Legal Medicine xxx (2014) xxx–xxx Contents lists available at ScienceDirect Legal Medicine journal homepage: www.elsevier.com/locate/legalmed Haptoglobin genotyping of Vietnamese: Global distribution of HPdel, complete deletion allele of the HP gene Mikiko Soejima a, Tetsuro Agusa b, Hisato Iwata b, Junko Fujihara c, Takashi Kunito d, Haruo Takeshita c, Vi Thi Mai Lan e, Tu Binh Minh e, Shin Takahashi f, Pham Thi Kim Trang e, Pham Hung Viet e, Shinsuke Tanabe e, Yoshiro Koda a,⇑ a Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume, Japan Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan 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 b c 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 xxxx 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 frequency of HPdel in 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 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, HP1 and HP2, that give rise to the three common phenotypes 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 and of HP1 Both HP1 and HP2 have 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 ⇑ 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) HP-gene deletion allele (HPdel), which has an approximately 28 kb deletion extending from the HP promoter region to intron of the HP-related gene We identified HPdel by 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 HPdel homozygotes produce no HP protein and are phenotypically anhaptoglobinemic, while HPdel heterozygotes 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 HPdel in 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 Please cite this article in press as: Soejima M et al Haptoglobin genotyping of Vietnamese: Global distribution of HPdel, complete deletion allele of the HP gene Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.08.004 M Soejima et al / Legal Medicine xxx (2014) xxx–xxx 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 [12,20] Briefly the 20 ll PCR reaction contained about 0.1 ng to 10 ng genomic DNA, 10 ll of Premix Ex Taq™ (Perfect Real Time) (Takara), and the following primers and TaqMan probes at the indicated 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), HP50 probe (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 s, and annealing and extension at 60 °C for 30 s All oligonucleotides 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.) The DCT (difference in threshold cycles) of each sample was calculated as [CT of HP50 (control)] À [CT of HP2 (target)] The DDCT of each sample (DDCT sample) was obtained by [DCT of reference] À [DCT of sample] The DCt reference value was obtained using ng of genomic DNA from an HP2/HP2 individual The ratio of HP2:HP50 of each sample was calculated as 2ÀDDCT sample [20] Allele and genotype frequencies were calculated by the counting method and deviations from Hardy– Weinberg equilibrium (HWE) were assessed by using the v2 test 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 (50 flanking sequence of exon 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 measurements were performed for each sample The samples with the 2ÀDDCT sample in the range of À0.47–0.37 (mean ± SD; À0.05 ± 0.17) were determined to be HP2/HP2, while those with the 2ÀDDCT sample in the range from 0.64 to 1.54 (1.10 ± 0.16) were determined to be HP2/HP1 Samples with no HP2 signal were typed as HP1/HP1 Samples with amplification of the Hp50 and HPdel allelespecific regions and without amplification of the HP2 allele-specific region were genotyped as HP1/HPdel, while those with all three signals 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, HP1/HPdel, and 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 populations (approximately 0.294) or previous results (0.25–0.30) [3,21] The frequency of HPdel in various populations including that of Vietnamese is shown in Table As mentioned above, distribution of this allele seemed to be restricted to East and Southeast Asia [6,12,13,20,22] How did this result come about? As discussed in a previous paper, possible reasons are human migration and genetic drift [13] The frequencies of HPdel in 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 HPdel from somewhere in China to Korea, and Japan [13] The analysis 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 HPdel in Mongolians than in other East Asian populations (Table 1) The second explanation for the distribution is natural selection Some genetic variants that confer resistance 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 Frequency of the HPdel allele in various populations Populations Chromosomes HPdel Refs Number Frequency (%) 2130 12,404 568 314 1018 1962 19 212 17 31 57 0.9 1.7 3.0 2.5 3.0 2.9 [12,13] [6,7] [7] [7] [16] [22] Central Asians Tibetans Tamang Uyghurs 240 106 112 0 0 0 [13] [13] [13] Southeast Asians Indonesians Thais Vietnamese 210 400 586 12 1.0 1.5 2.0 [9] [17] This study South Asians Bangladeshi Tamils Sinhalese 102 104 102 0 0 0 [13] [13] [13] West Asians Turks 214 0 [13] 200 200 0 0 [7] [6] 202 246 1196 0 0 0 [7] [14] [15] East Asians Mongolians Japanese Han Chinese Koreans Koreans Taiwanese Europeans European-Africans EuropeanAmericans Africans Xhosans Ghanaians Gambians Please cite this article in press as: Soejima M et al Haptoglobin genotyping of Vietnamese: Global distribution of HPdel, complete deletion allele of the HP gene Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.08.004 M Soejima et al / Legal Medicine xxx (2014) xxx–xxx diseases 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 trypanosomiasis [3] Although HPdel homozygotes seem to live their lives without any symptoms except for having a risk of developing anaphylaxis after a blood transfusion, at least in our time, the individuals having HPdel may have higher susceptibility to some kinds of diseases in the restricted area [7] HPdel was 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 Himalayas 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 se357,385 with Han Chinese [27] Considering these findings, the absence of the HPdel in Tibetans and Tamang may suggest the possibility of a deleterious effect of the allele on highlanders for some reason(s) Alternatively, the HPdel in 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 populations [7] From a preventive medicine perspective, the genetic diagnosis of HPdel in 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 HPdel and summarized the geographical distribution of the HPdel in 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 References [1] 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HPdel, complete deletion allele of the HP gene Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.08.004 ... 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 , HP1 /HPdel, and HP2 /HPdel,. .. cite this article in press as: Soejima M et al Haptoglobin genotyping of Vietnamese: Global distribution of HPdel, complete deletion allele of the HP gene Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.08.004... cite this article in press as: Soejima M et al Haptoglobin genotyping of Vietnamese: Global distribution of HPdel, complete deletion allele of the HP gene Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.08.004