ORIGINAL ARTICLE Incidence and molecular basis of CD36 deficiency in Shanghai population Ruishu Li,*1 Zhenglei Qiao,*2 Bing Ling,1 Ping Lu,1 and Ziyan Zhu1
BACKGROUND: CD36 is a multifunctional membrane receptor and is expressed in several cell lines. Individuals who lack platelet (PLT) CD36 are at risk for immunization against this antigen, leading to several clinical syndromes. This study aimed to investigate the frequency and molecular basis of CD36 deficiency in Shanghai. STUDY DESIGN AND METHODS: Whole blood samples were collected from healthy blood donors, and the PLTs and monocytes were analyzed using flow cytometry to determine CD36 deficiency type. After genomic DNA was extracted, Exons 3 to 14 of CD36 gene including a part of relevant flanking introns were amplified. Direct nucleotide sequencing and sequence alignment were performed. The samples that showed mutations were confirmed by clonal sequencing. RESULTS: Of the 1022 healthy blood donors analyzed, 22 individuals failed to express CD36 on PLTs; two of them expressed no CD36 on their monocytes either. These results demonstrated that the frequencies of Type I (lacking CD36 expression on PLTs and monocytes) and Type II (lacking CD36 expression on PLTs only) CD36 deficiency among the study population were 0.2 and 2.0%, respectively. Nucleotide sequencing analysis revealed nine different mutations including six mutations that were not yet reported. The most frequent mutations among the study population were 329330delAC and 1228-1239delATTGTGCCTATT. CONCLUSION: The study findings have confirmed the fact that the frequency of CD36 deficiency in the Chinese population is slightly lower than that in other Asian countries. The identification of several new mutation types indicated the polymorphism of CD36 gene in the Shanghai population.
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D36, also named platelet (PLT) glycoprotein (GP)IV or GPIIIb, is expressed in a number of cells such as monocytes, endothelial cells, erythroblasts, adipocytes, skeletal and heart muscle cells, and mammary epithelial cells.1-4 The 88-kDa membrane GP is a Class B scavenger receptor capable of interacting with Types I and II collagen, thrombospondin, oxidatively modified low-density lipoprotein, Plasmodium falciparum–infected red blood cells, and long-chain fatty acids.5-9 Healthy individuals who lack CD36 are at risk of producing antibodies against this antigen, when they receive transfusions or pregnancy. The isoantibodies may cause PLT transfusion refractoriness, posttransfusion purpura, and early fetal loss.10-12 The frequency of CD36-negative phenotype varies among ethnic groups. It is extremely rare (0.3%) in whites and relatively high in Asians (3%10%) and Africans (7%).13,14 CD36 deficiency can be classified into two types: Type I is characterized by lacking CD36 expression in PLTs and monocytes, and Type II lacks ABBREVIATIONS: AA(s) = amino acid(s); GP = glycoprotein; pCD36 = platelet CD36; PEB = 0.02 mol/L phosphate-buffered saline containing 9 mmol/L ethylenediaminetetraacetic acid and 0.1% bovine serum albumin. From the 1Shanghai Institute of Blood Transfusion, Shanghai Blood Center; and the 2School of Life Science, East China Normal University, Shanghai, China. Address reprint requests to: Ping Lu, Shanghai Institute of Blood Transfusion, Shanghai Blood Center, Hongqiao Road 1191, 200051 Shanghai, China; e-mail: [email protected]. *These authors contributed equally to this work. This work was supported by grants from the Medical Research Fund of Shanghai Municipal Health and Family Planning Commission (No. 20124240) and Science and Technology Commission of Shanghai Municipality (No. 14430722800). [Correction statement added after online publication 24-Oct-2014: the funders were updated.] Received for publication June 2, 2014; revision received August 17, 2014, and accepted August 24, 2014. doi: 10.1111/trf.12890 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
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surface expression on PLTs only with normal phenotype in monocytes and macrophages.15 In this research, PLT and monocyte CD36 deficiency in healthy blood donors in Shanghai was studied to determine the frequency of this phenotype and molecular mechanisms of CD36 gene mutations.
MATERIALS AND METHODS
determine the monocyte CD36 phenotype. As negative controls, FITC-mouse IgG1 κ isotype and PE-mouse IgG1 κ isotype control (Clone P3.6.2.8.1, eBioscience) were used. Cells were analyzed on a flow cytometry. Monocytes were identified by the forward-scatter properties and were subsequently included in a gate. CD36 expression was analyzed on monocytes from pCD36-negative blood donors by comparing the negative and positive controls.
Blood donors Whole blood samples were collected from healthy donors (n = 1022) during routine blood donation in Shanghai Blood Center (Shanghai, China).
PLT preparation Whole blood samples were centrifuged at 400 × g for 10 minutes. PLT-rich plasma was obtained and transferred into a fresh tube and centrifuged at 200 × g for 10 minutes. The supernatant was centrifuged at 1200 × g for 10 minutes, followed by the addition of 0.02 mol/L phosphate-buffered saline containing 9 mmol/L ethylenediaminetetraacetic acid (EDTA) and 0.1% bovine serum albumin (PEB). The isolated PLTs were washed twice in PEB, resuspended in the same buffer, and stored at 4°C.
Peripheral blood mononuclear cell preparation Peripheral blood mononuclear cells (PBMNCs) were purified using standard Ficoll-Paque gradient centrifugation according to the instructions of the manufacturer (GE Healthcare, Shanghai, China). Briefly, 3.5 to 4 mL of FicollPaque gradient was pipetted into two 15-mL centrifuge tubes. The anticoagulated blood was diluted 1:1 in PEB and carefully layered over the Ficoll-Paque gradient. The tubes were centrifuged for 20 minutes at 1020 × g. The cell interface layer was harvested carefully, and the cells were washed twice in PEB (for 10 min at 640 × g followed by 10 min at 470 × g) and resuspended in PEB at a concentration of 106 cells/mL.
Antibody detecting Antibodies in serum samples of pCD36-negative blood donors were detected by monoclonal antibody solid PLT antibody test using a commercial kit (MASPAT, Sanquin, Amsterdam, the Netherlands) according to kit instruction.
Nucleotide sequencing analysis Genomic DNA was extracted from EDTA-anticoagulated blood using a blood DNA kit (TIANamp, Tiangen, Beijing, China). Exon 3 to Exon 14 of CD36 gene including a part of relevant flanking introns were amplified with specific primers (see Table S1, available as supporting information in the online version of this paper; reference sequence NG_008192). The polymerase chain reaction (PCR) system comprised 2.5 μL of genomic DNA, 5 μL of 10× PCR buffer, 3 μL of magnesium chloride (25 mmol/L), 2 μL of deoxynucleotide triphosphates (2.5 mmol/L), 1 μL of each primer (10 μmol/L), 0.5 μL of Taq DNA polymerase, and 35 μL of double-distilled water. The mixtures were incubated at 94°C for 5 minutes for initial denaturation of the target DNA and then subjected to 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds, and a final extension time of 7 minutes at 72°C was given. The PCR products were sequenced by Shanghai Sangon Biotechnology Company using the Sanger method, and a sequence alignment was performed.
Clonal sequencing Flow cytometry The PLTs isolated from all the whole blood samples were labeled at 4°C with the fluorescein isothiocyanate (FITC)conjugated anti-human CD36 monoclonal antibody (MoAb; Clone NL07, eBioscience, San Diego, CA) to determine the PLT CD36 (pCD36) phenotype. FITC-mouse immunoglobulin (Ig)G κ isotype control (Clone P3.6.2.8.1, eBioscience) was used as a negative control. The PLTs were analyzed by flow cytometry (FACSCalibur, BD, San Jose, CA). PBMNCs from pCD36-negative blood donors were simultaneously labeled with FITC-conjugated antihuman CD36 MoAb and phycoerythrin (PE)-conjugated anti-human CD14 MoAb (Clone 61D3, eBioscience) to 2
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The PCR products which contained mutations were purified using an agarose gel DNA extraction kit (TaKaRa Mini BEST, Version 3.0, Takara, Dalian, China) and ligated into the pGM-T vector using the pGM-T ligation kit (Tiangen). The plasmids were added into a tube containing competent TOP10 cells (Tiangen). After incubation on ice for 30 minutes, the tubes were placed in a 42°C water bath for 90 seconds without shaking, and they were then replaced on ice for 2 to 3 minutes. After addition of preheated LuriaBertani medium without antibiotics, the cells were incubated at 37°C with shaking at 150 rpm for 45 minutes, after which they were plated onto a Luria-Bertani agar plate containing ampicillin, isopropyl β-d-1thiogalactopyranoside, and X-gal. The plates were
STUDY ON CD36 DEFICIENCY IN SHANGHAI
incubated at 37°C overnight. White bacterial clones containing the target gene fragments were selected through blue-white screening. The plasmids were extracted using a plasmid purification kit (Version 3.0, Takara) and sequenced by Shanghai Sangon Biotechnology Company using the Sanger method.
RESULTS Frequency of CD36 deficiency in Shanghai and antibody detecting The expression of CD36 on PLTs collected from June 1, 2010, to July 26, 2011, were screened by flow cytometry. Of the 1022 individuals analyzed, 22 were negative for CD36 expression on PLTs, indicating that the incidence of CD36 deficiency was approximately 2.2% (Fig. 1C). PBMNCs from these pCD36-negative blood donors were then analyzed to determine their deficiency type. Two individuals failed to express CD36 on their monocytes as well
(Fig. 1F). This result demonstrated that the frequencies of Type I and Type II CD36 deficiency in the study population were 0.2 and 2.0%, respectively. In the serum samples of the 22 pCD36-negative blood donors, no antibody was detected by MASPAT (data not shown).
Molecular analysis of CD36 deficiency To reveal the molecular mechanism of CD36 deficiency, the coding region of CD36 gene was amplified by PCR and analyzed by direct sequencing. The samples that showed mutations were confirmed by clonal sequencing. There was no genetic mutation in nine individuals lacking CD36 expression on PLTs. Flow cytometry results indicated that they all belonged to Type II CD36 deficiency. In individuals with Type I CD36 deficiency, a two-nucleotide deletion (329-330delAC) was found in one donor (Donor 1), which was located in Exon 5 and caused frameshift at Amino Acid Residue 110 as previously reported.16 Moreover, a
Fig. 1. Flow cytometry analysis of CD36 expression on PLTs and monocytes of healthy blood donors. (A-C) Representative of PLTs tests. (A) Isotype control; (B and C) anti-CD36 with CD36-positive and CD36-negative PLTs, respectively. (D-F) Representative of monocytes tests. (D) Isotype control; (E and F) anti-CD36 and anti-CD14 with CD36-positive and CD36-negative monocytes. Volume **, ** **
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new mutation 371C>T in Exon 14 was identified in another individual (Donor 14), which led to missense amino acid substitution Pro124Leu. Among 11 Type II CD36-deficient individuals with mutations, a deletion of 12 nucleotides (1228-1239delATTGTGCCTATT in heterozygous form) was found to be located in Exon 13 in four donors (Donors 6, 8, 15, and 18). The deletion led to deletion of four amino acids (Ile-Val-Pro-Ile; Amino Acids (AAs) 410-413), which could impair the transportation of CD36 precursors from the endoplasmic reticulum to the Golgi apparatus.17 The two-nucleotide deletion (329330delAC) was also found in three individuals (Donors 4, 7, and 9) in the Type II CD36 deficiency group. Other mutation types associated with Type II CD36 deficiency identified were 14G>A (Arg5Gln), 1008G>T (Gly336Gly), 1156C>T (Arg386Trp), 1163A>T (Gln388Leu), 1226A>G (Tyr409Cys), and 1344insTCTT (frameshift at AA 448). More than 35 mutations in the coding sequence of CD36 gene that lead to CD36 deficiency have been described so far (Fig. 2). In this study, five new mutations were identified that were never previously observed (Fig. 3). These five mutations were located respectively in Exons 3, 12, 13, and 14, and they cause frameshift or substitutions in amino acids, which eventually could lead to changes in protein structure. These findings add value to the already existing mutations and provide a clearer understanding of the possible molecular mechanisms of CD36 deficiency.
DISCUSSION CD36 is a transmembrane GP of Class B scavenger receptor with multiple functions. Individuals with a deficiency of CD36 antigen on their PLTs and monocytes are at risk of immune reaction induced by transfusion or pregnancy, which leads to a series of alloimmune conditions. This study aimed to investigate the frequency of different types of CD36 deficiency among healthy blood donors in Shanghai. Relevant information on similar lines has recently been reported by Xu and colleagues.18 The study results showed a similar frequency of total CD36 deficiency to that reported by Xu and colleagues, which was lower than that in the Japanese population (2.2% vs. 4.6%).15 Moreover, the most frequent mutations found among the study population were 329-330delAC and 1228-1239delATTGTGCCTATT (Table 1), and these results were also consistent with that reported by Xu and colleagues. Both mutations had the frequency of 18.2% in the CD36 deficiency group. The dinucleotide deletion 329330delAC in Exon 5 causes a frameshift leading to the appearance of a translation stop codon at Position 396, which might affect the splicing of Exon 4.16 The mutation 1228-1239delATTGTGCCTATT located in the middle of Exon 13 results in a deletion of amino acids, which causes maturation and transport defects of CD36 precursor form.17 It was reported that 268C>T (Pro90Ser) represented the most frequent mutation among Asians, and a
TABLE 1. CD36 mutations found in 22 blood donors Age/sex 24/female 28/male 34/female 23/female 25/male 31/male
CD36 PLT/monocytes −/− −/+ −/+ −/+ −/+ −/+
Type I II II II II II
7 8
45/male 49/female
−/+ −/+
9 10 11 12 13 14 15
58/male 33/female 44/male 23/female 26/male 30/female 26/female
16 17 18 19 20 21 22
Subject 1 2 3 4 5 6
Mutations* 329-330del AC
Change in amino acid Frameshift at AA 110
Reference(s) Kashiwagi et al.16
329-330del AC
Frameshift at AA 110
Kashiwagi et al.16
1228-1239delATTGTGCCTATT
Deletion of Ile-Val-Pro-Ile
II II
329-330del AC 1228-1239delATTGTGCCTATT
Frameshift at AA 110 Deletion of Ile-Val-Pro-Ile
−/+ −/+ −/+ −/+ −/+ −/− −/+
II II II II II I II
329-330del AC 1156C>T 1344insTCTT
Frameshift at AA 110 Arg386Trp Frameshift at AA 448
Kashiwagi et al.,17 Tanaka et al.19 Kashiwagi et al.16 Kashiwagi et al.,17 Tanaka et al.19 Kashiwagi et al.16 Xu et al.18 This study
371C>T; 1008G>T 1228-1239delATTGTGCCTATT
Pro124Leu; Gly336Gly Deletion of Ile-Val-Pro-Ile
23/male 27/female 70/female
−/+ −/+ −/+
II II II
14G>A; 1226A>G
Arg5Gln; Tyr409Cys
1228-1239delATTGTGCCTATT
Deletion of Ile-Val-Pro-Ile
Kashiwagi et al.,17 Tanaka et al.19
31/female 31/male 37/male 24/male
−/+ −/+ −/+ −/+
II II II II
1163A>T
Gln388Leu
This study
* GenBank NM 001001548; the first mRNA nucleotide encoding CD36 protein is +.
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This study Kashiwagi et al.,17 Tanaka et al.19 This study
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TABLE 2. Mutations in the exons of human CD36 gene Exon number
mRNA nucleotides*
1 2 3
−356 to −184 −183 to −90 −89 to 120
4
121 to 281
5
282 to 429
6 7
430 to 609 610 to 701
8 9 10
702 to 748 749 to 818 819 to 1006
11
1007 to 1125
12
1126 to 1199
13
1200 to 1254
14
1255 to 1419
15
1420 to 2044
Change in nucleotide sequence
Change in amino acid sequence
del Exon1 del Exon2 14G>A del Exon3 268C>T 121-126delgCAAGTT 220C>T 319-324delGCTGAG 329-330delAC 367G>A 371C>T 411T>C 380C>T 560insT 619-624del ACTGCA/ins AAAAC 691-696delAAAGGT None 760T>C 845-849delACGTT 949insA 975T>G 1079T>G 1226A>G Del TTTAGAT 1140-1146delTTTACAA/insCCAAA 1150G>C 1155delA 1163C>T 1156C>T Del TATTACAGAG Dupl. 1204-1246 1218-1224del GAGGAAC 1228-1239del ATTGTGCCTATT
No expression of CD36 protein No expression of CD36 protein Arg5Gln No expression of CD36 protein Pro90Ser Unknown Gln74Term In-frame delAA 107-108 Frameshift at 110 Glu123Lys Pro124Leu Ala137Val Ser127Leu Frameshift at AA187 Frameshift at AA 207 In-frame del AA 231-232 Phe254Leu Frameshift at AA282 Frameshift at AA 317 Tyr325Term Leu360Term Tyr409Cys Skipping Exon 12 Frameshift at AA 380 Ala383Pro Frameshift atAA385 Gln388Leu Arg386Trp Skipping exon 13 Frameshift at AA 416 Frameshift at AA 406 Del IIe-Val-Pro-IIe
1237A>C 1344insTCTT
IIe413leu Frameshift at AA 448
Reference(s) Sato et al.21 Yanai et al.22 This study Sato et al.21 Yanai et al.22 Xu et al.18 Kashiwagi et al.16 Kashiwagi et al.16 Kashiwagi et al.23 This study Imai et al.24 Omi et al.25 Tanaka et al.19 Kashiwagi et al.17 Aitman et al.26 Hanawa et al.27 Aitman et al.26 Kashiwagi et al.28 Aitman et al.26 Lepretre et al.29 This study Tanaka et al.19 Aitman et al.26 This study Xu et al.18 Tanaka et al.19 Tamala et al.19 Kashiwagi et al.17 Kashiwagi et al.,17 Tanaka et al.19 Imai et al.24 This study
* GenBank NM 001001548; the first mRNA nucleotide encoding CD36 protein is +1.
high occurrence was shown in Japanese and Korean populations.17,20 However, neither this study nor the study by Xu and colleagues discovered this mutation in the Chinese population. In this study, some new CD36 mutations were identified by direct nucleotide sequencing and clonal sequencing (see above). 371C>T located in Exon 5 was associated with Type I deficiency, and the rest of the mutations were related to Type II deficiency. Most of these mutations were located in exons of CD36 gene and led to corresponding amino acid substitutions except for 1008G>T, which was a synonymous mutation. In addition, 1344insTCTT located in Exon 14 led to a frameshift at AA 448 and caused loss of stop codon. Up to now, more than 30 kinds of mutations have been discovered in the CD36 gene (Table 2). However, the study on gene level is not sufficient to unravel the molecular behavior on protein level. Further research is necessary to explore the mechanism of CD36 deficiency in these mutations. The spectrum of CD36 mutations differs in various populations. The study results show that CD36 deficiency
exists in Shanghai population, and they are at risk for antiCD36 immune reaction induced by transfusion. In light of these observations, it is necessary to detect anti-CD36 in suspected immune-mediated thrombocytopenia. Establishment of a donor registry of CD36-deficient individuals would help patients with these disorders. Our research on frequency and type of CD36 deficiency in Shanghai population will not only provide assistance for clinical diagnostic of alloimmune conditions, but also give proper guidance to the safety and rationality of clinical blood transfusion. Moreover, the study on molecular mechanism of CD36 deficiency may provide reference for further researches on physiologic functions of CD36 as well as CD36-associated diseases.
ETHICS STATEMENT The protocol was approved by the Medical Ethics Review Committee of Shanghai Blood Center (Permit Number SBC-IRB-2014-02). Volume **, ** **
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Fig. 2. cDNA nucleotide and amino acid sequences of CD36 with mutations indicated as follows: underline = substitutions; vertical bar = insertions. 6
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Fig. 3. Sequencing chromatogram of the new mutations. (A) 14G>A in Exon 3 (Donor 16); (B) 1344insTCTT in Exon 4 (Donor 11); (C) 371C>T in Exon 5 (Donor 14); (D) 1008G>T in Exon 10 (Donor 14); (E) 1163A>T in Exon 12 (Donor 20); (F) 1226A>G in Exon 12 (Donor 16). CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
REFERENCES 1. Silverstein RL, Asch AS, Nachman RL. Glycoprotein IV mediates thrombospondin-dependent platelet-monocyte and platelet-U937 cell adhesion. J Clin Invest 1989;84: 546-52. 2. Edelman P, Vinci G, Villeval JL, et al. A monoclonal antibody against an erythrocyte onto genic antigen identifies fetal and adult erythroid progenitors. Blood 1986;67:56-63. 3. Knowles DM II, Tolidjian B, Marboe C, et al. Monoclonal anti-human monocyte antibodies OKM1 and OKM5 possess distinctive tissue distributions including differential reactivity with vascular endothelium. J Immunol 1984; 132:2170-3. 4. Greenwalt DE, Watt KW, So OY, et al. PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV). Biochemistry 1990;29:7054-9. 5. Endemann G, Stanton LW, Madden KS, et al. CD36 is a receptor for oxidized low-density lipoprotein. J Biol Chem 1993;268:11811-6.
6. Brouwers A, Langlois M, Delanghe J, et al. Oxidized lowdensity lipoprotein, iron stores, and haptoglobin polymorphism. Atherosclerosis 2004;176:189-95. 7. Asch AS, Silbiger S, Heimer E, et al. Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding. Biochem Biophys Res Commun 1992;182:1208-17. 8. Barnwell JW, Ockenhouse CF, Knowles DM II. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells. J Immunol 1985;135: 3494-7. 9. Abumrad NA, el-Maghrabi MR, Amri EZ, et al. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. Homology with human CD36. J Biol Chem 1993;268:17665-8. 10. Ikeda H, Mitani T, Ohnuma M, et al. A new plateletspecific antigen, Naka, involved in the refractoriness of HLA-matched platelet transfusion. Vox Sang 1989;57:213-7. 11. Bierling P, Godeau B, Fromont P, et al. Posttransfusion purpura-like syndrome associated with CD36 (Nak) isoimmunization. Transfusion 1995;35:777-82. 12. Borzini P, Riva M, Nembri P, et al. CD36 autoantibodies and thrombotic diathesis, thrombocytopenia and repeated early fetal losses. Vox Sang 1997;73:46-8. Volume **, ** **
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13. Curtis BR, Ali S, Glazier AM, et al. Isoimmunization against CD36 (glycoprotein IV): description of four cases of neonatal isoimmune thrombocytopenia and brief review of the literature. Transfusion 2002;42:1173-9. 14. Lee K, Godeau B, Fromont P, et al. CD36 deficiency is frequent and can cause platelet immunization in Africans. Transfusion 1999;39:873-9. 15. Yamamoto N, Akamatsu N, Sakuraba H, et al. Platelet glycoprotein IV (CD36) deficiency is associated with the absence (type I) or the presence (type II) of glycoprotein IV on monocytes. Blood 1994;83:392-7. 16. Kashiwagi H, Tomiyama Y, Kosugi S, et al. Identification of molecular defects in a subject with type I CD36 deficiency. Blood 1994;83:3545-52. 17. Kashiwagi H, Tomiyama Y, Nozaki S, et al. Analyses of
gamma ligands. J Biol Chem 2002;277:15703-11. 22. Yanai H, Chiba H, Morimoto M, et al. Human CD36 deficiency is associated with elevation in low-density lipoprotein-cholesterol. Am J Med Genet 2000;93:299-304. 23. Kashiwagi H, Honda S, Tomiyama Y, et al. A novel polymorphism in glycoprotein IV (replacement of proline-90 by serine) predominates in subjects with platelet GPIV deficiency. Thromb Haemost 1993;69:481-4. 24. Imai M, Tanaka T, Kintaka T, et al. Genomic heterogeneity of type II CD36 deficiency. Clin Chim Acta 2002;321:97106. 25. Omi K, Ohashi J, Patarapotikul J, et al. CD36 polymorphism is associated with protection from cerebral malaria. Am J Hum Genet 2003;72:364-74.
genetic abnormalities in type I CD36 deficiency in Japan:
26. Aitman TJ, Cooper LD, Norsworthy PJ, et al. Malaria sus-
identification and cell biological characterization of two novel mutations that cause CD36 deficiency in man. Hum
ceptibility and CD36 mutation. Nature 2000;405:1015-6. 27. Hanawa H, Watanabe K, Nakamura T, et al. Identification
Genet 2001;108:459-66. 18. Xu X, Ye X, Xia W, et al. Studies on CD36 deficiency in South China: two cases demonstrating the clinical impact of anti-CD36 antibodies. Thromb Haemost 2013;110:1199206. 19. Tanaka T, Nakata T, Oka T, et al. Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations. J Lipid Res 2001;42:751-9. 20. Kashiwagi H, Tomiyama Y, Honda S, et al. Molecular basis of CD36 deficiency. Evidence that a 478C -->T substitution (proline90 -->serine) in CD36 cDNA accounts for CD36 deficiency. J Clin Invest 1995;95:1040-6. 21. Sato O, Kuriki C, Fukui Y, et al. Dual promoter structure of mouse and human fatty acid translocase/CD36 genes and unique transcriptional activation by
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peroxisome proliferator-activated receptor alpha and
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of cryptic splice site, exon skipping, and novel point mutations in type I CD36 deficiency. J Med Genet 2002;39:28691. 28. Kashiwagi H, Tomiyama Y, Nozaki S, et al. A single nucleotide insertion in codon 317 of the CD36 gene leads to CD36 deficiency. Arterioscler Thromb Vasc Biol 1996;16:1026-32. 29. Lepretre F, Vasseur F, Vaxillaire M, et al. ACD36 nonsense mutation associated with insulin resistance and familial type 2 diabetes. Hum Mutat 2004;24:104-10.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s Web site: Table S1. PCR and sequencing primers for CD36 gene
BACKGROUND: CD36 is a multifunctional membrane receptor and is expressed in several cell lines. Individuals who lack platelet (PLT) CD36 are at risk for immunization against this antigen, leading to several clinical syndromes. This study aimed to investigate the frequency and molecular basis of CD36 deficiency in Shanghai. STUDY DESIGN AND METHODS: Whole blood samples were collected from healthy blood donors, and the PLTs and monocytes were analyzed using flow cytometry to determine CD36 deficiency type. After genomic DNA was extracted, Exons 3 to 14 of CD36 gene including a part of relevant flanking introns were amplified. Direct nucleotide sequencing and sequence alignment were performed. The samples that showed mutations were confirmed by clonal sequencing. RESULTS: Of the 1022 healthy blood donors analyzed, 22 individuals failed to express CD36 on PLTs; two of them expressed no CD36 on their monocytes either. These results demonstrated that the frequencies of Type I (lacking CD36 expression on PLTs and monocytes) and Type II (lacking CD36 expression on PLTs only) CD36 deficiency among the study population were 0.2 and 2.0%, respectively. Nucleotide sequencing analysis revealed nine different mutations including six mutations that were not yet reported. The most frequent mutations among the study population were 329330delAC and 1228-1239delATTGTGCCTATT. CONCLUSION: The study findings have confirmed the fact that the frequency of CD36 deficiency in the Chinese population is slightly lower than that in other Asian countries. The identification of several new mutation types indicated the polymorphism of CD36 gene in the Shanghai population.
C
D36, also named platelet (PLT) glycoprotein (GP)IV or GPIIIb, is expressed in a number of cells such as monocytes, endothelial cells, erythroblasts, adipocytes, skeletal and heart muscle cells, and mammary epithelial cells.1-4 The 88-kDa membrane GP is a Class B scavenger receptor capable of interacting with Types I and II collagen, thrombospondin, oxidatively modified low-density lipoprotein, Plasmodium falciparum–infected red blood cells, and long-chain fatty acids.5-9 Healthy individuals who lack CD36 are at risk of producing antibodies against this antigen, when they receive transfusions or pregnancy. The isoantibodies may cause PLT transfusion refractoriness, posttransfusion purpura, and early fetal loss.10-12 The frequency of CD36-negative phenotype varies among ethnic groups. It is extremely rare (0.3%) in whites and relatively high in Asians (3%10%) and Africans (7%).13,14 CD36 deficiency can be classified into two types: Type I is characterized by lacking CD36 expression in PLTs and monocytes, and Type II lacks ABBREVIATIONS: AA(s) = amino acid(s); GP = glycoprotein; pCD36 = platelet CD36; PEB = 0.02 mol/L phosphate-buffered saline containing 9 mmol/L ethylenediaminetetraacetic acid and 0.1% bovine serum albumin. From the 1Shanghai Institute of Blood Transfusion, Shanghai Blood Center; and the 2School of Life Science, East China Normal University, Shanghai, China. Address reprint requests to: Ping Lu, Shanghai Institute of Blood Transfusion, Shanghai Blood Center, Hongqiao Road 1191, 200051 Shanghai, China; e-mail: [email protected]. *These authors contributed equally to this work. This work was supported by grants from the Medical Research Fund of Shanghai Municipal Health and Family Planning Commission (No. 20124240) and Science and Technology Commission of Shanghai Municipality (No. 14430722800). [Correction statement added after online publication 24-Oct-2014: the funders were updated.] Received for publication June 2, 2014; revision received August 17, 2014, and accepted August 24, 2014. doi: 10.1111/trf.12890 © 2014 AABB TRANSFUSION **;**:**-**. Volume **, ** **
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surface expression on PLTs only with normal phenotype in monocytes and macrophages.15 In this research, PLT and monocyte CD36 deficiency in healthy blood donors in Shanghai was studied to determine the frequency of this phenotype and molecular mechanisms of CD36 gene mutations.
MATERIALS AND METHODS
determine the monocyte CD36 phenotype. As negative controls, FITC-mouse IgG1 κ isotype and PE-mouse IgG1 κ isotype control (Clone P3.6.2.8.1, eBioscience) were used. Cells were analyzed on a flow cytometry. Monocytes were identified by the forward-scatter properties and were subsequently included in a gate. CD36 expression was analyzed on monocytes from pCD36-negative blood donors by comparing the negative and positive controls.
Blood donors Whole blood samples were collected from healthy donors (n = 1022) during routine blood donation in Shanghai Blood Center (Shanghai, China).
PLT preparation Whole blood samples were centrifuged at 400 × g for 10 minutes. PLT-rich plasma was obtained and transferred into a fresh tube and centrifuged at 200 × g for 10 minutes. The supernatant was centrifuged at 1200 × g for 10 minutes, followed by the addition of 0.02 mol/L phosphate-buffered saline containing 9 mmol/L ethylenediaminetetraacetic acid (EDTA) and 0.1% bovine serum albumin (PEB). The isolated PLTs were washed twice in PEB, resuspended in the same buffer, and stored at 4°C.
Peripheral blood mononuclear cell preparation Peripheral blood mononuclear cells (PBMNCs) were purified using standard Ficoll-Paque gradient centrifugation according to the instructions of the manufacturer (GE Healthcare, Shanghai, China). Briefly, 3.5 to 4 mL of FicollPaque gradient was pipetted into two 15-mL centrifuge tubes. The anticoagulated blood was diluted 1:1 in PEB and carefully layered over the Ficoll-Paque gradient. The tubes were centrifuged for 20 minutes at 1020 × g. The cell interface layer was harvested carefully, and the cells were washed twice in PEB (for 10 min at 640 × g followed by 10 min at 470 × g) and resuspended in PEB at a concentration of 106 cells/mL.
Antibody detecting Antibodies in serum samples of pCD36-negative blood donors were detected by monoclonal antibody solid PLT antibody test using a commercial kit (MASPAT, Sanquin, Amsterdam, the Netherlands) according to kit instruction.
Nucleotide sequencing analysis Genomic DNA was extracted from EDTA-anticoagulated blood using a blood DNA kit (TIANamp, Tiangen, Beijing, China). Exon 3 to Exon 14 of CD36 gene including a part of relevant flanking introns were amplified with specific primers (see Table S1, available as supporting information in the online version of this paper; reference sequence NG_008192). The polymerase chain reaction (PCR) system comprised 2.5 μL of genomic DNA, 5 μL of 10× PCR buffer, 3 μL of magnesium chloride (25 mmol/L), 2 μL of deoxynucleotide triphosphates (2.5 mmol/L), 1 μL of each primer (10 μmol/L), 0.5 μL of Taq DNA polymerase, and 35 μL of double-distilled water. The mixtures were incubated at 94°C for 5 minutes for initial denaturation of the target DNA and then subjected to 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds, and a final extension time of 7 minutes at 72°C was given. The PCR products were sequenced by Shanghai Sangon Biotechnology Company using the Sanger method, and a sequence alignment was performed.
Clonal sequencing Flow cytometry The PLTs isolated from all the whole blood samples were labeled at 4°C with the fluorescein isothiocyanate (FITC)conjugated anti-human CD36 monoclonal antibody (MoAb; Clone NL07, eBioscience, San Diego, CA) to determine the PLT CD36 (pCD36) phenotype. FITC-mouse immunoglobulin (Ig)G κ isotype control (Clone P3.6.2.8.1, eBioscience) was used as a negative control. The PLTs were analyzed by flow cytometry (FACSCalibur, BD, San Jose, CA). PBMNCs from pCD36-negative blood donors were simultaneously labeled with FITC-conjugated antihuman CD36 MoAb and phycoerythrin (PE)-conjugated anti-human CD14 MoAb (Clone 61D3, eBioscience) to 2
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The PCR products which contained mutations were purified using an agarose gel DNA extraction kit (TaKaRa Mini BEST, Version 3.0, Takara, Dalian, China) and ligated into the pGM-T vector using the pGM-T ligation kit (Tiangen). The plasmids were added into a tube containing competent TOP10 cells (Tiangen). After incubation on ice for 30 minutes, the tubes were placed in a 42°C water bath for 90 seconds without shaking, and they were then replaced on ice for 2 to 3 minutes. After addition of preheated LuriaBertani medium without antibiotics, the cells were incubated at 37°C with shaking at 150 rpm for 45 minutes, after which they were plated onto a Luria-Bertani agar plate containing ampicillin, isopropyl β-d-1thiogalactopyranoside, and X-gal. The plates were
STUDY ON CD36 DEFICIENCY IN SHANGHAI
incubated at 37°C overnight. White bacterial clones containing the target gene fragments were selected through blue-white screening. The plasmids were extracted using a plasmid purification kit (Version 3.0, Takara) and sequenced by Shanghai Sangon Biotechnology Company using the Sanger method.
RESULTS Frequency of CD36 deficiency in Shanghai and antibody detecting The expression of CD36 on PLTs collected from June 1, 2010, to July 26, 2011, were screened by flow cytometry. Of the 1022 individuals analyzed, 22 were negative for CD36 expression on PLTs, indicating that the incidence of CD36 deficiency was approximately 2.2% (Fig. 1C). PBMNCs from these pCD36-negative blood donors were then analyzed to determine their deficiency type. Two individuals failed to express CD36 on their monocytes as well
(Fig. 1F). This result demonstrated that the frequencies of Type I and Type II CD36 deficiency in the study population were 0.2 and 2.0%, respectively. In the serum samples of the 22 pCD36-negative blood donors, no antibody was detected by MASPAT (data not shown).
Molecular analysis of CD36 deficiency To reveal the molecular mechanism of CD36 deficiency, the coding region of CD36 gene was amplified by PCR and analyzed by direct sequencing. The samples that showed mutations were confirmed by clonal sequencing. There was no genetic mutation in nine individuals lacking CD36 expression on PLTs. Flow cytometry results indicated that they all belonged to Type II CD36 deficiency. In individuals with Type I CD36 deficiency, a two-nucleotide deletion (329-330delAC) was found in one donor (Donor 1), which was located in Exon 5 and caused frameshift at Amino Acid Residue 110 as previously reported.16 Moreover, a
Fig. 1. Flow cytometry analysis of CD36 expression on PLTs and monocytes of healthy blood donors. (A-C) Representative of PLTs tests. (A) Isotype control; (B and C) anti-CD36 with CD36-positive and CD36-negative PLTs, respectively. (D-F) Representative of monocytes tests. (D) Isotype control; (E and F) anti-CD36 and anti-CD14 with CD36-positive and CD36-negative monocytes. Volume **, ** **
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new mutation 371C>T in Exon 14 was identified in another individual (Donor 14), which led to missense amino acid substitution Pro124Leu. Among 11 Type II CD36-deficient individuals with mutations, a deletion of 12 nucleotides (1228-1239delATTGTGCCTATT in heterozygous form) was found to be located in Exon 13 in four donors (Donors 6, 8, 15, and 18). The deletion led to deletion of four amino acids (Ile-Val-Pro-Ile; Amino Acids (AAs) 410-413), which could impair the transportation of CD36 precursors from the endoplasmic reticulum to the Golgi apparatus.17 The two-nucleotide deletion (329330delAC) was also found in three individuals (Donors 4, 7, and 9) in the Type II CD36 deficiency group. Other mutation types associated with Type II CD36 deficiency identified were 14G>A (Arg5Gln), 1008G>T (Gly336Gly), 1156C>T (Arg386Trp), 1163A>T (Gln388Leu), 1226A>G (Tyr409Cys), and 1344insTCTT (frameshift at AA 448). More than 35 mutations in the coding sequence of CD36 gene that lead to CD36 deficiency have been described so far (Fig. 2). In this study, five new mutations were identified that were never previously observed (Fig. 3). These five mutations were located respectively in Exons 3, 12, 13, and 14, and they cause frameshift or substitutions in amino acids, which eventually could lead to changes in protein structure. These findings add value to the already existing mutations and provide a clearer understanding of the possible molecular mechanisms of CD36 deficiency.
DISCUSSION CD36 is a transmembrane GP of Class B scavenger receptor with multiple functions. Individuals with a deficiency of CD36 antigen on their PLTs and monocytes are at risk of immune reaction induced by transfusion or pregnancy, which leads to a series of alloimmune conditions. This study aimed to investigate the frequency of different types of CD36 deficiency among healthy blood donors in Shanghai. Relevant information on similar lines has recently been reported by Xu and colleagues.18 The study results showed a similar frequency of total CD36 deficiency to that reported by Xu and colleagues, which was lower than that in the Japanese population (2.2% vs. 4.6%).15 Moreover, the most frequent mutations found among the study population were 329-330delAC and 1228-1239delATTGTGCCTATT (Table 1), and these results were also consistent with that reported by Xu and colleagues. Both mutations had the frequency of 18.2% in the CD36 deficiency group. The dinucleotide deletion 329330delAC in Exon 5 causes a frameshift leading to the appearance of a translation stop codon at Position 396, which might affect the splicing of Exon 4.16 The mutation 1228-1239delATTGTGCCTATT located in the middle of Exon 13 results in a deletion of amino acids, which causes maturation and transport defects of CD36 precursor form.17 It was reported that 268C>T (Pro90Ser) represented the most frequent mutation among Asians, and a
TABLE 1. CD36 mutations found in 22 blood donors Age/sex 24/female 28/male 34/female 23/female 25/male 31/male
CD36 PLT/monocytes −/− −/+ −/+ −/+ −/+ −/+
Type I II II II II II
7 8
45/male 49/female
−/+ −/+
9 10 11 12 13 14 15
58/male 33/female 44/male 23/female 26/male 30/female 26/female
16 17 18 19 20 21 22
Subject 1 2 3 4 5 6
Mutations* 329-330del AC
Change in amino acid Frameshift at AA 110
Reference(s) Kashiwagi et al.16
329-330del AC
Frameshift at AA 110
Kashiwagi et al.16
1228-1239delATTGTGCCTATT
Deletion of Ile-Val-Pro-Ile
II II
329-330del AC 1228-1239delATTGTGCCTATT
Frameshift at AA 110 Deletion of Ile-Val-Pro-Ile
−/+ −/+ −/+ −/+ −/+ −/− −/+
II II II II II I II
329-330del AC 1156C>T 1344insTCTT
Frameshift at AA 110 Arg386Trp Frameshift at AA 448
Kashiwagi et al.,17 Tanaka et al.19 Kashiwagi et al.16 Kashiwagi et al.,17 Tanaka et al.19 Kashiwagi et al.16 Xu et al.18 This study
371C>T; 1008G>T 1228-1239delATTGTGCCTATT
Pro124Leu; Gly336Gly Deletion of Ile-Val-Pro-Ile
23/male 27/female 70/female
−/+ −/+ −/+
II II II
14G>A; 1226A>G
Arg5Gln; Tyr409Cys
1228-1239delATTGTGCCTATT
Deletion of Ile-Val-Pro-Ile
Kashiwagi et al.,17 Tanaka et al.19
31/female 31/male 37/male 24/male
−/+ −/+ −/+ −/+
II II II II
1163A>T
Gln388Leu
This study
* GenBank NM 001001548; the first mRNA nucleotide encoding CD36 protein is +.
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This study Kashiwagi et al.,17 Tanaka et al.19 This study
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TABLE 2. Mutations in the exons of human CD36 gene Exon number
mRNA nucleotides*
1 2 3
−356 to −184 −183 to −90 −89 to 120
4
121 to 281
5
282 to 429
6 7
430 to 609 610 to 701
8 9 10
702 to 748 749 to 818 819 to 1006
11
1007 to 1125
12
1126 to 1199
13
1200 to 1254
14
1255 to 1419
15
1420 to 2044
Change in nucleotide sequence
Change in amino acid sequence
del Exon1 del Exon2 14G>A del Exon3 268C>T 121-126delgCAAGTT 220C>T 319-324delGCTGAG 329-330delAC 367G>A 371C>T 411T>C 380C>T 560insT 619-624del ACTGCA/ins AAAAC 691-696delAAAGGT None 760T>C 845-849delACGTT 949insA 975T>G 1079T>G 1226A>G Del TTTAGAT 1140-1146delTTTACAA/insCCAAA 1150G>C 1155delA 1163C>T 1156C>T Del TATTACAGAG Dupl. 1204-1246 1218-1224del GAGGAAC 1228-1239del ATTGTGCCTATT
No expression of CD36 protein No expression of CD36 protein Arg5Gln No expression of CD36 protein Pro90Ser Unknown Gln74Term In-frame delAA 107-108 Frameshift at 110 Glu123Lys Pro124Leu Ala137Val Ser127Leu Frameshift at AA187 Frameshift at AA 207 In-frame del AA 231-232 Phe254Leu Frameshift at AA282 Frameshift at AA 317 Tyr325Term Leu360Term Tyr409Cys Skipping Exon 12 Frameshift at AA 380 Ala383Pro Frameshift atAA385 Gln388Leu Arg386Trp Skipping exon 13 Frameshift at AA 416 Frameshift at AA 406 Del IIe-Val-Pro-IIe
1237A>C 1344insTCTT
IIe413leu Frameshift at AA 448
Reference(s) Sato et al.21 Yanai et al.22 This study Sato et al.21 Yanai et al.22 Xu et al.18 Kashiwagi et al.16 Kashiwagi et al.16 Kashiwagi et al.23 This study Imai et al.24 Omi et al.25 Tanaka et al.19 Kashiwagi et al.17 Aitman et al.26 Hanawa et al.27 Aitman et al.26 Kashiwagi et al.28 Aitman et al.26 Lepretre et al.29 This study Tanaka et al.19 Aitman et al.26 This study Xu et al.18 Tanaka et al.19 Tamala et al.19 Kashiwagi et al.17 Kashiwagi et al.,17 Tanaka et al.19 Imai et al.24 This study
* GenBank NM 001001548; the first mRNA nucleotide encoding CD36 protein is +1.
high occurrence was shown in Japanese and Korean populations.17,20 However, neither this study nor the study by Xu and colleagues discovered this mutation in the Chinese population. In this study, some new CD36 mutations were identified by direct nucleotide sequencing and clonal sequencing (see above). 371C>T located in Exon 5 was associated with Type I deficiency, and the rest of the mutations were related to Type II deficiency. Most of these mutations were located in exons of CD36 gene and led to corresponding amino acid substitutions except for 1008G>T, which was a synonymous mutation. In addition, 1344insTCTT located in Exon 14 led to a frameshift at AA 448 and caused loss of stop codon. Up to now, more than 30 kinds of mutations have been discovered in the CD36 gene (Table 2). However, the study on gene level is not sufficient to unravel the molecular behavior on protein level. Further research is necessary to explore the mechanism of CD36 deficiency in these mutations. The spectrum of CD36 mutations differs in various populations. The study results show that CD36 deficiency
exists in Shanghai population, and they are at risk for antiCD36 immune reaction induced by transfusion. In light of these observations, it is necessary to detect anti-CD36 in suspected immune-mediated thrombocytopenia. Establishment of a donor registry of CD36-deficient individuals would help patients with these disorders. Our research on frequency and type of CD36 deficiency in Shanghai population will not only provide assistance for clinical diagnostic of alloimmune conditions, but also give proper guidance to the safety and rationality of clinical blood transfusion. Moreover, the study on molecular mechanism of CD36 deficiency may provide reference for further researches on physiologic functions of CD36 as well as CD36-associated diseases.
ETHICS STATEMENT The protocol was approved by the Medical Ethics Review Committee of Shanghai Blood Center (Permit Number SBC-IRB-2014-02). Volume **, ** **
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Fig. 2. cDNA nucleotide and amino acid sequences of CD36 with mutations indicated as follows: underline = substitutions; vertical bar = insertions. 6
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Fig. 3. Sequencing chromatogram of the new mutations. (A) 14G>A in Exon 3 (Donor 16); (B) 1344insTCTT in Exon 4 (Donor 11); (C) 371C>T in Exon 5 (Donor 14); (D) 1008G>T in Exon 10 (Donor 14); (E) 1163A>T in Exon 12 (Donor 20); (F) 1226A>G in Exon 12 (Donor 16). CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s Web site: Table S1. PCR and sequencing primers for CD36 gene
