Glucose-6-phosphate dehydrogenase de ﬁ ciency screening and gene analysis in blood donors of Guangdong province

Objectives: The characteristic of glucose-6-phosphate dehydrogenase (G6PD) de ﬁ ciency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce hemolysis in G6PD-de ﬁ cient RBCs. We intend to investigate the incidence of G6PD de ﬁ ciency in voluntary blood donors and to evaluate the transfusion risk associated with G6PD de ﬁ ciency in Guangdong province. Methods: G6PD enzyme was analyzed in 3042 donors and gene mutations were genotyped in G6PD-de ﬁ cient samples. Results: The G6PD-de ﬁ cient prevalence of voluntary blood donors was 6.97% (212/3042), 55.19% blood donors with G6PD de ﬁ ciency donated blood more than twice. Eighty- ﬁ ve cases of G6PD de ﬁ ciency were genotyped, and the common types of G6PD mutations were c.1376 G > T, c.1388 G > A, c.95 A > G, c.1024 C > T, and c.871 G > A. Conclusions: Due to the high prevalence of G6PD de ﬁ ciency in Foshan area, we recommended that the screening of G6PD de ﬁ ciency should be carried out for the regular blood donors to ensure the safety of blood users.


Introduction
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder affecting the ability of RBCs to handle oxidant stress, is the most common human enzymopathy, approximately 400 million people are affected worldwide [1]. G6PD deficiency results from deleterious variants in the housekeeping gene of G6PD, causing impaired response to oxidizing agents [1]. G6PD deficiency is very common in southern China. According to recent reports, the overall prevalence of G6PD deficiency in China is 2.10% at the national level, the top six common mutations are c.1388 G > A, c.1376 G > T, c.95 A > G, c.392 G > T, c.871 G > A, and c.1024 C > T, accounting for more than 90% of G6PD-deficient alleles in China [2]. The prevalence varies in different parts of China, and G6PD deficiency prevalence is estimated to be around 2.4% in Sichuan province [3], 4.08% in Dongguan of Guangdong province [4], and 7.28% in Guangxi province [5]. Most adults with G6PD deficiency usually do not have any symptoms. Blood donors with G6PD deficiency are usually unaware of their condition and are likely to join the blood donation program. The present regulations of China do not require G6PD deficiency screening before blood donation, blood bank also do not perform this test, and G6PD-deficient erythrocytes are likely to be transfused into blood recipients.
Generally, G6PD-deficient erythrocytes do not induce blood transfusion reaction in the recipients. However, patients requiring RBC transfusions may simultaneously have concurrent infections or receive oxidative medications, both of which can induce hemolysis in G6PD-deficient RBCs, intravascular hemolysis may affect kidney function and exacerbate other human diseases [6,7].
Guangdong is a high incidence area of G6PD deficiency [8,9], and there is no report about screening data of G6PD deficiency in blood donors for the large scale. In order to determine risk of blood transfusion, we have carried out this investigation for the blood donors in Foshan area of Guangdong province, and the G6PD deficiency was screened and genotypes of G6PD mutation were identified.

Subjects
A total of 3042 cases of blood donor samples were randomly selected from July 2012 to September 2012 in Foshan Central Blood Bank, including 1996 male cases and 1046 female cases. Their cumulative donation volume and donation times in the past 6 years were calculated. The study was approved by the Institutional Review Board of Foshan Central Blood Bank (Approval number: 20120104) and Chaozhou Central Hospital Affiliated to Southern Medical University (Approval number: 2012EA0406). All the study-related data were acquired from the information system of Foshan Central Blood Bank.

G6PD enzyme detection
EDTA-Na 2 anticoagulant blood was used for G6PD enzyme assay, enzyme activity was determined by commercially available kits (Guangzhou Kefang Medical Instrument Co., Ltd.) on Hitachi 7180 automatic biochemical analyzer, the operations followed the manufacturer's instructions. Normal reference range was 1300-3600 U/L for adults, the enzyme activity of G6PD less than 1300U/ L was considered to be deficient, but not all the samples less than 1300U/ L could be identified with G6PD gene mutation [3,10]. This method could detect all hemizygous males, homozygous females, and compound heterozygotes. Unfortunately, G6PD heterozygous females could not be accurately recognized using this quantitative biochemical method, and according to our previous study, only about half of heterozygous females could be recognized [10].

Molecular diagnosis of G6PD deficiency
The G6PD-deficient specimens were further subjected to molecular analysis. Genomic DNA was extracted from peripheral venous blood by whole blood DNA extraction kit (YANENG China Shenzhen Co, Ltd). A reverse dot blot assay (RDB) kit (HYBRIBIO Co, Ltd, China) was used for genotyping the mutations of G6PD-deficient specimens. The assay was capable of detecting 13 common mutations in the Chinese G6PD-deficient population, namely Gaohe (c. . The detailed information of the primers and probes was described previously [11].

DNA sequencing
Our RDB assay could only detect 13 common G6PD mutations in the Chinese, some rare mutations were not included in the detection panel. Therefore, the G6PD deficiency samples that could not be identified by above-mentioned detection kits were further tested by PCR and DNA sequencing. The target fragments ranging from exon 2 to exon 13 of the G6PD gene were designed according to the complete sequence of human G6PD DNA Gene Bank (Accession: NG_009015.2; GI: 575006674). The detailed information of primer pairs was described in our previous report [12]. PCR reaction was performed in an MJ Mini Personal Thermal Cycler (Bio-RAD Company) [12]. The PCR amplification products were sequenced by Invitrogen Biological Technology Company (Shanghai, China). Chromas and BLAST software (http://blast. ncbi.nlm.nih.gov/Blast.cgi) were applied to analyze the sequencing results.

Statistical analysis
Statistical analysis was conducted with SPSS 17.0 statistical software. The difference of G6PD deficiency between males and females were compared using Chi-square test; P < 0.05 was considered statistically significant.

Blood donation data analysis of G6PD-deficient blood donors
The mean donation times for G6PD-deficient donors was 3.23 times for the past 6 years, the mean total donation blood volume was 1032 ml, 55.19% (117/ 212) of the donors donated more than 2 times (Tables 1 and 2). One hundred thirty-seven male blood donors donated 468 times with a total volume of 156900 ml of blood, and 75 female blood donors donated 216 times with a total volume of 61900 ml of blood (Table 1). Eight blood donors donated more than 10 times with 60600 ml of blood (  (Table 3).

Discussion
RBC transfusions can be a critical life-saving measure for hospitalized and chronically transfused patients. In contrast to therapeutical drugs, there is substantial variability in RBC quality and hemoglobin dose among units of blood [1]. Blood donors are a heterogenous population for which genetic, biologic, and behavioral variables interact with various storage and manufacturing conditions, which contribute to variation in the hemoglobin dose of packed RBC units and their responses to oxidative stress [13]. Blood donor G6PD deficiency increases the susceptibility of RBCs to the storage lesion, resulting in decreased posttransfusion recovery of transfused RBCs and G6PD deficiency may increase the concern of hemolysis in the recipients [6,7,13]. Guangdong is a high incidence area of G6PD deficiency [8][9][10]12]. Up to date, blood products are still not required to be checked for G6PD deficiency in China. Generally, most patients with G6PD deficiency usually do not have any symptom, and some will volunteer to donate blood. There is no relative law and regulation for limiting these carriers to donate blood, but the impact of the donated blood on the recipients is not determined clearly yet. Theoretically, hemolysis could occur when the red blood cells with G6PD deficiency encounter oxidative agents or infection. It is clear that antimalarial drugs such as primaquine and quinoline, sulfonamides (sulfonamides isoxazolyl, sulfapyridine, etc.), antipyretics, and analgesics (acetanilide) could cause hemolysis in G6PD-deficient patients [14]. Some commonly used drugs such as acetaminophen, chloramphenicol, and streptomycin have been shown to induce hemolysis [14]. If the recipients received G6PD-deficient blood, and were administered these drugs mentioned above coincidently, the transfused blood could not take effect, and adversely, this act could produce transfusion reaction caused by intravascular hemolysis.
There have been reports about adverse reactions caused by infusion of G6PD-deficient blood [6,7,14]. Mimouni et al reported that two G6PD normal preterm infants were transfused with G6PD-deficient blood, and the hemolytic reaction occurred [6]. Another study showed that blood transfusion with G6PD-deficient erythrocytes in preterm infants of normal G6PD activity could cause hemolytic reaction and delayed total serum bilirubin decline after blood exchange, prolonged phototherapy time, and increased need of repeated blood transfusion [7]. Shanthala et al screened 2005 blood donors for G6PD deficiency in India, and 16 (0.8%) cases were G6PD deficient, 16 bags of G6PD deficiency blood from these 16 cases were investigated retrospectively, and 2 bags of the blood transfusion were found to be related to adverse reactions. One recipient showed itching and rash after receiving 50 ml of blood, possibly due to suspected allergic reaction [15]. The  second case was a neonate with ruptured myelomeningocele, received blood transfusion during operation, and presented with hematuria of suspected hemolytic reaction [15]. The complications associated with receiving G6PD-deficient red cells were: insufficient rise in hemoglobin, hemoglobinuria, and rise in bilirubin. Therefore, routine G6PD screening was strongly proposed in the high prevalence areas of G6PD deficiency in blood donors of India [16,17].
In this regard, there have been some studies in China mainland, which has been reported in the last century, a case of hemolytic reaction was reported after receiving G6PD-deficient blood [18]. The blood donors were screened for G6PD deficiency in Suining city of Sichuan province, the prevalence of G6PD deficiency was 8.3%, and 2.1% were severely deficient. Blood banks in this area established a G6PD-deficient database for these donors to avoid the transfusion of G6PD-deficient blood to G6PDdeficient patients [19]. A recent study showed that prevalence of G6PD deficiency in 1000 blood donors in Guangzhou was 5.1% [20]. This kind of research is very rare, and the study samples are limited, thus there is the need for further study of this issue.
G6PD deficiency prevalence of the blood donation population of Foshan was 6.79%, similar to the prevalence of Guangxi Province (7.28%) [5]. 55.19% of blood donors with G6PD deficiency donated blood more than 2 times in our study. Although G6PD-deficient blood donors were not banned from their blood donation, based on our current knowledge, they should not be encouraged, at least not included in the emergency program of the blood donors. We recommend to perform G6PD deficiency screening for blood donors in areas with a high prevalence of G6PD deficiency. It has not been routine practice to screen healthy blood donors for G6PD deficiency. Therefore, if for all donors G6PD deficiency screening is not practical, a screening for regular and emergency blood donors is recommended to screen out severe G6PD deficiency, and to avoid their further blood donation because of the risk of poor outcomes for the recipient [21,22]. Considering their enthusiasm for blood donation, they should be mobilized to donate platelets. Blood donated by G6PD-deficient donors should not be used for exchange transfusion of neonates, which may induce further hemolysis and jaundice [6,7,23,24]. If the G6PD status of donated blood is not clear, the blood should not be transfused in patients with infection or fever, and the recipients should avoid using oxidative drugs. This would also improve outcomes for chronically transfused recipients, who would most benefit from more effective transfusion products.
In this study, three cases had no identifiable mutation, this might be due to that our PCR-sequencing only covered part of the G6PD sequence, some joint sequence bounding exon and intron were missed out. Next generation sequencing might resolve this question. Another possible explanation was that three cases did not have G6PD gene mutation, they were recognized due to their borderline phenotypes.
Our research had certain shortcomings and limitations. Our study was based on a central blood bank, we could not get clinical data of blood recipients, these data would be valuable to evaluate the risk of receiving G6PD-deficient blood. Further study should be performed to reveal the whole picture of transfusion of G6PD-deficient blood.