleotide differences are more pronounced as compared to
DRB1*1301. While DRB1*1308 encodes a Phe (TTC) at codon 37,
DRB1*1340 encodes an Asn (AAC). Compared to DRB1*1332, that
encodes a Ser (AGC) at codon 57, DRB1*1340 encodes an Asp (GAT)
at this position. Allele DRB1*1336 differs from DRB1*1340 at codon
86; DRB1*1336 encodes a Gly (GGT) and DRB1*1340 encodes a Val
(GTG) at this position. The amino acids at these positions are import-
ant in HLA-peptide interactions, because they participate in pocket 7
and 9 structures (7). These substitutions are conservative and com-
mon among HLA-DRB1 molecules, what means that allele
DRB1*1340 most likely is functionally normal. Alleles DRB1*1308,
DRB1*1332 and DRB1*1336, all three described in Caucasoids (2) (in
an Asian Indian, a Slovenian and a North-American individual, re-
spectively), are rare. Considering the similarity of the nucleotide se-
quence, and the comparatively high frequency and wide geographic
distribution of DRB1*1301, allele DRB1*1340 most probably arose by
a gene conversion event or point mutation of allele DRB1*1301.
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The two samples were also HLA-DQA1 and -DQB1 typed by
PCR-SSOP (3, 4). From known linkage disequilibria, the most likely
haplotypes are DRB1*0402-DQA1*03011 or 0302-DQB1*0302, and
DRB1*1340-DQA1*0103-DQB1*0603. The two individuals are prob-
ably related, as indicated by sharing of the same rare allele, same
genotype, birth place and place of residence.
The northeastern Brazilian population is ethnically heterogen-
eous and admixed. Ancestry is roughly 60% European, 30% sub-
Saharan African and 10% Amerindian (8, 9). Therefore, in spite of
the light skin color of the two donors, allele DRB1*1340 might be
of African instead of European origin. Amerindian origin is un-
likely, since DR13 alleles have never been detected in non-admixed
Amerindian populations. Alternatively, this allele might have a local
and more recent origin. In any event, it is rare or absent from all
populations thus far studied, including the population of Joa
˜o Pes-
soa, from which a sample of 487 individuals has been HLA typed.
These specimens originated from a larger epidemiological study
conducted in Joa
˜o Pessoa (10).