Haptoglobin genotyping of Vietnamese: Global distribution of HP del ,complete deletion allele of the HP gene Mikiko Soejimaa, Tetsuro Agusab, Hisato Iwatab, Junko Fujiharac, Takashi Kuni
Trang 1Haptoglobin genotyping of Vietnamese: Global distribution of HP del ,
complete deletion allele of the HP gene
Mikiko Soejimaa, Tetsuro Agusab, Hisato Iwatab, Junko Fujiharac, Takashi Kunitod, Haruo Takeshitac,
Vi Thi Mai Lane, Tu Binh Minhe, Shin Takahashif, Pham Thi Kim Trange, Pham Hung Viete,
Shinsuke Tanabee, Yoshiro Kodaa,⇑
a
Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume, Japan
b
Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan
c
Department of Legal Medicine, Shimane University Faculty of Medicine, Enya 89-1, Izumo 693-8501, Japan
d
Department of Environmental Sciences, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan
e Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Vietnam National University, T3 Building, 334 Nguyen Trai Street, Thanh Xuan District, Hanoi, Viet Nam
f
Department of Environmental Conservation, Faculty of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama 790-8566, Japan
a r t i c l e i n f o
Article history:
Received 16 August 2014
Received in revised form 18 August 2014
Accepted 19 August 2014
Available online 27 August 2014
Keywords:
Gene deletion
Geographic distribution
Ancestry informative marker
Anaphylaxis
a b s t r a c t
The haptoglobin (HP) gene deletion allele (HPdel) is responsible for anhaptoglobinemia and a genetic risk factor for anaphylaxis reaction after transfusion due to production of the anti-HP antibody The distribution of this allele has been explored by several groups including ours Here, we studied the fre-quency of HPdelin addition to the distribution of common HP genotypes in 293 Vietnamese The HPdel was encountered with the frequency of 0.020 The present result suggested that this deletion allele is restricted to East and Southeast Asians Thus, this allele seems to be a potential ancestry informative marker for these populations
Ó 2014 Elsevier Ireland Ltd All rights reserved
1 Introduction
Haptoglobin (HP) is a plasma glycoprotein and is known to be
one of the acute phase reactants[1,2] It binds hemoglobin (Hb)
to prevent both iron loss and kidney damage due to oxidative
activity of Hb during intravascular hemolysis[3] Humans have a
genetic polymorphism of the protein due to two codominant
alleles, HP1and HP2, that give rise to the three common
pheno-types HP1-1, HP2-1, and HP2-2[3] According to this polymorphic
feature, HP had been used as a genetic marker in determinations of
parentage The HP gene locates on the long arm of chromosome 16
(16q22.3) and consists of five (HP1) or seven (HP2) exons HP2
appears to have been generated by a 1.7-kb intragenic duplication
of exons 3 and 4 of HP1 Both HP1and HP2have been found in every
population examined, although their frequencies vary considerably
among populations[3,4]
In addition to common polymorphisms, several rare variants of
the HP phenotypes have been reported [3] One of them is the
HP-gene deletion allele (HPdel), which has an approximately
28 kb deletion extending from the HP promoter region to intron
4 of the HP-related gene We identified HPdelby genetic analysis
of several Japanese cases with a negative result only for HP inheritance encountered in determinations of parentage performed
in forensic practice [5] This silent allele allows us to interpret many cases with ‘‘incompatible’’ heredity HPdelhomozygotes pro-duce no HP protein and are phenotypically anhaptoglobinemic, while HPdelheterozygotes have a lower amount of HP protein in their serum than those without HPdel [5,6] HPdel homozygotes have a risk of undergoing anaphylactic transfusion reactions if they produce HP antibodies[7] Because washed red blood cells and platelet concentrate are effective in preventing transfusion-related anaphylactic reactions [8], we have developed several simple methods to detect this allele before transfusion[7,9–11] A series
of studies by several groups including us have found HPdelin East and Southeast Asian populations but not in others[6,7,9,12–17] For better understanding of the distribution of this allele in order
to prevent serious problems in clinical practice and to determine whether it can be used as one of the ancestry informative markers,
we determined the HP genotypes of a Vietnamese population in this study
http://dx.doi.org/10.1016/j.legalmed.2014.08.004
1344-6223/Ó 2014 Elsevier Ireland Ltd All rights reserved.
⇑Corresponding author Address: Department of Forensic Medicine and Human
Genetics, Kurume University School of Medicine, Kurume 830-0011, Japan Tel.: +81
942 31 7554; fax: +81 942 31 7700.
E-mail address: ykoda@med.kurume-u.ac.jp (Y Koda).
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Legal Medicine
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / l e g a l m e d
Trang 22 Materials and methods
2.1 Samples
Blood samples of 293 local residents were randomly collected in
Hoa Hau and Liem Thuan in March 2006[18], Thanh Vanh and
Thach Hoa in September 2007[18], and Thach Hoa, Son Dong,
and Van Phuc in September 2008 All are communes located in
the Red River Delta, Vietnam Informed consent was obtained from
all participants, and this study was approved by the Ethical
Committee of Ehime University and Kurume University, Japan
2.2 HP genotyping
Genomic DNA was extracted from the blood of 293 subjects
using a QIAamp DNA mini kit (Qiagen, Chatworth, CA) as described
previously [19] HP genotypes were determined using
TaqMan-based real-time PCR methods as described previously
10 ng genomic DNA, 10ll of Premix Ex Taq™ (Perfect Real Time)
(Takara), and the following primers and TaqMan probes at the
indi-cated concentrations: HP2-F and -R primers (300 nmol/L), HP2
probe (83 nmol/L) for the detection of HP2, HP50-F and -R primers
(150 nmol/L), HP50probe (42 nmol/L) for the detection of the HP 50
region, HPdel-F and -R primers (300 nmol/L), and HPdel probe
(83 nmol/L) for the detection of HPdel The PCR temperature profile
was 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C
for 5 s, and annealing and extension at 60 °C for 30 s All
oligonu-cleotides for TaqMan-based real-time PCR were synthesized by
Biosearch Technologies (Tokyo, Japan) Fluorescence was measured
using an Mx3000P system (Agilent Technologies, Inc., Tokyo,
Japan) Data were collected and analyzed using MxPro™ Software
(version 4.00, Agilent Technologies, Inc.) TheDCT (difference in
threshold cycles) of each sample was calculated as [CTof HP50
(con-trol)] [CT of HP2 (target)] The DDCT of each sample (DDCT
sample) was obtained by [DCTof reference] [DCTof sample] The
DCt reference value was obtained using 5 ng of genomic DNA from
an HP2/HP2individual The ratio of HP2:HP50of each sample was
calculated as 2DD C T sample
[20] Allele and genotype frequencies were calculated by the counting method and deviations from Hardy–
Weinberg equilibrium (HWE) were assessed by using thev2test
3 Results and discussion
The real-time PCR method for determination of common HP
alleles was based on comparative threshold cycles (CT) of the
HP2-specific sequence (duplication junction) and a control
sequence (50flanking sequence of exon 1 of HP)[20] The previous
population genetic analyses revealed that HPdel is distributed
among East and Southeast Asians The countries of Southeast Asia
are geographically isolated from one another but are occupied by
ethnically similar peoples[3] The genetic variation of the FUT2
gene, which is rich in population specific polymorphisms, found
in the same samples supported this, and the variation in Vietnam
also seemed reflect the present geographic location and history
of human migration [19] Therefore, HPdel was expected to be
found in Vietnamese Real-time PCR was designed to detect HPdel
at the same time as other alleles, and at least duplicate
measure-ments were performed for each sample The samples with the
2DD C T sample
in the range of 0.47–0.37 (mean ± SD; 0.05 ± 0.17)
were determined to be HP2/HP2, while those with the
2 DD C T samplein the range from 0.64 to 1.54 (1.10 ± 0.16) were
deter-mined to be HP2/HP1 Samples with no HP2 signal were typed as
HP1/HP1 Samples with amplification of the Hp50and HPdel
allele-specific regions and without amplification of the HP2allele-specific
region were genotyped as HP1/HPdel, while those with all three sig-nals were genotyped as HP2/HPdel We did not detect an HPdel
homozygote in this study Accordingly, the HP genotypes of 293 Vietnamese consisted of 26 HP1/HP1, 111 HP1/HP2, 144 HP2/HP2,
4 HP1/HPdel, and 8 HP2/HPdel, and the allele frequencies were
HP1= 0.285, HP2= 0.695, and HPdel= 0.020 (Table 1) The genotype distribution frequencies were in accordance with the distribution expected based on Hardy–Weinberg equilibrium In addition, the allele frequency of HP1 was similar to those of neighbor popula-tions (approximately 0.294) or previous results (0.25–0.30)[3,21] The frequency of HPdelin various populations including that of Vietnamese is shown inTable 1 As mentioned above, distribution
of this allele seemed to be restricted to East and Southeast Asia
a previous paper, possible reasons are human migration and genetic drift[13] The frequencies of HPdelin Han Chinese, Koreans, and Japanese, also described previously, is lowest in Japanese and highest in Han Chinese (Table 1), which is consistent with the hypothesis that recent human migration from China brought the
HPdelfrom somewhere in China to Korea, and Japan[13] The anal-ysis of FUT2 variations in a Mongolian population suggested that Mongolians are an admixture of East Asians (Mongoloid) and West Asians (Caucasoid) based on the composition of the non- or weakly functional alleles of FUT2[23] This observation is also consistent with the lower frequency of HPdelin Mongolians than in other East Asian populations (Table 1) The second explanation for the distri-bution is natural selection Some genetic variants that confer resis-tance to various infectious diseases are now understood to have spread through human populations over time, leaving distinctive patterns in the human genome by natural selection because such
Table 1 Frequency of the HP del allele in various populations.
Populations Chromosomes HP del
Refs Number Frequency (%) East Asians
Mongolians 2130 19 0.9 [12,13]
Japanese 12,404 212 1.7 [6,7]
Han Chinese 568 17 3.0 [7]
Koreans 314 8 2.5 [7]
Koreans 1018 31 3.0 [16]
Taiwanese 1962 57 2.9 [22]
Central Asians
Southeast Asians Indonesians 210 2 1.0 [9]
Vietnamese 586 12 2.0 This
study South Asians
Bangladeshi 102 0 0 [13]
Sinhalese 102 0 0 [13]
West Asians
Europeans European-Africans 200 0 0 [7]
European-Americans
Africans
Ghanaians 246 0 0 [14]
Gambians 1196 0 0 [15]
Trang 3diseases may threaten life before the reproductive years The most
typical example is observed in malaria-related variants, sickle cell
hemoglobin, thalassemia, glucose-6-phosphate dehydrogenase
deficiency (G6PD), and the Duffy blood group[24] The common
HP alleles and HP serum levels have been shown to be associated
with various inflammatory-linked infectious diseases such as
malaria, tuberculosis, HIV, hepatitis C, and American
trypanosomi-asis[3] Although HPdelhomozygotes seem to live their lives
with-out any symptoms except for having a risk of developing
anaphylaxis after a blood transfusion, at least in our time, the
indi-viduals having HPdelmay have higher susceptibility to some kinds
of diseases in the restricted area [7] HPdelwas encountered in
neither Tibetans (2n = 240) nor Tamang (2n = 106) (Table 1) A
recent study reported that evidence for relatively recently (7000–
10,000 years ago) shared Y chromosome and mitochondrial DNA
haplotypes between Tibetans and Han Chinese[25] Another study
by analysis of mitochondrial DNA suggested that while the
Hima-layas acted as a geographic barrier to gene flow from the Indian
subcontinent to the Tibetan highland, they also served as a conduit
for gene flow between Central and East Asia[26] The variation of
our FUT2 data was also consistent with these observations That is,
both Tibetans and Tamang shared the common nonfunctional
allele se357,385with Han Chinese[27] Considering these findings,
the absence of the HPdelin Tibetans and Tamang may suggest the
possibility of a deleterious effect of the allele on highlanders for
some reason(s) Alternatively, the HPdelin these populations was
neutral and became extinct due to genetic drift
The incidence of IgA deficiency in Japanese was reported to be
about 1/30,000, which is lower than that in Europeans (1/2500)
However, the incidence of anhaptoglobinemia appears to be much
higher than that of IgA deficiency in East and Southeast Asian
pop-ulations[7] From a preventive medicine perspective, the genetic
diagnosis of HPdelin the residents of these areas may be desirable
because it is a germline mutation and is needed only once in a
lifetime
In conclusion, we demonstrated the frequencies of HP alleles
including HPdeland summarized the geographical distribution of
the HPdelin a Vietnamese population, a genetic risk factor for the
serious adverse reaction to transfusion This allele may be one of
the potential ancestry informative markers of East and Southeast
Asia
Acknowledgements
This work was supported by grants-in-aid for Scientific
Research from the Ministry of Education, Science, Culture and
Sports of Japan and grant from the Ishibashi Foundation for the
Promotion of Science The authors wish to thank the staff of the
CETASD, Hanoi University of Science and Dr Nguyen Minh Tue
from CMES for their help in sample collection The authors thank
Ms Katherine Ono for the English editing of this manuscript
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