FU Xiao-ling, CAI Xing-quan, XIA Lan

1. Department of Blood Transfusion, Hainan Women and Children’s Medical Center, Haikou 570311, China

2. Department of Blood Transfusion, Hainan General Hospital, Haikou 570311, China

Keywords:Blood group gene Gene frequency Rare blood group bank Li ethnic

ABSTRACT Objective: To screen the blood group system genes of Duffy, Lutheran, Kidd, Diego,Dombrock blood group systems of Li ethnic group in Hainan Province and provide laboratory data for the rare blood group database in this area. Methods: The alleles of Duffy, Lutheran,Kidd, Diego, Dombrock blood group systems of 300 voluntary participants of Li ethnic group in Hainan were detected by sequence-specific primer polymerase chain reaction , and the polymorphism was analyzed. Results: The allele frequencies of Duffy, Lutheran , Kidd,Diego, Dombrock blood group systems of Li ethnic groups in Hainan Province are 0.958 3 for Fya, 0.041 7 for Fyb, 0.835 0 for Aua, 0.165 0 for Aub, 0.450 0 for Jka, 0.550 0 for Jkb, 0.066 7 for Dia, 0.933 3 for Dib, 0.101 7 for Doa and 0.898 3 for Dob, respectively. The antigen incompatibility rates of Fya/Fyb, Aua/Aub, Jka/Jkb, Dia/Dib, Doa/Dob of Duffy, Lutheran,Kidd, Diego, Dombrock blood group systems were 7.67%, 23.76%, 37.25%, 11.67% and 16.60%, respectively. Conclusion: The gene frequencies of Duffy, Lutheran, Kidd, Diego,Dombrock blood group systems of Li ethnic group in Hainan Province are polymorphic, and the antigen incompatibility rates of alleles are higher, which is quite different from that of other nationalities in China and with unique ethnic distribution characteristics.It is of great significance to establish the rare blood group database in this region.?Corresponding author: CAI Xing-quan, Deputy Chief Technician E-mail: cxqhnhn@163.com.

1. Introduction

Human erythrocyte blood group is an important genetic marker.The frequency distribution of blood group antigen gene is different due to the differences of race and region[1-4].At present, The ABO and Rh blood group systems are the main focus of clinical attention.Relatively little attention has been paid to blood group systems such as Duffy, Lutheran, Kidd, Diego and Dombrock. However,there are great differences in the frequency distribution of these rare blood group alleles, and the antigen incompatibility rate of alleles is high, which is an important factor affecting the difficulty of blood typing and cross matching in patients with rare blood group who have repeated blood transfusion and multiple pregnancie. Therefore,it is an effective way to solve the transfusion safety of rare blood group patients and an important direction of precision transfusion therapy by exploring the gene distribution characteristics of rare blood groups in different regions and different ethnic populations,and establishing a rare blood group database. In this paper, five rare blood group systems (Duffy, Lutheran, Kidd, Diego and Dombrock)of Li ethnic group in Hainan Province were detected by PCRSSP method, and the polymorphism was analyzed, which lay the foundation for the establishment of rare blood group bank in this region.

2. Materials and methods

2.1 General information

A total of 300 blood samples were collected from Li ethnic group in Hainan Province, with a male to female ratio of 180:120 and an age range of 18-60 years. Inclusion criteria: Li ethnic volunteers in Limu Mountain area of Qiongzhong County, Hainan Province were selected, who were from Li ethnic group in Hainan Province within three generations, and without consanguineous relationship and in good health without family genetic diseases or concurrent diseases.All participants in the experimental study signed the informed study agreement, and the study was approved by the ethics committee of the hospital. 5 mL of EDTA anticoagulant blood was collected from each volunteer. The plasma and blood cells were separated by centrifugation at a centrifugal force of 2 000 g and 4 000 RPM for 5 min. All plasma and blood cells were stored in a refrigerator at -20 ℃.

2.2 Reagents and instruments

The blood group gene detection kit covers 31 RBC blood group antigens from 13 blood group systems, including Scianna, Colton,Kell, MNS, Lutheran, Dombrock, Kidd, Diego, Duffy, Knops, Indian and Vel, which provided by Jiangsu Zhongji Wantai Biomedical Co., LTD. The detected alleles and corresponding SNPS was in Table 1, and the length of amplified products of alleles was in Table 2. Nucleic acid extraction and purification Kit and BioReady rTaq polymerase were provided by Hangzhou Bori Company. Agarose was provided by Sheng Xing Biological Company. 10 × TBE Buffer and RealBand DNA Molecular Weight Marker were provided by Shanghai Shengong Company. Nucleic Acid Purifier QIAcube was provided by Qiagen Company. PCR Amplification Instrument HEMA9600, Centrifuge 14D and Gel Imaging System UV-3C were provided by Dark Horse Medical Devices Company. Electrophoresis apparatus DY-16 was provided by No.16 Company.

Tab 1 Alleles and corresponding SNP detected by the kit

Tab 2 PCR product lengths and corresponding alleles

2.3 Methods

2.3.1 Genomic DNA extraction

The DNA of blood samples was extracted according to the instructions (nucleic acid extraction and purification kit).Subsequently, the purity and concentration of DNA were determined,and the concentration value was adjusted to 30-50 ng/L, and the OD 260/280 ratio was 1.8-2.0. The DNA was stored at -20 ℃ for later use.

2.3.2 PCR amplificationThere are 7 microliters of primer mixture, 1 microliter of 0.3-05 U Taq enzyme, 1 microliter of DNA sample in the PCR reaction system which the total volume is 9 microliters. After 5 min at 95 ℃, the PCR amplification procedure was as follows: 95 ℃ for 3 s; 60 ℃for 30 s; 72 ℃for 90 s; 30 cycles were performed at, then followed by an extension of 5 min at 72 ℃and storage at 4 ℃.

2.3.3 Electrophoresis of PCR productsAfter mixing 1 microliter of 0.5×TBE solution and 5 microliter of PCR product thoroughly, The samples were added into the Wells of 2.5% agarose gel, and electrophoresis was carried out at a voltage of 100 V and a time of 45min. After electrophoresis, electrophoretic images were taken and stored.

2.3.4 Analysis of experimental resultsThe two antigenic primers were in different PCR reaction tubes.If there are two bands, it means heterozygous; if there is one band,it means homozygous; if there is no amplification, it means that the experiment fails, and repeatability test is needed for verification.According to the length of the gel electrophoresis band, the genotypes of the tested samples were specified according to the kit instructions (Figure 1).

Fig 1 Genotyping of thirteen rare blood group systems of a volunteer in Li ethnic group of Hainan Province by PCR-SSP

2.3.5 Statistical treatmentThe frequencies of rare blood group genes of Li ethnic group in Hainan Province were calculated according to genetic gene counting method. Hardy-weinberg coincidence was verified between the observed value and the expected value of gene phenotype (P＞0.05).χ2test was used to analyze the differences in gene frequency distribution of Duffy, Lutheran, Kidd, Diego and Dombrock blood group systems between Li ethnic group in Hainan and other ethnic groups in different regions. Only when P＜0.05 was considered statistically significant. Differences in gene frequency distribution of Duffy, Lutheran, Kidd, Diego and Dombrock blood group systems between Li ethnic group in Hainan and other ethnic groups in different regions was analyzed by χ2test, and only when P＜0.05 was considered statistically significant.The the antigen incompatibility rate (MP) was calculated according to the formula MP= 2ab (1-ab)[2], where a and b represent the frequencies of allele a and b,respectively.

3. Results

3.1 The allele frequencies of Duffy, Lutheran, Kidd, Diego and Dombrock blood group systems in

Li ethnic group in Hainan Province were as follows: Fya=0.9583,Fyb=0.0417; Aua=0.8350, Aub=0.1650, Jka=0.4500, Jkb=0.5500,Dia=0.0667, Dib=0.9333; Doa=0.1017; Dob=0.8983.The mismatching rates of Fya/Fyb, Aua/Aub, Jka/Jkb, Dia/Dib, Doa/Dobwere 7.67%,23.76%, 37.25%, 11.67%, 16.60%, respectively. The results of χ2test showed that the difference between the observed value and expected value of Kidd blood group system was statistical significance (P＜0.05), while the observed values and expected values of Duffy, Lutheran, Diego, Dombrock blood group systems were in accordance with Hardy-Weinberg law (P＞0.05).(Table 3).

3.2 Comparison of allele frequencies of Duffy, Lutheran,Kidd, Diego and Dombrock blood group systems between Li ethnic group in Hainan and other ethnic groups in China

In Duffy blood group system, the gene frequency of Li ethnic group in Hainan was Fya=0.958 3,which was significantly higher than that of Hui ethnic group in Xinjiang(0.872 7), Uygur ethnic group in Xinjiang(0.699 4), Kazak ethnic group in Xinjiang(0.757 6), Han ethnic group in Yuncheng(0.896 7), Han ethnic group in Xian(0.896 7)[5-7,11,15].The frequency of Fybgene was 0.041 7, significantly lower than the data of the above several ethnic groups, the difference was statistically significant (P＜0.05).Compared with the Fya/Fybgene frequencies of Tibetan ethnic group, Yi ethnic group in Sichuan, Han ethnic group in Yueyang, Han ethnic group in Dalian,Zhuang ethnic group in Guangxi, Dai ethnic group in inYunnan[8-10,12-14],the difference was not statistically significant.(Table 4).

The gene frequency of Lutheran blood group system in Li ethnic group in Hainan was Aua=0.8350,it was Significantly lower than that of Hui ethnic group in Xinjiang(0.909 1),Tibetan ethnic group(0.910 8),Han ethnic group in Chengdu(0.893 1)[5,8,10].The gene frequency of Aubwas 0.165 0,which was Higher than the data of Hui ethnic group in Xinjiang, Tibetan ethnic group, Han ethnic group in Chengdu, the difference was statistically significant (P＜0.05).Compared with the gene frequency of Aua/Aubof other ethnic groups[5,6], there was no significant difference. (Table 5).

In the Kidd blood group system,the gene frequency of Li ethnic group in Hainan was Jka=0.450 0. It was significantly lower than that of Tibetan ethnic group(0.551 3) and Man ethnic group in Harbin(0.565 0) [8,16].The gene frequency of Jkbwas 0.550 0,Which was Higher than that of Tibetan ethnic group and Man ethnic group in Harbin, and the difference was statistically significant (P＜0.05).Compared with the gene frequency of Jka/Jkbof other ethnic groups[5-10,17], the difference was not statistically significant. (Table 6).

In the Diego blood group system, the gene frequency of Li ethnic

group in Hainan was Dia=0.066 7. It was significantly higher than that of Hui ethnic group in Xinjiang(0.015 9),Tibetan ethnic group(0.034 2),Yi ethnic group in Sichuan(0.016 7), and Han ethnic group in Yueyang (0.498 3)[3,5,8,9].The gene frequency of Dibwas 0.933 3, it was significantly lower than that of Hui ethnic group in Xinjiang, Tibetan ethnic group, Yi ethnic group in Sichuan,and Han ethnic group in Yueyang. The difference was statistically significant(P＜0.05).Compared with the gene frequency of Dia/Dibof other ethnic groups[6,7,10,16], the difference was not statistically significant.(Table 7).

Tab 3 Allele frequency distribution of Duffy, Lutheran, Kidd, Diego and Dombrock blood group systems of Li ethnic group of Hainan Province (n=300)

Tab 4 Comparison of Duffy blood group allele frequency distribution between Li ethnic group of Hainan Province and other ethnic groups in China

Tab 5 Comparison of Lutheran blood group allele frequency distribution between Li ethnic group of Hainan Province and other ethnic groups in China

Tab 6 Comparison of Kidd blood group allele frequency distribution between Li ethnic group of Hainan Province and other ethnic groups in China

In the Dombrock blood group system,the gene frequency of Li ethnic group in Hainan was Doa=0.101 7.Compared with that of Uygur ethnic group in Xinjiang(0.234 2)and Kazak ethnic group in Xinjiang(0.221 9)[6,7], there is a significant difference.The gene frequency of Dobwas 0.898 3,which was higher than the data of Uygur ethnic group in Xinjiang and Kazak ethnic group in Xinjiang,and the difference was statistically significant (P＜0.05).Compared with the gene frequency of Doa/Dobof other ethnic groups, such as: Hui ethnic group in Xinjiang,Tibetan ethnic group, Yi ethnic group in Sichuan, Han ethnic group in Yueyang, Han ethnic group in Chengdu, Zhuang ethnic group in Guangxi and Man ethnic group in Harbin[3,5-10,13,16], the difference was not statistically significant.(Table 8).

Tab 7 Comparison of Diego blood group allele frequency distribution between Li ethnic group of Hainan Province and other ethnic groups in China

Tab 8 Comparison of Dombrock blood group allele frequency distribution between Li ethnic group of Hainan Province and other ethnic groups in China

4. Discussion

There are many types and amounts of blood group system antigens in human erythrocytes, but the blood group antigens expressed on the erythrocytes of each person are not the same. At present, clinical blood transfusion is conducted based on the principle of compatible infusion of ABO and Rh (D) blood group antigens, while the antigens of Duffy, Lutheran, Kidd, Diego and Dombrock blood group system were not routinely detected. This may have little impact on patients who receive blood transfusion for the first time, but it has a great impact on patients who receive repeated blood transfusion or rely on blood transfusion for a long time. Due to the inconsistent transfusion of blood group antigens, it was easy to stimulate the body to produce alloimmunity to produce irregular antibodies. Clinical screening for irregular antibodies, it is found that the common alloantibodies included not only antibodies of the Rh blood group system, but also included the irregular antibodies of Duffy, Lutheran, Kidd, Diego,Dombrock and other blood group systems. It may occur as a kind of antibodies or several resistance joint, and these immune antibodies can cause blood group identification difficulties and cross-matched blood incompatibility, clinically characterized by hemolytic transfusion reaction[18].Therefore, it is of great clinical significance to understand the differences of the distribution characteristics of gene frequency of Duffy, Lutheran, Kidd, Diego and Dombrock blood group systems in different ethnic groups establish a rare blood group database for finding suitable blood for patients with rare blood group in a short time.

This study showed that the characteristics of polymorphism in alleles frequency distribution of Duffy, Lutheran, Kidd, Diego and Dombrock of Li ethnic group in Hainan were similar to those reported in literature [2].The mismatch rates of the dual antigens Fya/Fyb, Jka/Jkb, Dia/Dib, Doa/Doband Aua/Aubwere relatively high.Theoretically, the higher antigen incompatibility rate, the higher the probability of stimulating the body to produce immune irregular antibodies during random blood transfusion and pregnancy. It would increase the risk of repeated blood transfusion patients and multiple pregnancies and lead to an increase in the incidence of immune hemolytic reaction, which would affect the effect of clinical transfusion therapy. Therefore, attention should be paid in clinical practices to the coordination of blood group antigens outside ABO and Rh blood group system to improve the efficacy of blood transfusion.

This study showed that the observed and expected values of gene frequency distribution of Duffy, Lutheran, Diego and Dombrock blood group system of Li ethnic group in Hainan Province were consistent with Hardy-Weinberg genetic equilibrium rule (P＞0.05).The difference between the observed value and expected value of Kidd blood group system was statistical significance (P＜0.05).This may be related to the fact that most of the people of Li ethnic group in Hainan affected by living environment and marriage customs. The gene heredity of Kidd blood group system has been changed and has its own distribution characteristics. This is of great significance to study the safety of clinical blood transfusion.

This study showed that the allele distribution of the five blood group systems of Duffy, Lutheran, Kidd, Diego and Dombrock in Li ethnic group in Hainan Province was significantly different from that of different ethnic group in other regions of China. Compared with other ethnic groups of China, such as Kazak ethnic group in Xinjiang, Han ethnic group in Yuncheng, Uygur ethnic group in Xinjiang, Hui ethnic group in Xinjiang and Han ethnic group in Xian[5-7,11,15],the difference of the gene frequencies of Fyaand Fybof Li ethnic group in Hainan Province was statistically significant(P＜0.05).While compared with the datas of Tibetan ethnic group, Yi ethnic group in Sichuan, Han ethnic group in Yueyang,Han ethnic group in Dalian, Zhuang ethnic group in Guangxi,Dai ethnic group in inYunnan[3,8-10,12-14],the difference was not statistically significant. Compared with other ethnic groups of China,such as Hui ethnic group in Xinjiang, Tibetan ethnic group,Han ethnic group in Chengdu, the difference of the gene frequencies of Auaand Aubof Li ethnic group in Hainan Province was statistically significant(P＜0.05).While compared with the datas of Uygur ethnic group in Xinjiang and Kazak ethnic group in Xinjiang, the difference was not statistically significant. Compared with Tibetan ethnic group and Man ethnic group in Harbin[8,16],the difference of the gene frequencies of Jkaand Jkbof Li ethnic group in Hainan Province was statistically significant(P＜0.05).While compared with Yi ethnic group in Sichuan, Kazak ethnic group in Xinjiang, Han ethnic group in Yueyang, Han ethnic group in Chengdu, Hui ethnic group in Xinjiang, Han ethnic group in Hebei[3,5-10,17],the difference was not statistically significant. Compared with Hui ethnic group in Xinjiang, Tibetan ethnic group, Yi ethnic group in Sichuan, Han ethnic group in Yueyang, the difference of the gene frequencies of Diaand Dibof Li ethnic group in Hainan Province was statistically significant(P＜0.05).While compared with Uygur ethnic group in Xinjiang, Kazak ethnic group in Xinjiang, Han ethnic group in Chengdu, Man ethnic group in Harbin[6-7,10,16],the difference was not statistically significant. Compared with Uygur ethnic group in Xinjiang and Kazak ethnic group in Xinjiang[6,7],the difference of the gene frequencies of Doaand Dobof Li ethnic group in Hainan Province was statistically significant(P＜0.05).While compared with Hui ethnic group in Xinjiang, Tibetan ethnic group, Yi ethnic group in Sichuan, Han ethnic group in Yueyang, Han ethnic group in Chengdu, Man ethnic group in Harbin, Zhuang ethnic group in Guangxi[3,5-10,13,16],the difference was not statistically significant. It can be seen that the inheritance and distribution of Duffy, Lutheran,Kidd, Diego and Dombrock rare blood group genes of Li ethnic group in Hainan have ethnic characteristics. This was due to the fact that most of the Li ethnic group in Hainan live in remote and economically backward areas, which are relatively closed,especially influenced by the living environment, local customs,intermarriage and other factors. It also showed that the differences in the distribution of blood group antigen genes are regional and ethnic specific[19,20].

With the deepening of Hainan free trade port construction, there are more and more people from different nationalities all over the world come to Hainan Province. In the future, medical institutions in Hainan will be faced with the Clinical blood transfusion requirements of patients with a variety of rare blood type systems. The distribution of antigen genes of rare blood group systems was different because of different regions and ethnic groups. Therefore, it is of great importance to establish a blood donor rare blood group bank for the safety of clinical blood transfusion in medical institutions under the environment of free trade port. It would help us find matching blood quickly for the patients with rare blood group systems. In addition,to minimize the incidence of immune transfusion reaction in clinical patients and withe scientific, reasonable and effective blood transfusion were an important direction of precise blood transfusion and the requirements of the development of blood transfusion technology[22].

At present, the database of rare blood donors in our province is mainly based on Rh (D) antigen negative data, and other rare blood group databases need to be further established and improved. The allele distribution of Duffy, Lutheran, Kidd, Diego and Dombrock in Li ethnic group in Hainan Province has unique characteristics.It would be helpful to study the allelic genetic background of rare blood group systems and provid reference data for the establishment of the database of various rare blood group systems in Hainan Free Trade Port, to provid the data of Li ethnic group in Hainan to the national database. Because the sample size of Li ethnic group in Hainan collected in this study was not very large, so the subsequent studies may expand the sample size to further screen for antigen genes of multiple rare blood group systems, and analyze the gene loci and gene mutations of blood groups by large sample. It would help to establish a more complete database of rare blood group systems.

Description of author contribution

Fu Xiao-ling was responsible for formulating the content and writing of the article, statistical analysis of the data and interpretation of the research results; Cai Xing-quan was responsible for the implementation and feasibility analysis of the experiment, and the supervision of the overall management of the article; Xia Lan was responsible for collection of samples, data collection and sorting.

All authors of this article have no conflicts of interest.