Download A novel DI*A allele without the band 3

Vox Sanguinis (2003) 84, 326 – 330
© 2003 Blackwell Publishing
ORIGINAL PAPER
A novel DI*A allele without the band 3-Memphis mutation in
Amazonian Indians
Blackwell Publishing Ltd.
W. Baleotti Jr,1 M. Rios,2* M. E. Reid,2 A. Fabron Jr,1 J. Pellegrino Jr,3 S. T. O. Saad3 & L. Castilho3
1
Hemocentro, Faculdade de Medicina, Marília, Sao Paulo, Brazil
New York Blood Center, New York, New York, USA
3
Hemocentro da UNICAMP, Sao Paulo, Brazil
2
Background and Objectives The blood-group antigens Dia and Dib are carried on
erythrocyte band 3 and are defined by a single amino acid substitution at position
854 (Leu for Dia and Pro for Dib). The Band 3-Memphis variant has a point mutation
(166A>G) in the SLC4A1 gene, which encodes the amino acid substitution Lys56Glu.
Two types of Band 3-Memphis, variants I and II, are distinguished by their susceptibility to covalent labelling with 4,4′-diisothiocyanato-1,2-diphenylethane-2,2′disulphonic acid (H2DIDS). Memphis II is more readily labelled than Memphis I or
normal band 3. It is reported that Memphis II is associated with Dia. In a study
designed to determine the frequency of the DI*A/DI*B and 166A>G polymorphisms
in different populations in Brazil, we found a new DI*A allele.
Materials and Methods We studied DNA samples from 70 Amazonian Indians, 71
individuals of Japanese descent, 93 random Brazilian blood donors and 84 blacks
with sickle cell disease. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses were performed on all samples, using MspI for DI*A/
DI*B (exon 19) and MnlI for 166A>G (exon 4). Exon 4 and exon 19 from four outliers
were sequenced.
Results Among Amazonian Indians, DI*A and 166G mutations both had a high
frequency (0·57 and 0·54, respectively). In individuals of Japanese descent, these
alleles were moderately frequent (0·07 and 0·19, respectively). We identified a new
allele with DI*A and 166A (56Lys) in four Amazonian Indians.
Received: 29 November 2002,
revised 13 February 2003;
accepted 17 February 2003
Conclusions Our results revealed that DI*A does not have a strict association with
166G. They also show the relevance of testing a cohort of different populations.
Key words: alleles, Band 3-Memphis, band 3, Brazilians, Diego, polymorphisms.
Introduction
The Diego blood-group system consists of 21 antigens carried
on band 3: it comprises two pairs of anti-thetical antigens –
Dia/Dib and Wr a/Wrb – and 17 other antigens of low incidence
[1]. Molecular studies on the gene encoding band 3 (SLC4A1,
Correspondence: Lilian Castilho, PhD, Hemocentro, Unicamp, Rua Carlos
Chagas, 480, Caixa Postal 6198, CEP 13081-970 Barão Geraldo, Campinas,
SP, Brazil
E-mail: [email protected]
*Present address: DETTD, CBER, FDA, Rockville, MD, USA
326
AE1) have revealed the molecular bases associated with
these antigens [2–7]. In the erythrocyte membrane, band 3 is
the major protein and transports anions and anchors the
erythrocyte lipid bilayer to the membrane skeleton [8–10].
The N-terminal 40 000-molecular weight (MW) domain binds
glycolytic enzymes and haemoglobin, and the glycosylated
C-terminal transmembrane 55-MW domain carries out anion
transport [11].
Band 3-Memphis is a variant of band 3 characterized by
slower migration than the normal band 3 on sodium dodecyl
sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
after treatment of red blood cells (RBCs) with pronase or
α-chymotrypsin. Band 3-Memphis results from a point
New DI*A allele among Brazilians
mutation – 166A>G – in the SLC4A1, leading to an amino
acid substitution, Lys56Glu, in the cytoplasmic domain
[12,13]. There are two types of Band 3-Memphis, variants
I and II, which are distinguished by their susceptibility
to covalent labelling with 4,4′-diisothiocyanato-1,2diphenylethane-2,2′-disulphonic acid (H2DIDS). Band 3Memphis II is more readily labelled than Band 3-Memphis I
or normal band 3 [14]. Spring et al. [15] reported that the
Memphis II variant of band 3 was associated with the Dia
antigen in samples tested, and that not all Band 3-Memphis
RBCs carry the Dia antigen (Memphis I). A subsequent study
confirmed the association of Band 3-Memphis II with the Dia
antigen and determined that the Diego polymorphism (DI*A/
DI*B) results from a point mutation at nucleotide 2561T>C
in exon 19, which causes the amino acid substitution
Leu854Pro [16]. RBCs with the Memphis form of band 3 have
no known morphological abnormalities [16].
Extensive serological and electrophoretic studies have
shown that the Dia antigen is rare among Caucasians and
African Blacks and is common among South Americans (up
to 54%) and southeast Asians (12%) [17]. The frequency of
Band 3-Memphis varies among different populations; it is
relatively common in Native Americans (up to 25%), African
Americans (15%) and Japanese (29%) [17–20].
Prior to the study described here, SLC4A1 analysis
performed on DNA from people whose RBCs express the
Dia antigen showed 2561T; 854Leu together with the Band
3-Memphis polymorphism 166G; 56Glu [15,16]. This finding,
in conjunction with electrophoretic and serological studies,
led to the assumption that DI*A was linked with the Band
3-Memphis mutation. In this study, we report the allelic
frequencies of DI*A and DI*B, and their degree of association
with Band 3-Memphis, among Brazilians, a population characterized by intense racial admixture. These polymorphisms
were analysed by polymerase chain reaction–restriction
fragment length polymorphism (PCR–RFLP) performed on
DNA samples from Amazonian Indians, Japanese living in
Sao Paulo, Brazilian blood donors, and African Brazilians
with sickle cell disease (SCD). During this study, we identified a novel allele with DI*A, but without the expected 166G
mutation (Band 3-Memphis), in four of 70 Amazonian
Indians.
Subjects and methods
Population studied
Peripheral blood samples from 318 people were collected in
EDTA after obtaining signed informed consent, as approved
by the Institutional Review Board. The samples were from
four ethnic groups: 70 Amazonian Indians (from Parakanã
tribe), 71 individuals of Japanese descent (representing a
racial mixture between Japanese and South American
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 84, 326 – 330
327
descendents), 93 random Brazilian blood donors (representing a high inter-racial mixture among Europeans,
South Americans and African Americans), and 84 African
Brazilians (representing admixture with native Brazilians)
with SCD.
The Parakanã Indians
The Parakanã Indians belong to the Tupi group, and did not
have contact with other populations until 1971. The tribe is
small and resides in three villages in the State of Pará
(Northern Brazil). This population has a restricted range of
polymorphisms for several genetic markers, including blood
groups [21].
Haemagglutination
Dia and Dib types were determined by haemagglutination
in gel cards (DiaMed AG, Morat, Switzerland) using two
commercial sources of anti-Dia antisera (Gamma Biologicals,
Houston, TX and Biotest SA, São Paulo, Brazil) and anti-Dib
patient sera.
DNA preparation and PCR amplification
DNA was extracted from leucocytes by using either the
phenol–chloroform method [22] or DNAzol (Gibco BRL®,
Gaithersburg, MD), according to the manufacturer’s recommendations. PCR assays were designed to amplify a segment
of 149 bp from exon 19 of SLC4A1 for the identification of
DI*A/DI*B alleles, and a segment of 84 bp from exon 4 of
SLC4A1 for the detection of Band 3-Memphis. The primers
used to determine DI*A/DI*B alleles were DiEX19S: 5′TGGCGCATGCACTTATTCAC-3′ (sense) and DiEX19R: 5′TTCCTGAAGATGAGCGGCAG-3′ (antisense). The primers to
determine the presence of 166G (band 3-Memphis) were
DiEX4S: 5′-TTCAGCTCACGACACCGAGG-3′ (sense) and
DiI4AS: 5′-GAGGCTGGGGTCCTCACCTT-3′ (antisense). Primer
sequences were designed based on the GenBank accession
number X77738. PCR amplification was carried out in a
thermal cycler (model 9700; Perkin Elmer, Foster City, CA),
using 100–200 ng of DNA, 50 pmol of each primer, 2 nmol
of each dNTP, 2·0 mM MgCl2, 1·0 U Taq DNA polymerase, and
buffer in a final volume of 50 µl. The PCR profile used in all
assays was as follows: 15 min at 95 °C; 35 cycles of 40 seconds
at 94 °C, 40 seconds at 62 °C, and 1 min at 72 °C; followed
by 10 min at 72 °C.
RFLP analysis
After amplification, 10 µl of PCR product was digested overnight with the appropriate restriction enzyme. MspI (MBI
Fermentas, Amherst, NY) was used to study the 2561C>T
328 W. Baleotti Jr, et al.
mutation in exon 19 associated with DI polymorphism, and
MnlI (MBI Fermentas) was used to study the 166A>G
mutation (Band 3-Memphis) in exon 4. Restriction enzyme
digestion was performed in a final volume of 20 µl under
conditions recommended by, and using buffers provided by,
the manufacturer. RFLP bands were analysed after electrophoresis in 2% agarose in Tris–acetate–EDTA buffer.
Sequence analysis
DNA from four Amazonian Indian samples that apparently
had a DI*A allele without the expected 166G, as judged
by PCR–RFLP analysis, were cloned and sequenced. PCR
products amplified from genomic DNA using the primers for
exon 4 (DiEX4S/DiI4AS) and exon 19 (DiEX19S/DiEX19) (as
described above) were purified by elution from 1% agarose
gels using a Qiaex II gel-extraction kit (Qiagen, Valencia, CA),
and cloned into a TA vector (Invitrogen, Carlsbad, CA).
Sequencing was performed on an ABI 373XL Perkin Elmer
Biosystems (PEB) sequencer, and the PEB Big Dye reagent BD
Half-term (GenPak, Perkin Elmer).
Results
Correlation between the DI*A and DI*B alleles with
Band 3-Memphis
The correlation of results from DI*A/DI*B and the Memphis
polymorphism 166A>G are shown in Table 1. The 318 samples
account for 636 alleles; 542 were DI*B, of which only 36
(6·6%) carried 166G (Band 3-Memphis). Interestingly, among
the Amazonian Indians, none with the DI*B allele had 166G,
while four with the DI*A allele were heterozygous for 166 A/
G. Therefore, it appeared that one of the DI*A alleles in each
of the four Indians did not have the expected 166G mutation.
Analysis of novel allele
Repeat samples were obtained from the four Amazonian
Indians with the unexpected DI*A allele. Haemagglutination
tests confirmed the Di(a+b–) status of their RBCs. Sequence
analysis of genomic DNA confirmed the interpretation of
heterozygous 166 A/G and homozygous 2561T (DI*A) (data
not shown). These results indicate that in each of these four
Amazonian Indians, one of their DI*A alleles had 166A
instead of the expected 166G, which is associated with Band
3-Memphis.
Distribution of DI*A/DI*B alleles and correlation
with the Diego phenotype
Distribution of allelic frequencies according to
ethnic group
The DI*A/DI*B PCR-MspI-RFLP and antigen-typing results
obtained on the 318 samples were in complete agreement
and are shown in Table 1. These results are consistent with
published prevalence data obtained from serological
studies [17].
The allelic frequencies are shown in Table 2. Both the DI*A
allele and the Band 3-Memphis 166 allele were frequent in
Amazonian Indians (allelic frequencies of 0·57 and 0·54,
respectively). The DI*A allele was less frequent in the other
ethnic groups: 0·07 in Japanese, 0·02 in Brazilian blood
Table 1 Results of Diego phenotyping, and DI and 166 polymerase chain reaction–restriction fragment polymorphism (PCR–RFLP) analyses
Diego
phenotype
DI*A/DI*B
alleles
Blood donors
(n = 93)
Di(a+b–)
Di(a+b+)
Di(a–b+)
DI*A/DI*A
DI*A/DI*B
DI*B/DI*B
0
0
86 (92·5)
0
2 (2·1)
4 (4·2)
0
1 (1·1)
0
0
3 (3·2)
90 (96·8)
African Brazilians
(SCD patients)
(n = 84)
Di(a+b–)
Di(a+b+)
Di(a–b+)
DI*A/DI*A
DI*A/DI*B
DI*B/DI*B
0
0
71 (84·5)
0
1 (1·2)
10 (11·9)
0
0
2 (2·4)
0
1 (1·2)
83 (98·8)
Individuals of
Japanese descent
(n = 71)
Di(a+b–)
Di(a+b+)
Di(a–b+)
DI*A/DI*A
DI*A/DI*B
DI*B/DI*B
0
0
47 (66·2)
0
7 (9·9)
14 (19·7)
1 (1·4)
1 (1·4)
1 (1·4)
1 (1·4)
8 (11·3)
62 (87·3)
Amazonian
Indians
(n = 70)
Di(a+b–)
Di(a+b+)
Di(a–b+)
DI*A/DI*A
DI*A/DI*B
DI*B/DI*B
0
0
17 (24·3)
4 (5·7)
26 (37·1)
0
23 (32·9)
0
0
27 (38·6)
26 (37·1)
17 (24·3)
221 (69·5)
68 (21·4)
29 (9·1)
Total
166A
n (%)
166A/G
n (%)
166G
n (%)
Total
n (%)
318
SCD, sickle cell disease.
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 84, 326–330
New DI*A allele among Brazilians
Table 2 Allelic frequencies
Allele
Nucleotide
DI A
2561T
DI B
2561C
Band 3
166 A
Band 3-Memphis
166G
Blood donors
SCD patients
Japanese
Amazonian Indians
0·02
0·01
0·07
0·57
0·98
0·99
0·93
0·43
0·96
0·91
0·81
0·46
0·04
0·09
0·19
0·54
SCD, sickle cell disease.
329
populations, among the Amazonian Indians none of the 60
DI*B alleles had the 166G mutation, whereas 76 of the 80 DI*A
alleles had the 166G mutation. It would be interesting to
determine the H2DIDS-binding status of RBCs and the electrophoretic mobility of RBC membranes from the Amazonian
Indians with the novel alleles. Such studies would be easier
to perform on RBCs from a person who is homozygous for the
mutation or, possibly, on transfected cells expressing variants
of band 3 encoded by combinations of DI and 166 alleles.
Of the 636 alleles tested, 542 were DI*B, of which 63 (6·6%)
carried 166G (Band 3-Memphis). Compared with Caucasians
and African Blacks, RBCs from people of Japanese descent
are serologically known to carry a high frequency of Dia.
Among the 71 Japanese included in this study (142 alleles),
10 were DI*A and 132 were DI*B; 27 were 166G. We observed
a correlation between DI A and 166G, and the presence of
166G with some of the DI*B alleles, which is consistent with
the findings of Spring et al. [15] and Black et al. [21]
Our findings highlight the importance of analysing different populations to achieve a better understanding of the
genetic basis associated with blood-group antigens.
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Fig. 1 Depiction of the Band 3 alleles showing the Diego and Band
3-Memphis associations.
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3-Memphis was present in 0·19 of Japanese, 0·04 of general
blood donors, and 0·09 of African Brazilians with SCD.
Conclusion
We report the frequencies of DI*A and DI*B alleles, in relation
to the Band 3-Memphis polymorphism, in different populations in Brazil. During this population study, unexpectedly
we found four Amazonian Indians who had the DI*A allele
associated with 166A instead of the expected 166G that
defines the Band 3-Memphis variant (Fig. 1) [15,16]. Red
cells from these four people typed Di(a+b–). The high
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© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 84, 326 – 330
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